Pathogen-host interactions in Pseudomonas aeruginosa respiratory ...

Pathogen-host interactions in Pseudomonas aeruginosa respiratory infections

February

2010

Mariette Barbier

Doctoral Thesis directed by : Dr. Sebastiá Albertí Serrano Universitat de les Illes Balears

Everything that can be counted does not necessarily count; Everything that counts cannot necessarily be counted. Albert Einstein

On the front page: Confocal microscopy observation of the interaction of Pseudomonas aeruginosa (stained in green) with the human bronchoepithelial cell line 16HBEo- labeled with a platelet activating factor receptor specific antibody (stained in red).

Acknowledgements

Acknowledgements Cada uno de nosotros tiene un camino, que va dibujando cada día con sus ilusiones y aspiraciones, anhelos y tropiezos, decisiones y desengaños. Mi camino empezó hace tan solo 25 años, y hacer este doctorado parecía la manera más lógica de seguirlo. Algunos persiguen el reconocimiento social, otros la recompensa económica, y algunos tan solo el conocimiento. Para mí, este doctorado ha sido un camino de humildad, en el que he tenido que volver a cuestionar constantemente mi manera de razonar y de trabajar, para aprender de todos y cada uno de los que han compartido conmigo este camino. Me he sentido pequeña, inútil a veces, impotente delante de problemas sin aparente solución e insignificante en este mundo de “científicos y sabios”. He descubierto un universo que me ha ilusionado con su energía y su complejidad, que me ha cautivado y envuelto a lo largo de estos años, pero que a la vez me ha desilusionado con su hipocresía, conformismo y decadencia. Durante estos años, he crecido como persona y como científica, a base de sacrificios, pero también gracias a los placeres y privilegios que concede la vida de becaria. He conseguido adquirir conocimiento, agudizar mi espíritu crítico, aprender nuevos idiomas, viajar, practicar respeto y aprender a trabajar en equipo. Para mí, todo esto vale mucho más que un título académico. La persona que estuvo en el origen de todo esto ha sido el Dr. Sebastián Albertí Serrano, también conocido como “el jefe”. Un simple e-mail enviado originalmente a Javier Benedí pidiendo una estancia en su laboratorio para hacer unas prácticas basto para que Sebastián aceptase acogerme en su grupo y guiarme. Siempre ha estado al lado

de

sus

becarios,

indicándonos

el

camino

a

seguir,

animándonos

y

presionándonos, para poder sacar día a día lo mejor de cada uno. Le estoy muy agradecida por su apoyo y su confianza que me ha mostrado a lo largo de los años, y seguiré teniéndole siempre la misma admiración. Pero nada durante esta tesis hubiese sido igual sin el apoyo de su grupo. Quiero dar a cada uno de los con quien he compartido horas detrás de las pipetas mi más profundo agradecimiento. Algunos me enseñaron la mayor parte de lo que he podido poner en práctica en la pollata estos últimos años. Catalina Crespí me introdujo al mundo de la inmunología, enseñándome a manejar cualquier técnica a mi disposición

Mariette Barbier

Pathogen-host interactions in P. aeruginosa respiratory infections

Acknowledgements

que involucrase anticuerpos. Debo todo lo que se sobre cómo manejar y mimar cultivos celulares a Marta Franco Capó, quien tuvo la inmensa paciencia para enseñarme a trabajar con ellas, a pesar de mis frecuentes “metiduras de pata”. María “de Soller” y Mercedes Urdiain, aparte de ser compañeras de laboratorio muy apreciadas, tuvieron la paciencia de enseñarme castellano y hacer que me sintiese más integrada en el grupo. Pero sobre todo, la persona con quien más cosas he compartido en este grupo y que ha estado a mi lado durante la integralidad de esta tesis ha sido Laura García Sureda. Claro está que tanto tiempo compartido crea tanto vínculos como roces, pero su apoyo y su amistad siguen siendo imprescindibles y muy valiosos. He aprendido mucho con ella, y espero seguir haciéndolo. Quiero dar igualmente las gracias a Inmaculada Martínez Ramos, por siempre saberme subir la moral, y por recordarme la ilusión que cada uno debería seguir guardando. Ha sido una grandísima compañera de laboratorio y amiga, con quien espero seguir trabajando mucho tiempo. I would like to give very special acknowledgement to Dr. Joanna Goldberg, who was a key player in this work together with the members of her group. I own her the whole study on EF-Tu identification, as well as fruitful discussions that allowed the progression of this project. I want also to thanks Josh Owings, for the interesting exchange of ideas concerning this project, as well as helping me not to feel too useless concerning the election and optimization of the harassing method of screening for phosphorylcholine used in this study. I am looking forward carrying on this exciting work with both of them. During those four years and a half of PhD, I had the opportunity to visit two different laboratories in Europe that participated greatly in the success of my work. I am very thankful to Dr. Kenneth Reid for welcoming me in his lab at the Oxford University (United Kingdom) and to Paul Townsend for his patience and good willing while teaching me the purification techniques for surfactant proteins. He gave me access to unimagined amounts of BAL, and to one of the most magical places on earth: the Bodleian library. I am also in debt with Dr. Vitor Martin dos Santos, who welcomed me in his lab at the HZI, in Braunschweig (Germany) and made possible the array experiment. I specially want to thanks Dr. Piotr Bielecki, who taught me almost any single thing I know about molecular biology, and who was the greatest labmate I ever had. I am very grateful with him to make my stay pleasant and unforgettable, as well as being a great friend with whom I look forward working again in the future.

Mariette Barbier


Acknowledgements

Me gustaría agradecerle a Jesús Blasquez su hospitalidad en las numerosas visitas que he hecho al CNB de Madrid. Le estoy muy agradecida por poner a mi disposición su librería de mutantes, equipos y material de laboratorio, así como por hacerme un hueco en su grupo y participar en que siempre me sintiera como en casa. Estas estancias se han hecho inolvidables gracias a la participación de todos los miembros de su grupo, sobre todo a Alejandro Couce Iglesias, Alexandro Rodríguez Rojas, Javier Ramos Guelfo, Alfredo Castañeda García, Thuy Do Thi y Chema. Me han sorprendido siempre de mil maneras y enseñado mucho. Son para mí unas personas excepcionales, que han ganado cada día mi mayor respeto y admiración. Siempre recordaré numerosos momentos inolvidables pasados con ellos, incluyendo un desafortunado incendio. Espero de todo corazón que podamos seguir colaborando en proyectos futuros. Estoy también muy agradecida con el Dr. Antonio Oliver, que me ha facilitado la gran mayoría de las cepas empleadas en este estudio, así como a los miembros de su equipo que siempre se han mostrado cooperativos. Entre todos ellos, me gustaría darles gracias especialmente a Tomeu por robarme las pipetas y el sitio de trabajo y devolvérmelo siempre con una sonrisa y algún que otro comentario de los suyos, a Xavi, por convertir largas horas de clase en momentos algo más divertidos y a Mariló, por sus conocimientos en métodos de estudio de la formación de biofilms, que me fueron de gran ayuda. Me gustaría igualmente agradecerles a Teresa de Francisco y Biel Martorell su ayuda y paciencia para la puesta a punto de los métodos de infección murinos, y análisis de fosfolípidos por espectrometría de masas respectivamente. Jáimerais tout particulièrement remercier a ceux qui ont été a mes cotés depuis le début, et surtout malgré la distance: ma famille. Merci de mávoir aidé dans mes études et dans la vie, en me facilitant la tâche et en me montrant le chemin. Je suis très orgueilleuse dávoir été élevée et dávoir grandis aux côtés de personnes aussi exceptionnelles. Même si ma condition de “stagiaire a temps complet et a durée indéterminée” nést pas toujours comprise, je sais que vous serez toujours là pour máppuyer. I would like as well to give special thanks to my proofreaders, who had the patience of going through the 168 pages constituting this thesis and pay attention to any single

Mariette Barbier


Acknowledgements

coma out of space. Thanks a lot Cédric, Alicja, Dani and Monique for spending time correcting my twisted style and converting this manuscript in your bedside book. Me gustaría tambien darles las gracias a estos compañeros que han hecho que todo pareciese ser más fácil y más agradable durante los últimos años. Gracias Tomeu por ser mi “taxista” durante tanto tiempo, y compartir tantas horas de coche. Me enseñastes a tener paciencia, a tomarme la vida con tranquilidad y a relativizar sobre muchas cosas. Gracias por compartir estos cafés y debates sobre música, cine, o política, y ser un gran amigo. Quiero agradecerles también a Paco Carillo y Jose Antonio, por haber sido unos maestros excepcionales, y participar en mi desahogo diario. Me habéis enseñado a ser más fuerte, a sobrepasarme, a liberarme de mis demonios y a crecer como persona. Para mí siempre sereís modelos a seguir. Estoy también en deuda con Paco Fernandez por transmitirme tanta paz, enseñarme a controlarme durante todo el tiempo de la escritura y haberme permitido considerar muchissimas cosas bajo otro punto de vista There are many people who inspired me along the way. Teachers at high school or at the University, such as Mme Defalco, Mme Balzaretti and Mr Peyret, colleagues and friends, such as the “Scottish usual suspects” with whom I spent unforgettable moments and incited me to carry on with my studies. Among all of them, I want to give very special thanks to Jose, who shared with me everything during this thesis, supported me, and has been my principal source of inspiration for the last years. I am greatly in debt with him, since he shared with me every single bad and good moment during those years, including the late hours at work, the bad moods, the deceptions and the frustrations, and always found a way to make me smile again. Overall I am very grateful to him for opening my mind, and being the great person he is. Para acabar, me gustaría dar las gracias a la Conserjería de Hacienda e Innovación que me otorgó una beca predoctoral sin la cual la realización de esta tesis no hubiese sido posible. Gracias por ayudarme de

manera económica, pero también

facilitándome los desplazamientos al extranjero que han sido muy enriquecedores. Cabe destacar que representan la unidad administrativa más colaborativa y eficiente con la que he tenido el placer de tratar, lo que constituye un alivio en el desconcertante

mundo

de

la

administración

española.

Estoy

también

muy

agradecida al Gobierno español y al Ministerio de Educación y Ciencia por financiar las expensas materiales de estos estudios.

Mariette Barbier


Table of content

Table of content Acknowledgements ...................................................................................... I Table of content ............................................................................................ V Abbreviations ............................................................................................... IX

1. Introduction ............................................................................. 1 1.1.

Pseudomonas aeruginosa ................................................................ 3

1.1.1. Taxonomy and characteristics .................................................................................. 3 1.1.2. Infections caused by P. aeruginosa ......................................................................... 4

1.2.

Biology of P. aeruginosa respiratory infections............................... 6

1.2.1. P. aeruginosa virulence factors ................................................................................ 7 1.2.1.1.

Lipopolysaccharide ........................................................................................... 7

1.2.1.2.

Capsule .............................................................................................................. 10

1.2.1.3.

Pilus and flagellum ........................................................................................... 12

1.2.1.4.

Type III secretion system .................................................................................. 14

1.2.1.5.

Soluble factors .................................................................................................. 15 1.2.1.5.1.

Proteases ............................................................................................. 16

1.2.1.5.2.

Toxins .................................................................................................... 17

1.2.1.5.3.

Phenazines and P. aeruginosa pigments ...................................... 17

1.2.1.6.

Quorum sensing................................................................................................ 18

1.2.1.7.

Biofilm formation ............................................................................................... 18

1.2.1.8.

Molecular basis of P. aeruginosa adaptability ........................................... 20

1.2.1.9.

Phosphorylcholine as adhesin of major respiratory pathogens .............. 21 1.2.1.9.1.

Structure and function...................................................................... 22

1.2.1.9.2.

Genetics of ChoP expression .......................................................... 24

1.2.1.9.3.

Regulation of ChoP expression ....................................................... 25

1.2.1.9.4.

ChoP in P. aeruginosa ...................................................................... 26

1.2.2. Immune response to P. aeruginosa respiratory infections .................................28 1.2.2.1.

Humoral innate immunity................................................................................ 29 1.2.2.1.1.

Pulmonary surfactant ....................................................................... 29

1.2.2.1.1.1. Structure of SP-A and SP-D ................................................ 30 1.2.2.1.1.2. Receptors for hydrophilic surfactant proteins ............... 32 1.2.2.1.1.3. Immune functions of hydrophilic surfactant proteins .. 34

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Table of content

Bactericidal effect ...................................................................... 34 Opsonization ................................................................................ 34 Immunomodulation of the cellular immune system ............. 35 1.2.2.1.1.4. In vivo relevance of immune functions of hydrophilic surfactant proteins during bacterial infections .............................................................................. 36 1.2.2.1.2. 1.2.2.2.

Other antimicrobial factors ............................................................. 37

Cellular innate immunity ................................................................................. 39 1.2.2.2.1.

Airway epithelial cells ....................................................................... 39

1.2.2.2.2.

Alveolar macrophages .................................................................... 42

1.2.2.2.3.

Neutrophils .......................................................................................... 43

2. Objectives ............................................................................. 47 2.1.1. Determination of ChoP expression and role in P. aeruginosa pathogenesis ..............................................................................................................49 2.1.2. Role of SP-A in the interaction of P. aeruginosa with the airway epithelial cells ...............................................................................................................................50

3. Materials and Methods ......................................................... 51 3.1.

Bacterial strains, cellular cultures and specific reagents ............ 53

3.1.1. Bacterial strains...........................................................................................................53 3.1.2. Cell cultures.................................................................................................................54 3.1.3. Bronchoalveolar lavage...........................................................................................55 3.1.4. Human surfactant protein A purification ..............................................................55

3.2.

Bacterial adhesion and internalization assays ............................. 57

3.3.

Immunodetection assays ............................................................... 60

3.3.1. Detection of ChoP and EF-Tu by Western blot analysis .....................................60 3.3.2. Flow cytometry analysis ............................................................................................61 3.3.3. Immunofluorescence microscopy .........................................................................61 3.3.4. SP-A binding assays ...................................................................................................62

3.4.

3.3.4.1.

Immunoblot ....................................................................................................... 62

3.3.4.2.

ELISA.................................................................................................................... 63

Identification of the 43 kDa ChoP-associated protein ................. 64

3.4.1. Two-dimensional gel electrophoresis .....................................................................64 3.4.2. Coimmunoprecipitation assays ..............................................................................65

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3.4.3. Protein identification by µLC/MS and MS/MS ......................................................65

3.5.

Molecular tools ................................................................................ 66

3.5.1. Transcriptome analysis ..............................................................................................66 3.5.2. Southern blot analysis ...............................................................................................67

3.6.

Other methods ................................................................................. 69

3.6.1. Murine model of lung infection ...............................................................................69 3.6.2. Bacterial survival and competition assays ............................................................69 3.6.3. Alginate quantification.............................................................................................69

4. Results .................................................................................... 71 4.1.

Determination of ChoP expression and role in P. aeruginosa pathogenesis ................................................................................... 73

4.1.1. Expression of the ChoP epitope among P. aeruginosa isolates from acute and chronic infections ..............................................................................................73 4.1.2. Identification of the 43-kDa ChoP-containing protein .......................................74 4.1.3. Surface location of ChoP epitope .........................................................................78 4.1.4. Effect of a PAF-R antagonist on the adhesion to and invasion of bronchial epithelial cells by P. aeruginosa ...........................................................80 4.1.5. Effect of PAF-R antagonist on P. aeruginosa lung infection in vivo .................82 4.1.6. Identification of the genes involved in ChoP association to EF-Tu in P. aeruginosa ..................................................................................................................83 4.1.6.1.

Screening for P. aeruginosa genes involved in ChoP biosynthesis and expression genes in other microorganisms ......................................... 83

4.1.6.1.1. Screening for homologs to lic-1, betT, pmtA, pcs or pptA............... 83 4.1.6.1.2. Screening of the genes involved in choline and ChoP metabolism in P. aeruginosa ................................................................. 84 4.1.6.2.

Screening for genes differentially expressed at 25ºC and 37ºC in PAO1 by microarrays ....................................................................................... 85

4.1.6.3.

ChoP screening in a transposon library ....................................................... 86

4.1.7. Study of the conservation of PA4178 in P. aeruginosa clinical isolates by Southern blot...............................................................................................................87

4.2.

Role of SP-A in P. aeruginosa interaction of with airway epithelial cells .................................................................................. 89

4.2.1. SP-A purification and detection .............................................................................89 4.2.2. Role of alginate exopolysaccharide in the resistance of P. aeruginosa to BAL and SP-A bactericidal effects .........................................................................91

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4.2.3. Role of alginate production in SP-A binding to P. aeruginosa .........................93 4.2.4. Effects of SP-A on the invasion of lung epithelial cells by P. aeruginosa ........95 4.2.5. p63 mediates P. aeruginosa internalization by airway epithelial cells ............98

5. Discussion ............................................................................ 101 Determination of ChoP expression and role in P. aeruginosa pathogenesis ............................................................................................ 103 Role of surfactant protein A in P. aeruginosa interaction with airway epithelial cells.............................................................................. 107

6. Conclusions ......................................................................... 111

7. References ........................................................................... 115

8. Appendix ............................................................................. 145 8.1.

Reagent composition .................................................................... 147

8.2.

Supplementary results ................................................................... 149

8.2.1. Table of the P. aeruginosa genes homologs to genes involved in ChoP biosynthesis and expression in other microorganisms ......................................149 8.2.2. Arrays data................................................................................................................151 8.2.2.1.

Genes with increased expression levels in PAO1 grown at 25ºC versus 37ºC ...................................................................................................... 151

8.2.2.2.

Genes with decreased expression levels in PAO1 grown at 25ºC versus 37ºC ...................................................................................................... 154

Mariette Barbier


Abbreviations

Abbreviations ADP ....................................... Adenosine diphosphate AHL ........................................ Acyl-homoserine lactones ATCC ..................................... American Type Culture Collection APTase .................................. Adenosine triphosphatase aRNA ..................................... Antisense RNA BAL......................................... Bronchoalveolar lavage BCIP-NBT ............................... 5-Bromo-4-chloro-3-indolyl phosphate/Nitro blue tetrazolium BPI .......................................... Bactericidal-permeability increasing protein BSA ........................................ Bovine serum albumin CCTα ..................................... Phosphocholine cytidylyltransferase cDNA .................................... Complementary DNA ChoP ..................................... Phosphorylcholine CF .......................................... Cystic Fibrosis CFTR ...................................... Cystic fibrosis transmembrane regulator cfu ......................................... Colony-forming unit CKAP4 ................................... Cytoskeleton-associated protein 4 CLIMP63 ................................ Cytoskeleton-linking membrane protein COPD .................................... Chronic obstructive pulmonary disease CRD ....................................... Carbohydrate recognition domain CRP ........................................ C-reactive protein CSF ........................................ Colony stimulating factor DAPI....................................... 4’-6-diamidino-2-phenylindole DMG ...................................... Dimethylglycine DNA ....................................... Desoxyribonucleic acid DNase ................................... Deoxyribonuclease DPPC ..................................... Dipalmitoylphosphatidylcholine EDTA ...................................... Ethylenediaminetetraacetic acid EF-Tu ...................................... Elongation factor Tu ELISA ...................................... Enzyme-linked immunosorbent assay ERGIC-63 .............................. Endoplasmic Reticulum-Golgi Intermediate Compartment-63 ETA ......................................... Exotoxin A FDR ........................................ False discovery rate FPLC ...................................... Fast protein liquid chromatography GB .......................................... Glycine-betaine GRO ...................................... Growth regulated oncogene GTPase .................................. Guanosine triphosphatase

Mariette Barbier


Abbreviations

HEPES..................................... 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HIV ......................................... Human immunodeficiency virus ICAM-1 .................................. Intercellular adhesion molecule-1 Ig ............................................ Immunoglobulin IL ............................................. Interleukin INF-γ ...................................... Interferon γ pI ............................................ Isoelectric point LB ........................................... Luria Bertani LBP ......................................... LPS-binding protein LPS ......................................... Lipopolysaccharide mAb ...................................... Monoclonal antibody MAPK..................................... Mitogen-activated protein kinase MBP ....................................... Mannose binding protein MCP ...................................... Monocyte chemoattractant protein MHC ...................................... Major histocompatibility complex mRNA .................................... Messenger RNA MS/MS .................................. Tandem mass spectrometry µLC/MS ................................ Microcapillary liquid chromatography mass spectrometry NF- κB .................................... Nuclear factor-κB NUSE ...................................... Normalized unscaled standard errors PAF ........................................ Platelet activating factor PAF-R ..................................... Platelet activating factor receptor PAGE ..................................... Polyacrylamide gel electrophoresis PAR ........................................ Proteinase-activated receptor PC .......................................... Phosphatidylcholine PBS ......................................... Phosphate Buffered Saline pfp ......................................... Percentage of false-positives RLE ......................................... Relative log expression RNase .................................... Ribonuclease RPMI ...................................... Roswell Park Memorial Institute SDS ......................................... Sodium dodecyl sulfate SP ........................................... Surfactant protein TE ............................................ Tris-EDTA TLR .......................................... Toll-like receptor TNF-α ..................................... tumor necrosis factor α TRITC ...................................... Tetramethyl rhodamine iso-thiocyanate TSA ......................................... Trypticase soy agar uTP ......................................... Uridine-5'-triphosphate

Mariette Barbier


Immune response to P. aeruginosa respiratory infection

1. Introduction

Introduction

Pseudomonas aeruginosa

1.1. Pseudomonas aeruginosa 1.1.1. Taxonomy and characteristics Pseudomonas aeruginosa is a Gram negative bacillus with unipolar mobility that was first isolated from infections in 1882 by Gessard, who called it Bacillus pyocyaneus [1]. This bacterium belongs to the class of gamma proteobacteria and to the family of Pseudomonadaceae [2]. In 2000, the genome of one of the type strains, PAO1 (isolated from a patient with an infected burn wound in 1955) was sequenced. P. aeruginosa has one of the largest bacterial genomes ever sequenced, with around 6.25 Mbp, and 5,570 predicted genes on the chromosome which provides insights into the basis of the genetic complexity and ecological versatility of the bacterium [3]. This bacterium is a straight rod, slightly curved, between 1.5 and 3 µm long and 0.5 to 0.8 µm large, and mobile due to the presence of a polar flagellum. This bacterium is a common inhabitant of soil and water, and is able to colonize multiple environmental niches utilizing many compounds as energy sources. Its nutritional requirements are simple and it has a predilection for growth in moist environments. It is strictly aerobic but is able to use nitrate as electron receptor in anaerobic conditions [4]. This bacterium produces oxidase and catalase, and grows in MacConkey medium as lactose nonfermenting colonies. P. aeruginosa grows at ambient temperature and at temperatures up to 42ºC, unlike most of the species of this genus; however, its optimal growth temperature is 37ºC. Another characteristic of P. aeruginosa is the production of pigments, such as pyocyanin (blue-green), pyoverdin (fluorescent yellow-green), pyorubin (brown-red) and pyomelanin (dark brown), of which combinations give the bacterial colonies a particular color.

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Introduction


1.1.2. Infections caused by P. aeruginosa P. aeruginosa is considered to be essentially an opportunistic pathogen that has emerged as one of the major opportunistic human pathogen during the past decades, maybe as a result of its broad resistance to antibiotics and disinfectants that select this bacterium against other environmental bacteria [5-6]. As opportunistic pathogen, P. aeruginosa causes infections in immunocompromised patients, such as burn patients, transplant recipients, neutropenic patients and patients with human immunodeficiency virus (HIV) [7-8]. P. aeruginosa causes a wide range of acute infections. As a matter of fact, P. aeruginosa is responsible for 10% of community acquired infections caused by Gram negative organisms, and for 10% of all nosocomial infections [9]. This bacterium is the most frequent cause of nosocomial and community acquired blood stream infections, and the second cause of nosocomial pneumonia (isolated in 21% of the cases) after Staphylococcus aureus [10]. In addition, P. aeruginosa is the most frequently identified pathogen in patients under mechanical ventilation and is associated to a high mortality rate in ventilator associated pneumonia compared with other pathogens [11]. P. aeruginosa also has emerged as an important source of burn wound sepsis, being isolated in more than half of the patients with burn wound infection [12]. More infrequently, P. aeruginosa may cause urinary tract infections, dermatitis, keratitis, soft tissues infections, bacteremia, bone and joint infections as well as a wide variety of systemic infections [13-15]. P. aeruginosa also chronically infects patients with significant underlying diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) or bronchiectasis. CF is an inherited life-threatening disease involving a genetic mutation that disrupts the cystic fibrosis transmembrane regulator (CFTR) protein, resulting in poorly hydrated and thickened mucous secretions in the lungs. Mutations in CFTR affect predominantly the Caucasian population, with an incidence rate in US of 1 for every 3,000 Caucasian new born. Overall, approximately 30,000 Americans have CF, making it one of the most common life-shortening inherited diseases. Furthermore, the CF lung is highly susceptible to P. aeruginosa infections and this microorganism plays a critical role in the development and progression of pulmonary disease in these patients [16]. Chronic P. aeruginosa respiratory infections affect between 54 and 80% of adults with CF [17-18], and recent studies described that 97% of the children with CF are already colonized

Mariette Barbier


Introduction


with P. aeruginosa by the age of 3 [19]. Chronic airway inflammation with recurrent P. aeruginosa infections is the major cause of morbidity and mortality in these patients [20]. Indeed, these infections are highly difficult to eradicate, partly due to the resistance of the bacterium to antibiotics and ultimately lead to pulmonary failure and death of the patient. P. aeruginosa is also associated to COPD. This disease represents the 5th leading cause of death in the world. COPD is characterized by airflow limitation both progressive and associated with an abnormal inflammatory response of the lungs to noxious particles or gases. The evolution of the disease is marked by frequent acute exacerbation that cause significant worsening of symptoms. P. aeruginosa is isolated in 8-13% sputum isolates of COPD patients with advanced airflow obstruction and in 18% of COPD patients under mechanical ventilation [21]. A recent study by Martinez-Solano et al. demonstrated that P. aeruginosa chronically infects patients with COPD [22]. Clonally related P. aeruginosa isolates were obtained in sequential exacerbation episodes of the disease in the same patients suggesting that P. aeruginosa establishes a chronic infection in the lung of those patients. Furthermore, during infection each clone diversified, showing an increased mutation rate, increased antibiotic resistance, and reduced production of proteases. Those isolates presented characteristics typical of P. aeruginosa isolates from chronic lung infections, such as a lower cytotoxicity, or higher biofilm production. P. aeruginosa plays also an important role in the evolution of bronchiectasis, an irreversible airway dilatation with chronic bronchial infection and inflammation. It is isolated in around 20% of the patients and participates in the worsening of the symptoms [23].

Mariette Barbier


Introduction

Biology of P. aeruginosa respiratory infections

1.2. Biology of P. aeruginosa respiratory infections P. aeruginosa is one of the major respiratory pathogens; however, the exact source and mode of transmission of P. aeruginosa is often unclear, mainly due to the ubiquitous presence of the microorganism in the environment. It is widely distributed and can grow in many aqueous habitats including soil, surface waters, sewage, plants and various foods. However, in hospitals, spread usually occurs from patient to patient through contact with hospital personnel, or by direct contact with contaminated reservoirs, foods or water [24-25]. Bacterial infections of the respiratory mucosa represent a dynamic interaction, to which both host and bacterial factors contribute. For that reason, the relative contribution of host and microbial determinants need to be considered, since bacterial presence in the lower respiratory tract can be due to both failure of host defense mechanisms and bacterial virulence. In this chapter will be discussed the virulence factors and mechanisms of P. aeruginosa involved in the process of infection and establishment of the bacterium in the respiratory tract, together with the mechanisms displayed by the host to control the infection and eliminate the microorganism.

Mariette Barbier


Introduction

Biology of P. aeruginosa respiratory infection

1.2.1. P. aeruginosa virulence factors P. aeruginosa virulence factors have been extensively studied to understand their function during the process of establishment of respiratory infections. However, new approaches still contribute to a better understanding of the factors that participate in P. aeruginosa virulence. For example, a recent study by Frisk et al. compared P. aeruginosa transcriptomes at different stages of in vitro lung epithelial cell infection. Out of the 162 genes differentially expressed at different infection times compared to non infecting bacteria, less than 14 % had been already associated to P. aeruginosa virulence. Among others, genes involved in membrane transport or encoding transcriptional regulators were shown to be upregulated, which may indicate their possible implication in P. aeruginosa virulence [26]. In another study by Bielecki, gene expression profiles of several P. aeruginosa isolates proceeding from burn or CF patients were compared to gene expression profiles of the same isolates forming in-vitro biofilms or recovered from planktonic cultures or lettuce infections [27]. Comparison of the results obtained in each condition indicates that different gene sets are involved. Furthermore, many of the genes overexpressed in both CF patients and burned patients are common and have not been related yet to P. aeruginosa pathogenesis. This study and similar ones provide insight into the genetic organisation responsible for P. aeruginosa virulence by identifying the function of previously uncharacterised operons. For that reason, a deeper study of the extended genome of P. aeruginosa is necessary for the understanding of the versatility of this pathogen during the process of infection. However, some virulence factors have been already well studied and their implication in P. aeruginosa virulence will be described in this chapter.

1.2.1.1.

Lipopolysaccharide

Lipopolysaccharide (LPS) is the major compound of P. aeruginosa outer membrane and greatly contributes to the structural integrity of the bacteria. The structure of P. aeruginosa LPS is typical of Gram negative bacteria and consists of the lipid A, the inner core, the outer core and the O-antigen (Figure 1 A). Lipid A is the hydrophobic anchor of LPS and is formed of a basic structure containing an N- and O-acylated diglucosamine bisphosphate backbone. Lipid A from laboratoryadapted P. aeruginosa strains has been shown to be penta-acylated in 75% of the cases, whereas lipid A is mainly hexa-acylated in strains proceeding from CF patients

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Introduction


[28]. Bound to lipid A is a relatively conserved inner core structure which contains two D-manno-oct-2-ulosonic acid residues and two L-glycero-D-manno-heptose residues [29], often phosphorylated with a 7-O-carbamyl group bound to the second heptose residue [30] (Figure 1 B). It has been shown that P. aeruginosa LPS inner core is essential for bacterial viability, since a mutation in the waaP gene that phosphorylates the first heptose group is lethal for the bacteria [31].

A

B

O-Antigen

Hexa-acylated Lipid A

Oligosaccharide subunit n

Core

Penta-acylated Lipid A

Lipid A

Figure 1: LPS structure in P. aeruginosa. Structure of P. aeruginosa LPS (A) and of possible forms of lipid A (B) [28, 32].

The outer core of P. aeruginosa LPS is usually synthesized as two different isoforms or glycoforms that contain an N-alanylated galactosamine residue, three D-glucose residues and one L-rhamnose residue, the position of which differs in the two glycoforms. There is extensive O-acetylation of the hydroxyl groups in the outer-core sugars, and the terminal rhamnose residue is often acetylated when not substituted with another monosaccharide. Attached to the outer core is a repetitive glycan polymer, or O-antigen. It is formed of repeated units containing 3–4 individual monosaccharides (except for serogroup O7, which contains disaccharide repeat units). Typical sugars within the P. aeruginosa LPS O-side chains include N-acyl derivatives of different amino sugars along with rhamnose.

Mariette Barbier


Introduction


The O-antigen portion of P. aeruginosa LPS is responsible for conferring serogroup specificity, which is defined by antibodies specific to the different variants of this antigen. Isolates from chronically infected patients are generally unable to synthesize O-antigen side chains, and their LPS is defined as “rough”, as opposed to “smooth”, which defines O-antigen containing LPS. The genes encoding LPS biosynthetic enzymes of P. aeruginosa have not been as extensively studied as those of other pathogenic Gram negative microorganisms, such as Escherichia coli [33]. However, lipid A biosynthetic pathway is fairly conserved between Gram negative bacteria. LpxA, LpxC, LpxD, LpxB, and LpxK are involved in lipid A synthesis, catalyzing modifications of the sugar core and addition of lipid tails [34]. The product of the gene pagL is involved in lipid A 3-O-deacylation and participates in the modulation of LPS interaction with Toll-like receptor 4 (TLR-4) and host immune response [35]. galU which encodes a uridinetriphosphate-glucose-1-phosphate uridylyltransferase

and

algC,

encoding

a

bifunctional

phosphomannomutase/phosphoglucomutase, together with an operon made of the genes pyrG, kdsA and eno, were shown to be involved in core biosynthesis [36]. The products of the genes waaP, wapP and wapQ are involved in inner core phosphorylation and are necessary for bacterial resistance to antibiotics; the gene waaL encodes a protein responsible for linking the O-antigen to the core [37]. Oantigen biosynthesis has been related to 11 distinct biosynthetic loci, in which the enzyme coded by the gene wpbM was shown to be essential for O-antigen biosynthesis [38-39]. Lipid A is recognized by TLR-4 [40] and can also be bound with high affinity by LPSbinding protein (LBP), a 58–60 kDa glycosylated polypeptide. LPS also interacts with MyD88 and TRIF, and lead to activation of transcription factors, notably nuclear factorκB (NF-κB), which promotes production of inflammatory cytokines such as interleukin (IL) 1, IL-6, IL-8, and tumor necrosis factor α (TNF-α). The outcome of the host response to LPS is driven by the production of these immunomodulatory factors, which undergo complex transcriptional and translational regulation. P. aeruginosa LPS also participates in the process of interaction of the bacteria with epithelial and immune cells. P. aeruginosa LPS binds to CFTR on the surface of lung epithelial cells most likely through its core domain, provoking the internalization of the bacterium [41]. Those results were confirmed from studies with transgenic CF mice that demonstrated that this phenomenon results in the activation of a rapid NF-κB nuclear

Mariette Barbier


Introduction


translocation response in airway epithelial cells followed by production of IL-6, IL-8, CXC1 and intercellular adhesion molecule-1 in wild-type mice, but not in CF mutants [42-43]. Furthermore, LPS is a highly immunogenic molecule that has been targeted for years for vaccine development [44]. However, only the immunoglobulins (Ig) directed against Oantigens and not core are protective. It has been demonstrated that the loss of the Ochain in P. aeruginosa chronic rough LPS isolates is involved in a higher sensitivity of those strains to serum mediated bacterial killing [45].

1.2.1.2.

Capsule

P. aeruginosa is able to produce a capsule-like exopolysaccharide called alginate. Alginate is a negatively charged linear copolymer of partially O-acetylated β-1,4-linked D-mannuronic acid and its C5 epimer, α-L-glucuronic acid [46], which is synthesized under the form of a precursor (guanosine diphosphate-mannuronic acid) by the enzymes encoded by the genes algA, algC and algD [47]. The precursor is then polymerized and transported through the inner membrane by a combination of alg44 and alg8 gene products [48], and some of the mannuronate residues are epimerized to glucuronate residues by a C-5-epimerase (AlgG) [49]. Although the synthesis of alginate precursor is well understood, there are gaps of knowledge on how the polymerization, modification and export are coordinated. The gene products of algF, algI, algJ, algK, algL and algX participate in alginate modification, and AlgE is believed to be involved in its export through the outer membrane [50]. This process is controlled transcriptionally and post transcriptionally (Figure 2). The expression of the alginate biosynthetic operon (algD-algA) is mainly under the control of the algD promoter. A key element in the regulation of alginate production is AlgT, which induces the expression of AlgD and increases the expression of regulatory proteins that enhance algD transcription such as AlgR or AlgB [51]. AlgT inhibits the genes products of mucA and mucB. Stable mutations in those genes are frequent in mucoid strains since the inactivation of those genes leads to a deregulation of AlgT and an overproduction of alginate. More than 80% of mucoid P. aeruginosa strains isolated from CF patients harbor mutations in mucA [52].

Mariette Barbier


Introduction


algT (algU)

mucB (algN)

mucA

mucC mucD (algM) (algY) - 836 000

- 830 000

MucA

MucB

AlgT

argH

algZ

algR

algP

algB - 6 173 000

- 5 915 000

- 5 924 000

AlgZ

algQ

AlgR

kinB

AlgB

algD - 3 962 000

algT (algU)

- 500

- 200

Structural gene

Transcriptional activator

Constitutive promoter

Transcriptional inhibidor

Heat-shock promoter

Positive regulation

Transcription

Negative regulation

Gene name

- 830 000

Chromosomal location

Alternative gene name

Figure 2: Molecular basis of alginate production and regulation in P. aeruginosa [53].

Alginate is the major virulence factor of P. aeruginosa in CF lung infections [54]. The pulmonary function of patients with CF declines only when mucoid P. aeruginosa are isolated and associated lung pathology develops [55]. Alginate has been shown to provide the bacterium with a protection against aeration and continuous renewal of air in the lung could be a selection factor for mucoid strains in vivo [56]. Oxygen species released by polymorphonuclear leukocytes represent as well a pressure in vivo during lung inflammation, which leads to the selection of alginate overproducing mucA mutants [57].

Mariette Barbier


Introduction


Overproduction of capsule modifies electrochemical repulsion forces on the surface of the bacteria. Alginate is poorly immunogenic, shields the highly immunogenic elements present on the cell surface, and protects the bacterium against opsonization by the complement (C3b) or antibodies, and against phagocytosis [58]. Alginate is involved as well in modifications of the bacterial adherence to epithelial cells, and induces the secretion of pro-inflammatory cytokines, mucins and lyzozyme by airway epithelia [59].

1.2.1.3.

Pilus and flagellum

P. aeruginosa expresses a polar type IV pilus of a diameter of around 5.2 nm and an average length of 2.5 µm, which consists in a homopolymer formed of thousand copies of a 15 kDa protein subunit called pilin [60]. The pilus also contains an intrachain disulfide loop of 12 to 17 semi-conserved amino acid residues at the C-terminus of pilin, which corresponds to the pilus epithelial cell-binding domain [61]. Pilus biosynthesis involves several pil loci, which harbor the genes necessary for pilus assembly, the main one being the pilE locus, which contains the genes pilA, -B, -C, -D, -R and –S [62]. Type IV pili biogenesis involves a large number of proteins (12 or more), including the major pilin subunit, a specific inner-membrane prepilin peptidase that cleaves the N-terminal signal peptide, a specific adenosine triphosphatase (ATPase) that powers pilus assembly, an integral inner-membrane protein that recruits the ATPase from the cytoplasm and an integral outer membrane secretin that is necessary for the emergence of type IV pili on the bacterial surface [63]. The pilus provides the bacterium with a unique form of locomotion called twitching mobility. Twitching motility is a flagella-independent form of bacterial translocation over moist surfaces, which occurs by the extension, tethering, and then retraction of polar type IV pili, which operate in a manner similar to a grappling hook. Monoclonal antibodies (mAb) that bind specifically to the distal end of the pilus are highly effective at inhibiting pili adherence to human epithelial cells and isolated glycosphingolipid receptors, whereas mAb specific for the pili base have no effect on the pili binding. P. aeruginosa pili bind specifically to the carbohydrate moiety of the glycosphingolipids asialo-GM1 and asialo-GM2 through the recognition of the disaccharide sequence GalNAcβ(1-4)Gal [60]. Interestingly, asialo-GM1 was shown to be overexpressed in patients with CF, which suggests that P. aeruginosa pilus-asialo-GM1 interaction in CF patient could be critical for the progression of the infection [64]. P. aeruginosa pili bind as well to lactosyl ceramide and ceramide trihexoside, but to a much weaker extent

Mariette Barbier


Introduction


[65]. The pilus is also responsible for triggering NF-κB and the expression of proinflammatory cytokines through the activation of a complex including the receptor TLR2 [66]. P. aeruginosa also expresses a single polar flagellum, which exhibits the typical conserved structure established for many Gram negative pathogens (Figure 3) [67]. It is composed of a basal body with a L-ring associated with the LPS layer, a P-ring associated with the peptidoglycan layer, the M-ring embedded in the plasmatic membrane and the S-ring directly attached to the plasma membrane. The basal body is associated to a motor switch, responsible for generating the clock-wise rotation of the filament, the origin of bacterial mobility. The outer part of the flagellum is composed of a hook, and a filament formed of the protein FliC. The filament ends with a capping protein, FliD. Most P. aeruginosa isolates express one of two types of flagella (a or b) based on the deduced amino acid sequences of the major structural gene fliC, encoding the flagellin filament [68]. Flagellum biosynthesis and regulation is a complex process involving more than 40 different genes clustered in three different non-continuous regions of the chromosome. Flagellar genes are usually expressed in planktonically growing organisms and motile environmental strains are often associated with the initial stages of P. aeruginosa infections. However, P. aeruginosa isolates from chronically infected patients have been found to exhibit an RpoN mutant phenotype, lacking expression of both flagella and pili. The majority of environmental isolates and clinical isolates proceeding from acute infections are motile, whereas 39% of strains isolated from chronically infected patients are nonmotile [69]. The flagellum is involved in early stages of infection and activates both IL-6 and IL-8 production by binding to asialoGM1, TLR-2 and TLR-5 on the apical surface of airway epithelial cells, and NF-κB signaling [70]. P aeruginosa flagellum also binds to human mucins through the cap protein FliD and to MUC-1, a cell surface–associated mucin [71]. Recently, Zhang et al. demonstrated that P. aeruginosa flagellum is also involved in the resistance of the bacterium to the bactericidal effects of surfactant protein (SP) A [72]. As a matter of fact, a mutant of the gene flgE, encoding the hook structure of the flagellum, was cleared in wild-type mice but persisted in the lung of SP-A deficient mice. This effect was independent on SP-A mediated opsonization, but was due to a higher permeabilization of the bacterial membrane. The loss of flagellum was related to a poorer capacity of the bacterium to produce LPS, which resulted in a destabilized

Mariette Barbier



Introduction

outer membrane. A controversial study described that flagellin also increases matrilysin expression, a protease required in epithelial repair and for α-defensins activation [73]. Matrilysin levels are markedly elevated in the CF lung; however, mucoid chronic isolates from CF patients fail to produce flagellin, which suggest that flagellin is not the main factor responsible for variations in matrilysin expression levels in the chronic lung.

FliD Filament FliC

FlgL FLgK

Hook-filament junction

FlgE Hook

FlgG FlgH FlgF FlgI

Basal body Mot B MotA

Motor / Switch

FlhA FlhH FlhB

Outer membrane Peptidoglycan

FliE/FlgB/FlgC FlgF FliG Inner membrane FliM FliN

Figure 3: P. aeruginosa flagellar structure [74]

1.2.1.4.

Type III secretion system

Once adhered to epithelial cells, P. aeruginosa activates its type III secretion system, which is a major determinant of virulence, and allows the bacterium to inject toxins into the host cell. The type III secretion apparatus is composed of approximately 20 proteins, most of which are located in the inner membrane and form the “needle complex”, a 10

Mariette Barbier



Introduction

x 60 nm external needle inserted within a cylinder traversing both bacterial membranes and the peptidoglycan [75]. At a genetic level, P. aeruginosa type III secretion system consists of 43 coordinately regulated genes encoding the secretion and translocation machinery, regulatory functions, type III effectors and effector-specific chaperones [76], organized in 5 different operons on the genome of P. aeruginosa, except for exoU, acquired by horizontal transfer and present on a pathogenicity island [76]. All type III secretion system genes are under the direct transcriptional control of ExsA, which controls the expression of pscN, the popN to pcrR operon, the exsD-pscB-pscL operon, and the exsC to exsA operon. In addition, ExsA activates the transcription of genes encoding the secreted proteins ExoS, ExoT, and ExoU. This system requires pilin-mediated bacterial-epithelial contact and is responsible for the secretion of 4 known effector proteins with cytotoxic effects, ExoS, ExoT, ExoU and ExoY which interfere with eukaryotic signal transduction and result in cell death and alteration of the immune response. ExoU is a potent intracellular phospholipase and has been shown to cause irreversible damage to cell membranes and rapid necrosis [77]. ExoS and ExoT are proteins with guanosine triphosphatase (GTPase) activating protein activity and adenosine diphosphate (ADP) ribosyltransferase activity [76]. Those enzymes interfere with cytoskeletal functions, and provoke the loss of tight junctions by the disruption of small GTPases, required for cell polarity, tight junctions integrity and epithelium repair, and expose the basolateral surface of the epithelium [78]. ExoS also stimulates TNF-α production through its direct interaction with TLR-2 and TLR-4 [77, 79]. ExoY is an adenylate cyclase that increases lung epithelial cyclic adenosine monophosphate, which in turn inhibits phagocytosis and disrupts pulmonary endothelial barrier function. While much of the type III secretion system-mediated damage is probably caused by the translocated effectors, there is increasing evidence that insertion of the needle complex itself can contribute to host cell injury, possibly by allowing ion influx and/or by activating the innate immune response through activation of caspase-1-dependent cleavage of IL-1β.

1.2.1.5.

Soluble factors

Many receptors that trigger the adherence and internalization of pathogens are localized on the basolateral membrane [80]. Tight junctions are responsible for the

Mariette Barbier


Introduction


sealing of the paracellular space and avoiding pathogens interaction with basolateral receptors. P. aeruginosa produces several extracellular and cell-associated molecules called invasins such as rhamnolipids, elastase, alkaline protease and exoenzymes, that are able to disrupt tight junctions and expose the underlying receptors on the epithelial cell surface [81]. Some of those factors have been extensively studied and have shown to be very important in the process of establishment of respiratory infections by P. aeruginosa and can be classified as extracellular proteases, toxin, phenazines and pigments.

1.2.1.5.1. Proteases P. aeruginosa secretes various types of proteases, such as elastases, alkaline protease, and protease IV. There are two kinds of elastases produced by P. aeruginosa: LasA, a metalloprotease of 27 kDa, and LasB, a metalloprotease of 33 kDa. LasA possesses a low level of elastolytic activity, but is important as an enzyme that enhances the elastolytic activity of LasB by cleaving elastin at unknown sites [82]. This enzyme seems to play an important role in burn infections, since lasA mutants are avirulent in burn mouse model compared to the parental strain [83]. LasB prevents the proteinase-activated receptor (PAR) 2 activation by cleaving its N-terminal domain. Elastases activate the mitogen-activated protein kinase (MAPK) pathway, increasing IL-8 expression [84] and degrade immunoregulatory proteins such as monocytes chemotactic protein-1 and damage epithelium [85]. They participate as well in the protection of the bacterium against the humoral immune system by cleaving IgA [86], surfactant proteins A and D [87] and antibacterial peptides [88]. P. aeruginosa secretes as well an alkaline protease AprA with a molecular mass of approximately 50 kDa that participates in the degradation of the complement components C1q and C3, as well as cytokines and chemokines [89], suggesting that AprA could potentially function as an immunomodulatory agent during infection. Protease IV, a serine protease with a molecular mass of approximately 26 kDa, has been related to surfactant protein A and D cleavage and impairment of surfactant functions [90]. Several studies suggest that this enzyme plays an important role in P. aeruginosa pathogenesis in patients with CF or keratitis [91-92]. Recently, Kida et al. isolated a new exoprotease (LepA) distinct from known proteases which activates NF-

Mariette Barbier


Introduction


κB-driven promoter through human PAR-1, -2 or -4, and could participate in the modulation of immune response to P. aeruginosa infections [93]. Furthermore, extracellular proteases and phospholipase C elaborated by P. aeruginosa also function to supply the bacterium with aminoacids and phosphate respectively. This process is very important since the availability of nitrogen and phosphate in the lung is limited [94].

1.2.1.5.2. Toxins Most clinical P. aeruginosa isolates secrete exotoxin-A (ETA), a potent inhibitor of protein synthesis via ADP-ribosilation of elongation factor 2. ETA is encoded by the gene toxA, and has been shown to increase permeability of the airway epithelia by inhibiting the repletion of tight junction proteins and partially contributes to apoptosis [95-96]. ETA also kills airway epithelial cells causing mitochondrial dysfunction, superoxide production and desoxyribonucleic acid (DNA) degradation [97]. Various other cytotoxins and hemolysins produced by the bacterium participate to the process of disruption of the epithelial barrier. The cytotoxin called leukocidin is a poreforming protein that has a cytotoxic effect on most eukaryotic cells [98]. The two hemolysins, a phospholipase (PlcH) and a lecithinase, act synergistically to breakdown lipids and lecithin [99]. PlcH activity provides the bacterium with phosphate and choline, both needed for bacterial metabolism. P. aeruginosa produces as well rhamnolipids, which lead to an improper assembly and interactions of the tight junctions strands, which results in the release of the barrier that normally prevents the access of P. aeruginosa to the paracellular space and allows the passive invasion of the epithelium [81, 100]. Rhamnolipids are also responsible for ciliostasis of airway epithelia [101].

1.2.1.5.3. Phenazines and P. aeruginosa pigments There are other compounds that participate in disrupting the epithelium, such as the family of phenazines. The most studied are the blue pigment pyocyanin, 1hydroxyphenazine and phenazine-1-carboxylic acid. Those molecules penetrate biological membranes, impair cilliary function, disrupt the respiratory epithelium and exert proinflammatory effects [101-103]. During infection, phenazines participate as well in nutrient acquisition. The bacteria compete with the host transferrin and lactoferrin for

Mariette Barbier


Introduction


iron using siderophores [104]. In P. aeruginosa, pyoverdin and pyochelin are the two main siderophores and sequester iron from the environment, permitting the growth of the pathogen in a relatively iron-limited environment [105]. Interestingly, the expression of the genes encoding for systems of acquisition of iron and phosphate has been shown to be modified during epithelial cells colonization in vitro by P. aeruginosa compared to non-infecting control bacteria [26].

1.2.1.6.

Quorum sensing

During lung infections, P. aeruginosa has developed a mechanism to coordinate expression of genes important for adaptation to the environment. This response is controlled by quorum sensing systems, a complex regulatory circuit involving cell-to-cell signaling through the production of small diffusible molecules, acyl-homoserine lactones (AHL), also called auto-inducers. In P. aeruginosa two quorum sensing systems called Las and Rhl, control the expression of more than 100 genes in a cell-densitydependent manner. Once a sufficient amount of autoinducer molecules has accumulated, these signaling molecules bind to their cognate transcriptional activators LasR and RhlR. LasR regulates the transcription of many genes, including several virulence genes, such as lasA, lasB, and toxA, whereas RhlR enhances the transcription of lasB, and the rhamnolipid synthesis genes rhlAB. However the AHL are not only responsible for the regulation of bacterial adaptation to the lung but also modulate eukaryotic cell functions. AHL were shown to induce mucus secretion, vasodilatation and edema through the induction of cyclooxygenase 2 and prostaglandine E2 production [106]. P. aeruginosa autoinducers were also shown to be responsible for the induction of apoptosis of neutrophils and macrophages but not lung epithelial cells [107], and to stimulate IL-8 production by those cells by activating NF-κB [108].

1.2.1.7.

Biofilm formation

One of the characteristic features of P. aeruginosa chronic lung infection is the formation of biofilms, structured communities embedded in an extracellular polymeric matrix that coats a wide range of surfaces, including mucosal surfaces. Biofilms develop in a complex and well-coordinated manner that involves sensing and responding to signals, such as bacterial cell density, nutrient availability and energy sources present in

Mariette Barbier


Introduction


the environment. The switch toward the biofilm mode of growth is often considered to be a survival strategy for bacteria. The first step involved in biofilm formation is the attachment of planktonic bacteria to a surface through weak Van der Waals forces, a process that involves P. aeruginosa flagella and type IV pili (Figure 4 A) [109]. Adhesion to the surface becomes irreversible and bacteria loose mobility (Figure 4 B). The genes involved in alginate production (algC, algD and algU) are upregulated by quorum sensing and an exopolysaccharide matrix is formed (Figure 4 C and D). Biofilms follow a process of maturation and then dissemination to colonize new surfaces (Figure 4 E). Biofilms are resistant to the infiltration of polymorphonuclear cells, as well as opsonization by complement or immunoglobulin and phagocytosis, and make bacterial persistence more favorable [110]. Several studies demonstrated that alginate production in biofilms inhibits phagocytosis by alveolar macrophages [111] and that opsonic antibodies produced by CF patients are unable to mediate biofilm opsonophagocytic killing [112]. Biofilm formation participates as well in bacterial resistance to antibiotics. Planktonic cultures of P. aeruginosa are more sensible to antibiotics and the combination of 2 or 3 different antibiotics is often required to eliminate biofilms in vitro [113]. In the environment, biofilm formation provides protection to P. aeruginosa from a wide range of environmental challenges, such as ultraviolet and acid exposure, metal toxicity or dehydration [114]. Furthermore, the bacteria inside the mature biofilm express fewer virulence factors because of their stationary state of growth [115] and of quorum sensing regulation [116], and are less stimulatory to the mucosa because of their binding to the epithelium through indirect contact. These factors facilitate both the colonization of bacteria and their extended survival even under unfavorable conditions. Together, alginate production and biofilm formation by mucoid strains of P. aeruginosa contribute significantly to the resistance of these organisms against diverse treatment regimens and host defenses.

Mariette Barbier



Introduction

A

B

C

D

E

Figure 4: P. aeruginosa biofilm formation [117].

1.2.1.8.

Molecular basis of P. aeruginosa adaptability

One remarkable capacity of P. aeruginosa is that the isolates of the bacteria proceeding from chronic infections present a wide spectrum of colony variants. Those isolates can be mucoid, dwarf, nonmotile, non-flagellated, LPS-deficient, auxotrophic, or resistant to commonly used antibiotics [118-119]. A lower expression of surface antigens such as LPS or pilus, which are potent activators of host immunity, facilitates the evasion of the recognition of the bacterium by the immune system. It is likely that this wide range of phenotypes, often related to therapy failure, is a result of

the

continuous

adaptation

of

the

microorganism

to

the

changing

and

heterogeneous conditions of the deteriorated lung tissue in these patients [120]. Oliver et al. described various types of mutations of the different genes participating to the DNA mismatch repair system (mutS, mutL and uvrD) of some P. aeruginosa strains isolated from the lungs of chronically infected patients. Because of the inactivation of their DNA repair system, those strains display a hypermutable (mutator) phenotype. Mutator strains present a mutation rate between 100 and 1000 times higher than the standard mutation rate described for the species. Those hypermutable strains have been described to represent around 1% of natural populations of E. coli, Salmonella

Mariette Barbier


Introduction


spp, Helicobacter spp, Neisseria meningitidis, and S. aureus [121-124]. However, in patients with COPD or bronchiectasis and patients with CF, the mutator P. aeruginosa strains represent 53% and 36% respectively of the stains isolated from these patients [125-126]. It has been shown that this genotype contributes to the establishment of a wide spectrum of phenotypes, and particularly contributes to the resistance to host immunological defenses [127] or to antibiotics [125-126, 128-129], and may confer a selective advantage for bacteria, especially in stressful and fluctuating environments such as the lung [130-131].

1.2.1.9.

Phosphorylcholine as adhesin of major respiratory pathogens

One of the strategies used by microorganisms to interact with eukaryotic epithelial cells is the expression of bacterial substances that mimic host molecules and provide the bacterium with a key mechanism to interact specifically with host receptors. Phosphorylcholine (ChoP) production in pathogens has been described to fulfill this function. ChoP function in P. aeruginosa pathogenesis has not been studied yet. However, it is an important bacterial compound present in major respiratory pathogens, and has been demonstrated to be involved in the establishment of respiratory infections and the persistence of those microorganisms in the respiratory tract. To better understand the work presented in this thesis, the structure, functions and metabolic pathways of synthesis of ChoP will be introduced. ChoP has been detected in Gram positive bacteria such as Streptococcus spp. (associated to teichoic and lipoteichoic acids [132]), Lactococcus spp, and Bacillus spp [133], Mycoplasma fermentans (associated to membrane phosphoglycolipids [134]) and Gram negative bacteria such as Haemophilus influenzae (associated to LPS [135]), Salmonella typhimurium [136], or Neisseria spp (associated to the pilin of pathogenic species and LPS of commensal Neisseria [137-138]). ChoP has been as well described in eukaryotic pathogens such as Leishmania major, Trypanosoma cruzy or Schistosoma mansoni [139], in which ChoP is mainly associated to glycoprotein glycans or glycolipids, fungi and filarial nematodes [140].

Mariette Barbier


Introduction


1.2.1.9.1. Structure and function ChoP is as well common in eukaryotic cells. It is closely related to phosphatidylcholine (PC), a major phospholipid component of the eukaryotic plasma membrane and is structurally related to the platelet activating factor (PAF) or 1-O-alkyl-2-acetyl-snglycero-3-phosphorylcholine (Figure 5). This compound is produced by a wide range of eukaryotic cells, including endothelial cells, neutrophils and macrophages. PAF plays an important role in proinflammatory responses through its interaction mediated by the ChoP motive of PAF with the platelet activating factor receptor (PAF-R) present on various eukaryotic cell types. This receptor is a G-coupled protein of 39 kDa, with 7 αhelicoidal

transmembrane

domains

separated

by

polar

domains.

PAF-R

is

overexpressed during inflammation and the binding of PAF to this receptor plays an important role in the regulation of the immune response [141].

Among others, PAF

union to this receptor mediates the degranulation of granulocytes, monocytes, and macrophages, as well as the release of inflammatory cytokines and toxic oxygen metabolites through the activation of the MAPK response system. PAF-R activation is also responsible for the phosphorylation of various proteins by tyrosine kinase, which plays an important role in PAF and leukotriene B4 production regulation in neutrophils. Interaction of PAF with this receptor leads as well to the liberation of intracellular calcium through a phospholipase C dependent pathway [142].

A

B O O

O P

N+ O

O-

H3C

O

O

OO

P

N+ O

OFigure 5: Chemical structure of ChoP and PAF. Chemical structure of the bacterial ChoP motive (A) and host PAF (B). The ChoP epitope of PAF is highlighted in the blue square.

Mariette Barbier



Introduction

In 1990, Zimmerman et al. described for the first time PAF-R as a receptor for cellassociated phospholipids, mediating intercellular adhesion [143]. Few years later, Cundell et al. demonstrated that this type of interactions was not only specific to eukaryotic cells but could as well involve bacteria-eukaryotic cells interactions [144]. PAF-R was shown to be a receptor for various microorganisms such as Streptococcus pneumoniae that binds to interleukin activated endothelial and epithelial cells through this receptor. Furthermore, this study demonstrated that the bacterial load of this bacterium during lung infection was reduced in rabbits treated with a PAF-R antagonist compared to untreated control. PAF-R antagonist was also shown to be efficient to protect from mortality sickle cell mice, which express higher levels of PAF-R, during pneumococcal infection [145]. In addition, PAF-R deficient mice were shown to be more resistant to pneumococcal pneumonia, presenting a delayed and reduced mortality, as well as a reduced dissemination of the infection and inflammation compared to wild type mice [146]. Further work demonstrated that ChoP present on LPS of H. influenzae mediates the interaction of the bacterium with the PAF-R present on the surface of lung epithelial cells [147-148]. It was shown that this interaction triggers the cell response associated with PAF-R activation, such as increased levels of calcium and inositol phosphate [149], and that invasion of the bacterium was less efficient in eukaryotic epithelial cells treated with PAF-R antagonist or glucocorticoids lowering PAF-R expression [150]. Apart from its function as bacterial ligand, ChoP is related to the sensitivity to C-reactive protein (CRP) mediated killing of H. influenzae [151-152]. ChoP has been described as the main ligand for CRP. Binding of CRP to H. influenzae and Neisseria strains that express pili has been shown to be an effective mean of complement mediated killing and can be competitively inhibited by soluble ChoP [153-154]. ChoP is also involved in the resistance of this bacterium to the bactericidal effects of βdefensins and the antimicrobial peptide LL-37/hCAP18 [155]. Studies demonstrated that ChoP content increases in a dose dependent manner in response to treatment with the linear antimicrobial peptide LL-37 in H. influenzae, indicating that ChoP provides a selective advantage for the survival of this bacterium [155]. Another interesting fact is that the regulation of ChoP is linked in some microorganisms such as non-typeable H. influenzae

to

biofilm

formation.

Lipooligosaccharides

extracted

from

biofilm

communities of H. influenzae showed a higher ChoP content than planktonic cultures [156], and the expression of ChoP in H. influenzae has been linked to biofilm maturation [157].

Mariette Barbier


Introduction


Furthermore, a recent study suggests that PAF-R is involved in phagocytosis. Neutrophils of PAF-R deficient mice demonstrated a diminished phagocytosing capacity of P. aeruginosa [158]. The same results were observed in PAF-R deficient mice infected with Klebsiella pneumoniae, which showed higher load of bacteria in the lung and a lower percentage of neutrophils with engulfed bacteria compared to wild-type infected control [159].

1.2.1.9.2. Genetics of ChoP expression The mechanisms responsible for acquisition, biosynthesis, transfer, or the phase variation of ChoP have been described in different bacteria. In many of them, choline is a precursor for ChoP biosynthesis. This compound is usually acquired from the medium by the mean of transport systems such as BetT, a proton-motive-force-driven, high-affinity transport system for choline, first described in E. coli, but also present in H. influenzae [160-161]. In S. pneumoniae and H. influenzae, another transporter, LicB, is necessary for choline uptake and ChoP expression. Once inside the bacterium, choline enters the glycine-betaine (GB) degradation pathway, or is used as precursor for ChoP biosynthesis. Up to now, three different pathways have been described for ChoP biosynthesis and incorporation on bacteria (Figure 6). The first pathway described for ChoP biosynthesis is the lic-1 pathway, which is found in H. influenzae and S. pneumoniae [162-163] (Figure 6 A). Commensal Neisseria synthesize as well ChoP using a pathway homologous to the lic-1 pathway. The genes for this pathway, however, are absent in pathogenic Neisseria [137]. ChoP can be synthesized as well through a reaction catalyzed by the product of the gene pmtA, which is responsible for three sequential reactions of methylation of phosphatidylethanolamine that lead to the formation of ChoP (Figure 6 B). This pathway was found in Rhodobacter sphaeroides, Sinorhizobium meliloti, Bradyrhizobium japonicum, and P. aeruginosa [164167]. Another way of synthesis of ChoP is the pcs pathway which involves Pcs, a PC synthase, and allows the synthesis of ChoP from DCP-diacylglycerol in S. meliloti and P. aeruginosa [165, 167] (Figure 6 B).

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Introduction


In pathogenic Neisseria, the gene pptA encoding the pilin phosphorylcholine transferase A is responsible for the incorporation of ChoP on the pilus of those bacteria (Figure 6 C).

Figure 6: Metabolic routes involved in ChoP metabolism and expression. The lic-1 pathway (A) is present in microorganisms such as S. pneumoniae and H. influenzae. Both PmtA and Pcs pathways have been described in S. meliloti and P. aeruginosa while only PmtA pathway was described in R. sphaeroides, and B. japonicum for ChoP metabolism (B). The pptA pathway (C) is present in pathogenic Neisseria.

1.2.1.9.3. Regulation of ChoP expression In many organisms ChoP expression can be variable. In H. influenzae and Neisseria, the ChoP phenotype undergoes a phase variation. In H. influenzae, ChoP expression is regulated by multiple tandem repeats of the tetramer 5´-CAAT-3´ within the coding region of lic1, allowing the bacteria to quickly adapt to the environment [168]. The same genetic regulation of ChoP is seen in commensal Neisseria, which harbor between 5 and 13 5´-CAAT-3´ repeats in the lic1 locus [169]. A different mechanism was found in N. meningitidis, which shows a homopolymeric guanosine tract located within

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Introduction


the gene pptA. This repeat has variable lengths in different strains, from 8 to 11 bp and controls frameshift of pptA [138].

1.2.1.9.4. ChoP in P. aeruginosa In P. aeruginosa, ChoP is an intermediate product in the catabolism of host molecules such as PC and sphingomyelin (Figure 7). Those two products are degraded by P. aeruginosa hemolytic phospholipase C (PlcH) to form ChoP [170], which is transported through the outer-membrane to the periplasm where it is phosphorylated by phosphorylcholine phosphatase PchP to choline and phosphate [171]. The choline is then transported across the inner membrane to the cytoplasm [172-173], where it is further degraded to GB by BetAB and used as source of carbon, nitrogen or energy, and as osmoprotectant. GB is further demethylated to form dimethylglycine (DMG), monomethylglycerine (sarcosine) and glycine [174-175]. This degradation pathway is regulated by phosphate availability, since plcH can be induced by phosphate limitation in a PhoB-dependent manner, but also the transcription of plcH can be induced by GB and DMG via the transcriptional regulator GbdR [176]. Over twelve years ago, Weiser et al. demonstrated the presence of ChoP on an unidentified 43 kDa protein of 12 P. aeruginosa clinical isolates [177]. They demonstrated that P. aeruginosa reactivity with anti-ChoP antibodies was high at ambient temperature, and decreased when temperatures rose up to physiological temperatures. It was unclear at that time whether this modification contributed to the ability of P. aeruginosa to colonize the airway tract via interaction with PAF-R since the expression of ChoP was only detected at lower growth temperatures ( 6 per group) were intratracheally inoculated with the ChoP+ strain PAHM4 (A) or the ChoP- strain PAHM9 (B). Both strains were administered with saline (control) or saline solutions of PAF-R antagonist (0.25 or 0.5 µg), and the numbers of bacterial cells in lung homogenates were determined at 24 h. Results that are significantly different from those in untreated controls are denoted by an asterisk (2-tailed t-test).

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Results

Determination of ChoP expression and virulence in P. aeruginosa

4.1.6. Identification of the genes involved in ChoP association to EF-Tu in P. aeruginosa Both in vitro and in vivo approaches demonstrated that ChoP plays an important role in P. aeruginosa attachment and invasion of airway epithelial cells. To characterize in detail the function of ChoP in P. aeruginosa pathogenesis, it is crucial to determine the molecular basis of its association to EF-Tu and expression on the bacterial cell surface. The identification of the genes involved in ChoP association to EF-Tu represents the first step to construct isogenic mutants, essential for the comparison of ChoP+ and ChoPstrains with the same genetic background. For that reason, three different approaches were used to identify the possible genes involved in ChoP biosynthesis and association to EF-Tu.

4.1.6.1.

Screening for P. aeruginosa genes involved in ChoP biosynthesis and expression genes in other microorganisms

The genetics of ChoP expression have been described in various microorganisms. In this first approach, the genes in P. aeruginosa with homology to the genes involved in ChoP uptake, biosynthesis, metabolism and/or expression in other microorganisms were screened. The sequences of those genes and of their translation products were compared with P. aeruginosa PAO1 genome using the algorithms BLAST-n and BLAST-p respectively, on http://blast.ncbi.nlm.nih.gov/Blast.cgi. All sequences giving a BLAST score higher than 40 bits of with a percentage of identity superior to 30 were selected for SDS-PAGE and Western blot analysis.

4.1.6.1.1. Screening for homologs to lic-1, betT, pmtA, pcs or pptA The lic-1 operon has been shown to be responsible for the association of ChoP to the LPS and teichoic/lipoteichoic acids of H. influenzae and S. pneumoniae respectively. To determine if those genes were involved in ChoP expression in P. aeruginosa, the genome of the bacterium was compared with the genes forming the lic-1 operon (licA, licB, licC and licD) in S. pneumoniae and Haemophilus spp. A total of 28 genes that presented homologies with the genes from the lic-1 locus were selected.

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The same analysis was performed to identify homologs of the genes betT, responsible for ChoP intake in other microorganisms, for which the sequence of the gene betT in E. coli was compared to the genome of PAO1. A total of 14 with homologies to the gene betT were selected. The gene pmtA was also found to be involved in ChoP biosynthesis in R. sphaeroides, S. meliloti, B. japonicum, and P. aeruginosa. The sequences of this gene in each of these microorganisms were blasted against PAO1 genome to detect possible homologs and paralogs in P. aeruginosa. The same was done with the sequence of psc, responsible for ChoP biosynthesis in S. meliloti and P. aeruginosa. 12 genes with homologies to pmtA and 7 with homologies to pcs were selected. In N. meningitidis, the gene pptA is responsible for the association of ChoP to pilin. The sequence of this gene was compared to PAO1 genome and gave 1 match. Overall a total of 34 genes, listed in the appendix 8.2.1, were selected out of this analysis. The mutants for the corresponding genes in the PA14 transposon mutant library were tested for ChoP expression at 25ºC by Western blot, as described in the chapter 3.3.1. The mutants for 8 of the genes selected were not available in the PA14 transposon library and none of the tested mutant presented alterations of ChoP association to EF-Tu at 25ºC.

4.1.6.1.2. Screening of the genes involved in choline and ChoP metabolism in P. aeruginosa Because of the potential overlap of choline metabolism and ChoP synthesis, mutants from the PA14 transposon library lacking the different genes involved in choline metabolism in P. aeruginosa such as plcH, plcP, plcR, plcN, and gdbR coding for enzymes involved in the metabolism of choline through the glycine-betaine pathway were tested for ChoP expression at 25ºC and 37ºC. As with other selected mutants, none of them presented modifications in ChoP expression at both temperatures compared to the wild-type strain PA14. Those results indicate that none of these genes is individually required for ChoP expression in P. aeruginosa.

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4.1.6.2.

Screening for genes differentially expressed at 25ºC and 37ºC in PAO1 by microarrays

The second approach used to identify the genes involved in ChoP incorporation on EFTu was based on the fact that ChoP is associated to EF-Tu at temperatures lower than 33ºC in PAO1. For that reason, the gene expression profiles of the strain PAO1 at 25ºC and 37ºC were analyzed by microarrays. mRNA extracted from independent cultures of PAO1 in mid-exponential phase was amplified and labeled before being spotted onto 6 Affymetrix P. aeruginosa GeneChips (Figure 20). After normalization, the analysis of the data revealed that a total of 353 genes were differentially regulated between both temperatures with a pfp < 0.05. When comparing the expression profile of PAO1 grown at 25ºC compared to 37ºC, a total of 173 genes were upregulated at 25ºC and 180 downregulated. The complete results for the transcriptome comparison of PAO1 at both temperatures are detailed in the appendix 8.2.2.

A

B

Figure 20: Scan of spotted aRNA on PAO1 microarrays. Scan result of two representative chips hybridized with biotinylated aRNA from PAO1 grown at 25ºC (A) and 37ºC (B).

The mutants for the genes overexpressed at 25ºC in the PA14 transposon mutant library were tested for ChoP expression at 25ºC and 37ºC by Western blot, to detect possible genes involved in ChoP incorporation on EF-Tu at low temperatures. However, the mutants for the genes overexpressed at 37ºC were also screened at both temperatures

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to determine if any of them would be responsible for ChoP catalysis or removal from EFTu at 37ºC. Out of the 353 genes selected for the screening, 265 were available in the PA14 transposon mutant library. However, none of the mutant tested presented alteration of ChoP expression at 25ºC or 37ºC, suggesting that none of these genes is responsible alone for ChoP incorporation or removal of EF-Tu.

4.1.6.3.

ChoP screening in a transposon library

Since the methods mentioned above failed to identify genes involved in ChoP incorporation on EF-Tu, a systematic screening of the ordered transposon mutant library in PA14 was performed. Several attempts were made to develop a high-throughput assay to analyze ChoP expression, such as colony immunoblots, dot blot, ELISA and flux cytometry. However the most reliable method to determine ChoP expression remained SDS-PAGE separation of proteins from whole cell lysates, followed by Western blot using ChoP-specific mAb. As ChoP is expressed normally at temperatures lower than 33ºC, each mutant from the PA14 transposon library was screened for the lack of ChoP expression at 25ºC. Out of the 5,593 mutants present in the library, 5,514 were screened and only one, referred as PA14∆4178, showed no ChoP expression at 25°C. ChoP expression was also tested at 37°C in this mutant and was shown to be negative (Figure 21). Similar results were obtained with a PAO1 transposon insertion mutant in the same gene obtained from the University of Washington Genome Center by Josh Owings (from Dr. J.B. Goldberg´s laboratory at the University of Virginia, US). PAO1 tag locus for this gene is PA4178 and it encodes a “hypothetical protein” with a domain of methyltransferase. This gene is 744 bp long and situated at the position 4673963 – 4674706 on the chromosome of P. aeruginosa and has similar GC contents (65 %) compared to PAO1 (66.6 %).

25ºC

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Figure 21: ChoP expression in PA14 and PA4178 at 25ºC and 37ºC. Representative Western blot analysis of ChoP epitope expression in PA14 and PA14∆4178 grown at 25°C and 37°C. Molecular size marker is indicated on the left.

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4.1.7. Study of the conservation of PA4178 in P. aeruginosa clinical isolates by Southern blot In order to determine if the gene on the locus PA4178 is conserved in different unrelated clinical isolates and is not a characteristic of the type strains PAO1 and PA14, the presence of this gene was studied by Southern blot in different P. aeruginosa clinical isolates. A DNA probe synthesized from the gene PA4178 in PAO1 was hybridized against the digested DNA of 20 unrelated clinical P. aeruginosa isolates, of which 8 were proceeding from acute infections and 12 from chronic infections. A band was detected for each of the strains studied, which indicates that the gene PA4178 is conserved in P. aeruginosa (Figure 22). Furthermore, the size of the fragment detected in PA14∆4178 was around 1,000 bp larger than the band detected in PA14, consistent with the insertion of the MAR1xT7 transposon of 994 bp.

19329 6223 5148 4254 3372 2690 1882 1489 925 -

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Figure 22: Detection of the gene PA4178 in different clinical P. aeruginosa isolates by Southern blot. Southern blot for the detection of the gene PA4178 in 9 representative P. aeruginosa clinical isolates proceeding from acute infections (lane 1 to 5) or chronic isolates (lane 6 to 11), the type strains PAO1 and PA14 and the PA14∆4178 mutant (lane 12, 13 and 14 respectively). Molecular size markers are indicated on the left in bp.

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Results

Role of SP-A in P. aeruginosa interaction with the airway epithelial cells

4.2. Role of SP-A in P. aeruginosa interaction of with airway epithelial cells 4.2.1. SP-A purification and detection In order to study the effect of SP-A in the interaction of P. aeruginosa with the airway epithelial cells, SP-A was purified from BAL proceeding from patients with alveolar proteinosis. Determination of sample optical density at 280 nm during BAL elution through the maltosyl-agarose column allowed the identification of two main overlapping peaks (Figure 23 A). To determine the components of each peak, a representative fraction of each of them was analyzed by SDS-PAGE and Coomassie blue staining (Figure 23 B).

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Fractions (ml) Figure 23: Isolation of SP-A from BAL. A. Sample optical density at 280 nm during BAL elution through the Superose-6 column for SP-A purification. B. SDS-PAGE and Coomassie staining of representative fractions of the two major peaks obtained during SP-A purification. Molecular weight markers are indicated on the left.

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In the first peak, a band was detected at around 30-34 kDa, corresponding to the monomeric form of SP-A, and in the second peak, two main bands were detected, at 30-34 kDa and at 60-68 kDa, which correspond to the monomeric and dimeric forms of SP-A. The fractions corresponding to both peaks were pooled and concentrated by filtration. To determine the content of the pool, the sample was examined by SDS-PAGE and Coomassie stain or by Western blot analysis using specific polyclonal anti-human SP-A antibodies (Figure 24 A and B, respectively). The analysis by SDS-PAGE revealed that SP-A is present in the pooled fractions as a mix of both monomeric and dimeric forms of SP-A of 30-34 and 60-68 kDa. Overall, a total of 10.02 mg of SP-A were purified out of 9 liters of BAL, with a purity of around 90%. Furthermore, SP-A was detected specifically by Western blot using a polyclonal anti-human SP-A antibody, in both purified protein and BAL, without showing any cross-reactivity with other structurally related proteins present in BAL such as SP-D.

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Figure 24: Purification and detection of SP-A by SDS-PAGE and Western blot analysis. SDS-PAGE (A) and Western blot analysis with polyclonal anti-human SP-A antibody (B) of purified human SP-A from BAL. Molecular size markers are indicated on the left.

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4.2.2. Role of alginate exopolysaccharide in the resistance of P. aeruginosa to BAL and SP-A bactericidal effects SP-A is responsible for direct permeabilization of many Gram negative pathogens, including P. aeruginosa. To determine if alginate production is a crucial factor in P. aeruginosa resistance to bactericidal effects of SP-A, PAO1 and its isogenic hyper alginate producer mutant PAOMA were incubated with a pool of 10x concentrated human BAL. After one hour of incubation at 37ºC, PAO1 viability was significantly reduced (68.9% ± 6.1%,) whereas the survival of PAOMA was significantly less affected (18.7% ± 5.1% p=0.0032) (Figure 25 A). Various components of human BAL could be responsible for P. aeruginosa loss of viability, including SP-A, SP-D or defensins. To investigate if SP-A, which is the major surfactant protein in the lung, is involved in the differences of resistance to killing by BAL between PAO1 and PAOMA, bacterial cells from both strains were incubated with different concentrations of purified SP-A and its bactericidal effects on the two strains were determined (Figure 25 B). PAO1 showed a significant dose-dependent sensibility to SP-A bactericidal effects compared to untreated control (p = 0.0006 for 40µg/ml SPA). The lowest SP-A concentration that exerted a significant bactericidal effect on PAO1 in these experimental conditions was 10 µg/ml. By contrast, PAOMA viability was not affected by SP-A at the tested concentrations. These results were confirmed by a competition assay in which PAO1 and PAOMA were incubated together with a concentration of 20 µg/ml SP-A and the proportion of cfu of each strain was determined by plating at different incubation time. The results shown in Figure 25 C indicate, that even though the proportion of PAO1 was higher at the beginning of the experiment, the proportion of PAO1 decrease in time while the proportion of PAOMA increase, thereby indicating a lower susceptibility of PAOMA to the bactericidal effects of SP-A than PAO1. To provide further evidence that high alginate production, a frequent phenotype in P. aeruginosa isolates from chronic infections, is a determinant factor in the resistance of P. aeruginosa to bactericidal effects of SP-A, 14 clinical isolates proceeding from acute or chronic infections were incubated with 20g µ/ml SP-A. After one hour, the strains presenting a highly mucoid phenotype (between 7.3 and 26.0 µg of alginate per 109 cfu) presented a higher survival rate compared to the poorly mucoid strains (between

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1.4 and 3.9 µg of alginate per 109 cfu) (p=0.0018) (Figure 25 D). Altogether, these results indicate that alginate confers a resistance to SP-A mediated killing of P. aeruginosa.

A

B

C

D

Figure 25: Role of alginate in P. aeruginosa resistance to SP-A bactericidal effects. A. Relative survival of PAO1 and PAOMA after incubation with a pool of 10x concentrated human BAL for 1 h at 37ºC. Data are representative of three independent experiments. Results that are significantly different are denoted by an asterisk (p = 0.0664) (2-tailed t-test). B. Relative survival of PAO1 (open circles) and PAOMA (closed circles) after incubation with different concentrations of SP-A during 1 h. Data represent three different experiments. Results that are significantly different are denoted by an asterisk (*: p < 0.05; ***: p < 0.001)(2-tailed t-test). C. Competition assay between PAO1 and PAOMA incubated with 20 µg/ml SP-A in veronal buffer. Results indicate the percentage of each strain of the total of cfu recovered by plating after 0, 2, 4, 6 and 8 h. Data are representative of three different experiments. D. Relative survival of different P. aeruginosa clinical isolates after 1h of incubation with 20 µg/ml SP-A in veronal buffer compared to untreated control. Lines represent the means of each group. The range of alginate production expressed in µg/109 cfu for the strains studied in each group is indicated under the graph. Data represent at least three independent experiments. p < 0.01 for the comparison between both groups of isolates (2-tailed t-test).

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4.2.3. Role of alginate production in SP-A binding to P. aeruginosa To get further insights into the molecular basis of the effect of the presence of alginate on the binding of SP-A to P. aeruginosa, the binding of SP-A to bacterial cells of the strains PAO1 and PAOMA was studied. For this purpose, PAO1 and PAOMA were incubated in presence of BAL or a sub-lethal concentration of SP-A (8 µg/ml) for 1 h, and bound SP-A was eluted and subjected to Western blot analysis using a specific polyclonal anti-human SP-A. The hyper alginate producer strain PAOMA bound significantly less SP-A than PAO1, either with BAL (Figure 26 A), or with purified SP-A (Figure 26 B). To confirm this result, the quantity of SP-A remaining in BAL after incubation with PAO1 and PAOMA was analyzed by ELISA using specific polyclonal anti-human SP-A. The results indicate that PAO1 depleted significantly more SP-A (39.57% ± 7.49%) than PAOMA (60.31% ± 4.08%) (p = 0.0291) (Figure 26 C).

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A

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75kDa –

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Figure 26: Effect of alginate on SP-A binding to P. aeruginosa. A. Detection of the binding of SP-A from BAL to PAO1 and PAOMA. Both strains were incubated in presence of BAL for 1 h (lane 1 and 3, respectively) or veronal buffer as negative control (lane 2 and 4, respectively). Proteins were eluted and the amount of SP-A bound to bacterial cells was detected by Western blot analysis using specific polyclonal anti-human SP-A antibodies. Monomer (32 kDa) and dimer (64 kDa) forms of SP-A are indicated with arrows in the sample of BAL incubated without bacteria (lane 5). Data representative of three independent experiments are shown. Molecular weight markers are indicated on the left. B. Detection of the binding of purified SP-A to PAO1 and PAOMA. Both strains were incubated in presence of 8 µg/ml of purified SP-A for 1 h (lane 1 and 3, respectively) or veronal buffer as negative control (lane 2 and 4, respectively). Proteins were eluted and the amount of SP-A bound to bacterial cells was detected by Western blot analysis using specific polyclonal anti human SP-A antibodies. Monomer (32 kDa) and dimer (64 kDa) forms of SP-A are indicated with arrows in the sample of SP-A incubated without bacteria (lane 5). Representative results of three independent experiments are shown. Molecular weight markers are indicated on the left. C. Depletion of SP-A from BAL by PAO1 and PAOMA. BAL was incubated for 1 h with bacterial cells of the strains PAO1 (open circles) and PAOMA (closed circles) or without bacteria (open squares). The amount of SP-A remaining in BAL after incubation was detected by ELISA using specific polyclonal anti human SP-A antibodies. Representative results of three independent experiments are shown.

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4.2.4. Effects of SP-A on the invasion of lung epithelial cells by P. aeruginosa The function of SP-A in opsonization, bacterial killing and immunomodulation have been widely studied. However, little is known about the function of SP-A in the interaction of respiratory pathogens with airway epithelial cells. To study if SP-A affects the capacity of interaction of the bacterium with lung epithelial cells, standard invasion assays were performed using the human immortalized bronchoepithelial cell line 16HBEo-. The capacity of invasion of bronchoepithelial cells by PAO1 and PAOMA was tested in presence of a sub-lethal concentration of SP-A (8 µg/ml). As expected, PAO1 invasion capacity was significantly higher than PAOMA (p = 0.0004) (Figure 27 A). Furthermore, SP-A significantly reduced PAO1 invasion by 39.59% ± 9.91% (p = 0.0040), and PAOMA invasion by 66.4% ± 6.61% (p = 0.0100). To confirm that the effect of SP-A on bacterial invasion was not only specific of PAO1 and its derived mutant PAOMA, the capacity of invasion of bronchoepithelial cells by two P. aeruginosa clinical isolates, PAA2 and PAC20 was tested in presence of a sublethal concentration of SP-A (8 µg/ml). Both PAA2 and PAC20 invasion capacity were also reduced, by 70.3% ± 3.78% (p = 0.0130) and 32.48% ± 2.48% (p = 0.0035) respectively (Figure 27 A). The appropriate controls demonstrated that the viability of the strains was not affected by SP-A at the concentration used in this assay (8 µg/ml) (data not shown). To determine if the effect of SP-A on P. aeruginosa invasion capacity of lung epithelial cells is characteristic of this bacterium or also affects other opportunistic Gram negative pathogens, K. pneumoniae strain 52-145 was incubated with a sub-lethal concentration of SP-A (8 µg/ml). The invasion capacity of the strain was not affected by SP-A (p = 0.2562) (Figure 27 A), which indicates that SP-A effect on the invasive capacity of P. aeruginosa could be specific for this bacterium. To determine if the binding of SP-A to P. aeruginosa is responsible for SP-A mediated inhibition of P. aeruginosa invasion of bronchoepithelial cells, standard invasion assays were performed by incubating 16HBEo- monolayers with PAO1 and PAOMA cells preopsonized with a sub-lethal concentration of SP-A (8 µg/ml). Results depicted in Figure 27 B indicate that the binding of SP-A to P. aeruginosa does not affect the capacity of invasion of either strain (p = 0.6849 and p = 0.1403 respectively). In order to determine if the SP-A mediated inhibition of P. aeruginosa invasion of bronchoepithelial cells was due to the direct interaction of SP-A with bronchoepithelial

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cells, 16HBE monolayers were pre-treated with 8 µg/ml SP-A and incubated with untreated PAO1 and PAOMA. SP-A reduced significantly the invasion capacity of both strains by 34.75% ± 7.14% (p = 0.0452) and 64.81% ± 7.02% (p = 0.0193) respectively, which suggests that SP-A inhibits P. aeruginosa invasion of lung epithelial cells through direct interaction with the epithelium (Figure 27 C).

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A

B

C

Figure 27: Effects of SP-A on the invasion of lung epithelial cells by P. aeruginosa A. Invasion of the bronchoepithelial cells 16HBEo- by P. aeruginosa in the presence of SP-A. PAO1, its isogenic hyper alginate mutant PAOMA and two P. aeruginosa clinical isolates proceeding from acute infection (PAA2) or chronic infection (PAC20) were incubated with the bronchoepithelial cells 16HBEo- in presence of PBS as control (white bars) or a sub-lethal concentration of SP-A (8 µg/ml) (black bars). Intracellular cells were quantified by plating. Results that are significantly different from those in controls are denoted by an asterisk (*: p < 0.05; **: p < 0.01) (2-tailed t-test). Data are representative of three independent experiments. B. Invasion of the bronchoepithelial cells 16HBEo- by P. aeruginosa pre-opsonized with SP-A. PAO1 and its isogenic hyper alginate mutant PAOMA were pre-incubated with PBS as control (white bars) or a sub-lethal concentration of SP-A (8 µg/ml) (black bars), washed and incubated with the bronchoepithelial cells 16HBEo. Intracellular cells were quantified by plating. Data are representative of three independent experiments. C. Invasion of the bronchoepithelial cells 16HBEo- pre-opsonized with SP-A by P. aeruginosa.

The

bronchoepithelial cells 16HBEo- were pre-incubated with PBS as control (white bars) or SP-A (8 µg/ml) (black bars), washed and infected with PAO1 and its isogenic hyper alginate mutant PAOMA. Intracellular cells were quantified by plating. Results that are significantly different from controls are denoted by an asterisk (*: p < 0.05) (2-tailed t-test). Data are representative of three independent experiments.

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4.2.5. p63 mediates P. aeruginosa internalization by airway epithelial cells The data obtained suggest that P. aeruginosa and SP-A share a common receptor on lung epithelial cells. Several specific receptors have been identified for SP-A interaction with epithelial cells including SPR-210, CD91-calreticulin, BP-55, SPAR and p63 [210-211, 213, 215]. However, p63 which was recently identified on the surface of human type II pneumocytes as the responsible for the SP-A mediated surfactant production inhibition effect of SP-A on type II cells, is the receptor for SP-A present on the surface of human airway epithelial cells better characterized. In order to determine if p63 is involved in the SP-A mediated inhibition of P. aeruginosa invasion of bronchoepithelial cells, it was first determined by immunofluorescence whether this receptor is present on the surface of those cells. The human immortalized type II pneumocytes A549 cell line was used as positive control for the presence of p63. The presence of p63 on both cells lines was determined on intact cells using specific antibodies directed against p63, with a secondary antibody labeled with TRITC. In parallel, actin was stained using Alexa fluor 488 Phalloidin. The observation of the cells using fluorescence microscopy determined that p63 is expressed on the surface of both cell lines (Figure 28 A). To study the involvement of p63 in the SP-A mediated inhibition of P. aeruginosa invasion of airway epithelial cells, standard invasion assays were performed using a specific monoclonal anti-human p63 blocking antibody or an isotopic antibody, directed against the receptor CD44 present on epithelial cells [319] as control. Invasion of PAO1 was significantly reduced by a 53.30% ± 7.60% in 16HBEo- (p = 0.0009), and by a 32.15% ± 8.89% in A549 cells (p = 0.0342) (Figure 28 B) in the presence of the anti-p63 antibody, whereas the anti-CD44 antibody did not affect the rate of invasion of PAO1. Altogether, these results indicate that p63, present on the airway epithelial cell surface, is a receptor for P. aeruginosa internalization by those cells.

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A

B

Figure 28: p63 expression on lung epithelial cells and blockade of P. aeruginosa internalization by those cells by anti-p63 antibody. A. 16HBEo- cells (top picture) and A549 cells (bottom picture) were labeled with a specific primary antibody directed against p63 and a secondary antibody labeled in red with TRITC. Cytoskeleton was stained green with Alexa fluor 488 Phalloidin. Pictures were taken with at 100 fold magnification and are representative of three independent experiments. B. Inhibition of P. aeruginosa internalization by lung epithelial cell 16HBEo- (top graph) and A549 (bottom graph) by p63 blocking antibodies. Monolayers were pretreated with a specific monoclonal antibody directed against p63 or with an isotopic antibody directed against CD44 as control. Data represent three independent experiments and significant results are denoted by an asterisk (2-tailed t-test).

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5. Discussion

Discussion

Determination of ChoP expression and role in P. aeruginosa pathogenesis

Determination of ChoP expression and role in P. aeruginosa pathogenesis Expression of ChoP undergoes phase variation in some microorganisms [135, 169, 177]. In P. aeruginosa, expression of ChoP is modulated by temperature: at 22°C expression is high, whereas at 37°C expression is negligible. The ability of P. aeruginosa to downregulate ChoP expression at 37°C may be crucial in its capacity to cause invasive acute infections. It is likely that in these infections the absence of ChoP contributes to the virulence of the microorganism evading humoral clearance mechanisms, such as the serum killing mediated by C-reactive protein that occurs in H. influenzae [153]. The results obtained in this study are consistent with this hypothesis, because the ChoP expression at 37°C was undetectable in most of the isolates collected from acute infections. In contrast, the expression of ChoP at 37°C was higher among the chronic infection isolates. Interestingly, within the group of the chronic infection isolates, the highest rate of ChoP+ isolates at 37°C was found in the group of the mutator strains (data not shown). In these strains, which are defective in mismatch repair systems, the rate of mutation is very high and changes allowing for the adaptation to the lung environment appear frequently [320]. Altogether, these results suggest that ChoP expression may favor the persistence of the microorganism in the airway tract, as in H. influenzae, where ChoP contributes to survival in the respiratory tract [153] and could represent an important adaptation to the environment of the deteriorated lung encountered by the microorganism during the chronic infection process. These results, obtained using different approaches, have demonstrated that the 43 kDa ChoP-containing protein is EF-Tu. Unlike the bacterial structures that have been shown to contain ChoP in other respiratory pathogens, such as S. pneumoniae [321] and H. influenzae [135], this epitope is found on a protein in P. aeruginosa, as well as in pathogenic Neisseria [177]. The presence of the ChoP epitope on EF-Tu was unexpected, because this would be the first example of a cytoplasmic protein containing ChoP and is in contrast to the surface localization of the ChoP epitope in other pathogens [135, 177, 321]. However, immunodetection assays using intact cells detected the presence of ChoP on the outer surface of P. aeruginosa. Furthermore, using specific antibodies against EF-Tu, the presence of this protein was also detected on the bacterial surface. Surface location of EF-Tu, a component of the protein

Mariette Barbier


Discussion


synthesis machinery, is unusual among microorganisms, although it has been reported that under stress conditions EF-Tu becomes membrane associated in E. coli. Furthermore, the EF-Tu molecule, originally thought to be restricted to the cytoplasm of bacteria, has also been shown to be associated with the cell envelopes of many microorganisms, including the cell wall of Mycobacterium leprae, the membrane of Mycoplasma pneumonia, as well as the periplasm of Neisseria gonorrhoeae and E. coli [322-326]. Interestingly, EF-Tu was found to be surface exposed at either 22°C or 37°C in all P. aeruginosa strains tested, suggesting that the incorporation of choline into EF-Tu is modulated by an unknown mechanism that operates at 37°C in the chronic infection isolates but not in the acute isolates. In H. influenzae, the locus involved in the regulation of the ChoP expression is lic1 [162]. Homologs to the lic1 genes have been identified in S. pneumoniae [163] and Neisseria [137] but none of the possible homologs to these genes in the genome of P. aeruginosa is responsible for the association of ChoP to EF-Tu. The same occurs with the genes pptA, psc, pmtA and BetT, already described to be involved in ChoP synthesis and incorporation in other microorganisms and which possible homologs in P. aeruginosa do not show alteration of ChoP association to EF-Tu. The different approaches used in this study to identify possible genes involved in ChoP incorporation on EF-Tu led to the identification of a unique locus, PA4178. The gene present at this locus, referred in this work as choP, is conserved in all the P. aeruginosa clinical strains tested and encodes a putative protein with a possible catalytic domain of transferase. This class of enzymes is usually involved in the transfer of methyl or phosphate groups from a donor molecule to an acceptor, which correlates with the possible function of this gene in the transfer of the ChoP motive onto the protein EF-Tu. However, real time PCR and transcriptome analysis showed that choP expression is not temperature dependent, which indicates that other mechanisms are responsible for phase variation of ChoP in P. aeruginosa. A detailed analysis of the gene sequence and its hypothetical promoter region in several clinical isolates has not revealed genetic patterns such as the CAAT repeats responsible for ChoP phase variation in H. influenzae and non pathogenic Neisseria or the guanosine tract of the gene pptA in N. meningitides (data not shown). These results suggest that, although in the screening of the PA14 transposon mutant library only one gene was found to be involved in ChoP incorporation of EF-Tu, choP is possibly not the only gene involved in ChoP expression in P. aeruginosa. This result could be explained in three different ways. First, a large number of genes annotated on the genome of PAO1 were not screened in this study, partly due to the unavailability of the remaining transposon mutants. Those

Mariette Barbier


Discussion


genes, probably essential in PA14, could be involved in ChoP incorporation on EF-Tu. Second, as mutants of individual genes were screened in this study, the possibility that the regulation of ChoP incorporation to EF-Tu relies on the expression of several genes could not be studied. Finally, the method of screening used in this study only allows the detection of negative and positive ChoP association to EF-Tu and does not represent a reliable tool to quantify moderate changes in ChoP expression levels. In fact, ongoing investigations will be required to characterize the genetic basis for incorporation of choline into EF-Tu in P. aeruginosa and to understand the different mechanisms that operate in acute and chronic infection isolates. The ability to adhere to and invade the epithelial lining is thought to be an important step in the respiratory pathogenesis of P. aeruginosa. A number of bacterial ligands and host receptors have been associated with adherence to and invasion of epithelial cells by P. aeruginosa [20, 53]. The results obtained show that the adhesion or invasion of the ChoP+ P. aeruginosa strains is reduced by the PAF-R antagonist, suggesting that PAF-R is a cellular receptor for the ChoP+ strains. However, the contribution of this interaction to P. aeruginosa airway chronic pathogenesis is still unclear. It has been proposed that ChoP is a determinant of the ability of H. influenzae to colonize and persist within the nasopharyngeal environment, perhaps by mediating bacterial adherence to and invasion of the host epithelia. Evidence for this hypothesis includes the finding that H. influenzae isolated from human respiratory secretions are enriched for variants that would be predicted to express ChoP [153]. On the other hand, it has been reported that ChoP promotes the establishment of stable biofilm communities of non-typeable H. influenzae [327]. One could speculate that the presence of ChoP+ variants among the P. aeruginosa chronic infection isolates might represent evidence for the role of ChoP in the colonization and persistence in the airway tract, promoting the biofilm formation in the lungs of chronically infected patients. It is predicted that there would be a selection for the ChoP+ variants that are able to infect and evade the early host defense mechanisms in the initial steps of the infection. These ChoP+ variants, which initially would represent a small part of the population, would be able to adhere to the epithelial cells and promote biofilm formation and chronic persistence in the airway tract. The results obtained from in vivo experiments support this hypothesis. PAF-R antagonist reduced significantly the bacterial loads in lung homogenates of mice infected with a ChoP+ chronic infection isolate. This observation was not due to the potential protective effects of the antagonist during airway infection, because a reduction of the bacterial loads in lung homogenates of mice infected with a ChoP+

Mariette Barbier


Discussion


isolate was not observed. In summary, this work shows that another important respiratory pathogen uses the ChoP moiety to interact with airway epithelium via PAF-R. In P. aeruginosa, this moiety is particularly predominant among the chronic infection isolates that may represent an adaptation of the pathogen to the inflamed airway tract where the expression of PAF-R is elevated, such as those found in patients with CF, bronchiectasis, or COPD, and may provide a mechanism to promote persistence in the airway tract. This indicates that the ChoP/PAF-R interaction may be of particular importance in the elevated incidence of P. aeruginosa in these patients and may be a new target for the development of future therapies.

Mariette Barbier


Discussion

Role of SP-A in P. aeruginosa interaction with airway epithelial cells

Role of surfactant protein A in P. aeruginosa interaction with airway epithelial cells SP-A plays a crucial role in the innate humoral immune response of the lung. Two different alleles of the gene coding for SP-A are responsible for the production of the protein, which results as a mix of the two forms: SP-A1 and SP-A2. Both forms possess different affinities for sugar binding and could be responsible for different pathogens recognition [328]. In order to study in vitro and in vivo effects of SP-A, recombinant SPA1 and SP-A2 are widely used. However, a study demonstrated that the oligomerization state of recombinant SP-A is different from native SP-A and that the state of oligomerization might be critical for SP-A function such as ligand aggregation [329]. For that reason, this study was performed with purified native human SP-A, in order to determine the effect of surfactant protein on the interaction of P. aeruginosa with airway epithelial cells. Alginate is commonly produced by P. aeruginosa strains and represents an important factor for the resistance of the bacterium to opsonization by antibodies or complement and to phagocytosis by alveolar macrophages and neutrophils [58, 229]. This study proposes a novel function for alginate as virulence factor, to confer resistance to P. aeruginosa against the bactericidal effect of SP-A. The data show that at physiologically relevant concentrations of SP-A [190-191], non-mucoid strains are more susceptible to the bactericidal effects of SP-A than the mucoid strains. In fact, the non mucoid strain was susceptible to all concentrations of SP-A tested. This result is consistent with the data obtained in SP-A deficient mice, in which the capacity to clear P. aeruginosa from the lung was dramatically decreased during the first hours of infection [241]. Both results indicate that SP-A mediated killing of P. aeruginosa is critical during the early stages of infections in the lung. At low concentrations of SP-A, such as those found in the CF lungs [191], the non mucoid strain remains more susceptible to SPA bactericidal effects than the mucoid strain, which suggest that SP-A could represent a selection factor for mucoid strains of P. aeruginosa, a typical phenotype observed in chronic lung infections. This new factor sums up to the list of mechanisms associated to the selection of the alginate phenotype in the lung, such as the continuous renewal of air in the lung [56], iron limitation or the use of phosphatidylcholine as sole source of

Mariette Barbier


Discussion


carbon [330]. Furthermore, this study demonstrates that alginate provides resistance to the bactericidal effects of SP-A to mucoid P. aeruginosa strains impeding the binding of the protein to the bacterium. This effect seen in a significant number of clinical isolates participates in the protection of the bacterium against surfactant proteins. A similar mechanism has been described for other collectins such as C1q. In pathogens such as E. coli [331] and K. pneumoniae [332], capsule production was shown to be involved in blocking the binding of C1q to its ligand on the bacterial surface, as occurs for SP-A with P. aeruginosa alginate. This strategy represents a novel active resistance mechanism for P. aeruginosa to SP-A mediated bacterial killing and sums up to the list of already known elements for SP-A resistance, such as the genes pch and pst and the production of flagellum [72, 246], which affect membrane integrity, or enzyme production such as elastase and protease IV which participate in SP-A degradation [87, 90]. During the infection process of P. aeruginosa in the lung, SP-A plays an important role in phagocytic cells mediates bacterial phagocytosis and modulates immune response. However, the function of surfactant proteins in the interaction of P. aeruginosa with epithelial cells has been poorly studied. This study demonstrates that SP-A blocks P. aeruginosa internalization by airway epithelial cells. These results differ from those obtained in various studies which described that SP-A increases P. aeruginosa uptake by pulmonary professional phagocytes [225]. However, the data obtained are consistent with other works describing that in the cornea and in the lung, recombinant SP-D reduces P. aeruginosa [333] and K. pneumoniae invasion of epithelial cells respectively [334]. Nevertheless, the mechanism by which SP-D decreases epithelial cell invasion was not studied. Unexpectedly, the data obtained in this study show that the opsonization of P. aeruginosa with sub-lethal concentrations of SP-A does not affect the internalization of the bacterium by bronchoepithelial cells. By contrast, SP-A was shown to decrease the internalization of P. aeruginosa by airway epithelial cells through a direct interaction with those cells. Altogether these results suggest that SP-A and P. aeruginosa share a common receptor on the surface of airway epithelial cells, which can be blocked by free SP-A but not pathogen bound SP-A. In a recent study, Gardai et al. described that the effect of SP-A on macrophages depends on the SP-A domain interacting with the cells [211]. These authors demonstrate that when SP-A binds pathogens through the CRD domain, the protein also engages SIRP-a receptor on the surface of macrophages through its collagen domain. In the absence of macrophages, SP-A binds to CD91-calreticuline through its CRD domain. These observations, together

Mariette Barbier


Discussion


with the experimental evidences seen in this study suggest that SP-A blocks the interaction of P. aeruginosa with a receptor on airway epithelial cells through the CRD domain of the protein. Despite several receptors have been described on the surface of airway epithelial cells, the mechanisms of SP-A interaction with those receptors and the cellular response which results of receptor engagement have been poorly studied in human airway epithelial cells. p63 is the most well characterized receptor for SP-A in human airway epithelial cells. It is a 63 kDa transmembrane protein, characterized on the surface of murine and human type II pneumocytes and associated with the cytosqueleton. p63 participates in the cytosolic compartment in the association between microtubules and endoplasmic reticulum in monkey kidney cells but is also present on cells surface [335]. This protein acts as a receptor for tissue plasminogen activator on smooth muscle cells and a receptor for antiproliferative factor on epithelial cells [336-337]. Several studies described that SP-A binding through the CRD domain to p63 results in inhibition of the secretagogue-stimulated surfactant secretion from type II pneumocytes, suggesting that this receptor plays an important role in surfactant secretion and homeostasis in the lung [221-222]. The results obtained in this work indicate that p63 is as well a receptor for P. aeruginosa since P. aeruginosa internalization by airway epithelial cells was blocked by the binding of p63 blocking antibodies. These results suggest that the union of P. aeruginosa to p63 could play a regulation role in surfactant secretion by alveolar type II cells in the lung. This hypothesis is supported by various studies which described that mucoid P. aeruginosa decreases surfactant phospholipids levels by direct inhibition of mRNA synthesis of phosphocholine cytidylyltransferase (CCTα), the rate-regulatory enzyme required for DPPC synthesis, by reducing CCTα promoter activity in murine and cell infection models [248]. In addition, it is well described that patients chronically infected with P. aeruginosa present altered surfactant composition, including decreased phospholipids contents. Altogether, these data provide new insights in the understanding of the decreased levels of surfactant proteins in the lung of patients chronically infected with P. aeruginosa [191]. However, further studies are required to investigate the dynamics of P. aeruginosa union to this receptor, the consequences on surfactant production and the contribution to the outcome of the infection.

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Appendix

Reagent composition

1. Conclusions

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Conclusions

1. Phosphorylcholine expression is higher at 37ºC among P. aeruginosa isolates proceeding from chronic infections, in comparison with those from acute infections. 2. Coimmunoprecipitation,

microcapillary

liquid

chromatography

mass

spectrometry and tandem mass spectrometry determined that in P. aeruginosa the phosphorylcholine epitope is associated to the elongation factor Tu, a protein of 43 kDa present in the cytoplasm and involved in protein synthesis. 3. Immunofluorescence microscopy and flow cytometry showed that the phosphorylcholine epitope and the elongation factor Tu are located on the surface of P. aeruginosa. 4. A platelet activating factor receptor antagonist inhibits the adhesion and invasion to epithelial cells of phosphorylcholine-producing P. aeruginosa strains, but not of phosphorylcholine-deficient strains. 5. A platelet activating factor receptor antagonist decreases efficiently the bacterial load in the lungs of mice infected by phosphorylcholine-producing P. aeruginosa strains, but not of phosphorylcholine-deficient strains. 6. The gene choP present in the locus PA4178 on the chromosome of PAO1 is involved in phosphorylcholine incorporation to the elongation factor Tu and is conserved among P. aeruginosa clinical isolates. 7. Alginate protects P. aeruginosa against surfactant protein A bactericidal effects, reducing surfactant protein A binding to mucoid P. aeruginosa strains. 8. Opsonization of P. aeruginosa with surfactant protein A does not affect the invasive capacity of the bacterium of bronchoepithelial cells. 9. SP-A inhibits P. aeruginosa internalization by airway epithelial cells through direct interaction of the protein with the cells. 10. Anti-p63 antibodies block the invasion of airway epithelial cells by P. aeruginosa, which suggest that p63 acts as a common receptor for SP-A and P. aeruginosa on the surface of airway epithelial cells.

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Appendix

Reagent composition

2. References

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Appendix References

Reagent composition

8. Appendix

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Appendix

Reagent composition

8.1. Reagent composition

Coating buffer

Laemmli sample buffer

35 mM .................. NaHCO3

20 % ...................... Glycerol

15 mM .................. Na2CO3

10 % ...................... β-mercaptoethanol 4 % ........................ SDS

Denaturation solution

30 mM ................... Tris-HCl pH 6.8 0.1 % ..................... Bromophenol blue

1.5 M .................... NaCl 0.5 M .................... NaOH

Loading buffer 50 % ...................... Glycerol

Depurination solution

0.1 M ..................... EDTA

250 mM ................ HCl

0.1 % ..................... SDS 0.1 % ..................... Bromophenol blue

Fragmentation buffer 200 mM ................ Tris-Acetate, pH 8.1

Luria Bertani medium

500 mM ................ Potassium acetate

10 g ....................... Tryptone

150 mM ................ Magnesium acetate

5 g ....................... Yeast extract 10 g ....................... NaCl Up to 1L ................ Distilled water

Killer Filler reagent 135 mM ................. NaCl

pH adjusted 7.2

5 mM ..................... Na2HPO4 1.5 mM .................. KH2PO4

Neutralization solution

10 mM ................... NaOH

1.5 M .................... NaCl

1% .......................... Casein

0.5 M .................... HCl

1% ......................... BSA 20 mg .................... Phenol Red 360 mg .................. NaN3 pH adjusted 7.4

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Appendix

Reagent composition

Phosphate Buffered Saline

SSC-20x buffer

135 mM ................. NaCl

3 M ....................... NaCl,

5 mM ..................... Na2HPO4

0.3 M .................... Sodium citrate

1.5 mM .................. KH2PO4 pH adjusted at 7.4

TE buffer 10 mM ................... Tris-HCl pH 7.5

Pre-hybridizing solution

1 mM ..................... EDTA

0.5 M .................... NaCl 5 % ........................ Blocking reagent (Amersham)

Trypticase soy agar 17 g ...................... Tryptone 3 g ........................ Soytone

Primary wash buffer 3 M ........................ Urea 2 % ........................ SDS 7.5 mM .................. NaCl 0.75 mM ................ Sodium citrate

2.5 g ..................... Dextrose 5 g ......................... NaCl 2.5 g ..................... K2HPO4 15 g ...................... Agar Up to 1L ............... Distilled water

Up to 1L................. Distilled water

Veronal buffer 5 mM .................... Sodium Barbital

Secondary wash buffer 300 mM ................. NaCl 30 mM ................... Sodium citrate

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0.145 M ................. NaCl 0.5 mM ................. MgCl2 0.15 mM .............. CaCl2


Table of the P. aeruginosa genes homologs to genes involved in ChoP biosynthesis and expression in other microorganisms

Appendix

8.2. Supplementary results

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34 34 34 30 35 32 35 32 30 30 32 30 31 37 32 32 32 36 32 35 30 33 31 30 32 34 32 34 75 49 44 30 33 30 33 31 35 34 30 32 32 51 38 34 32 34 38 31 32

62 46 60 42 42 60 46 57 53 50 60 52 62 62 48 53 52 59 42 43 44 50 50 55 39 51 58 61 87 68 64 55 52 50 48 45 51 46 62 48 60 62 54 45 50 55 50 50 49

licA licA licA licA licA licA licA licA licB licB licB licB licB licB licB licB licB licB licB licB licC licC licC licC licC licC licC licD betT betT betT betT betT betT betT betT betT betT betT betT betT betT pmtA pmtA pmtA pmtA pmtA pmtA pmtA


ChoP 25º Cc

Gene homolog

Hypothetical protein PA2372 Probable gamma-glutamyltranspeptidase precursor Transcription elongation factor NusA Probable amino acid-binding protein Hypothetical protein PA1114 Hypothetical protein PA1227 Probable binding protein component of ABC dipeptide transporter Hypothetical protein PA4929 Probable binding protein component of ABC transporter Hypothetical protein PA2090 Hypothetical protein PA1060 Probable transporter Probable transporter Probable transporter Probable MFS dicarboxylate transporter Probable restriction-modification system protein Multidrug efflux protein Hypothetical protein PA3358 3-oxoacyl-(acyl carrier protein) synthase II Hypothetical protein PA2530 Hypothetical protein PA1784 Probable nucleotidyl transferase UTP-glucose-1-phosphate uridylyltransferase Hypothetical protein PA1090 Predicted kinase Glucose-1-phosphate thymidylyltransferase Hypothetical protein PA3649 Probable phosphatidate cytidylyltransferase Choline transporter BetT Probable choline transporter Probable choline transporter Hypothetical protein PA5211 Probable carbohydrate kinase Probable TonB-dependent receptor Probable amino acid permease Hypothetical protein PA0343 Cytosine permease Probable major facilitator superfamily Type III secretion system protein Probable transcriptional regulator Flagellar biosynthesis protein FliP Probable amino acid permease Dimethyladenosine transferase Aminotransferase Ubiquinone/menaquinone biosynthesis methyltransferase Phospholipid methyltransferase Probable transcriptional regulator 3-demethylubiquinone-9 3-methyltransferase Protein-L-isoaspartate O-methyltransferase

Positive (%)b

nusA fabF1 galU rmlA betT1 codB pscR fliP ksgA ubiE pmtA ubiG pcm

Identity (%)a

Gene name

PA2372 PA0361 PA4745 PA3858 PA1114 PA1227 PA5317 PA4929 PA3889 PA2090 PA1060 PA1882 PA1519 PA4719 PA5530 PA2735 PA2018 PA3358 PA2965 PA2530 PA1784 PA0597 PA2023 PA1090 PA0486 PA5163 PA3649 PA2536 PA5375 PA3933 PA5291 PA5211 PA3579 PA0192 PA4072 PA0343 PA0438 PA4233 PA1693 PA3341 PA1446 PA1147 PA0592 PA4088 PA5063 PA0798 PA0547 PA3171 PA3624

Protein

Gene locus in PAO1

8.2.1. Table of the P. aeruginosa genes homologs to genes involved in ChoP biosynthesis and expression in other microorganisms

Appendix

PA3487 PA2118 PA4664 PA0412 PA0774 PA3164 PA2799 PA3346 PA3857 PA2541 PA2089 PA0224 PA4517

a

Table of the P. aeruginosa genes homologs to genes involved in ChoP biosynthesis and expression in other microorganisms

pldA ada hemK pilK pcs -

Phospholipase D O6-methylguanine-DNA methyltransferase Probable methyltransferase Methyltransferase PilK Hypothetical protein PA0774 3-phosphoshikimate 1-carboxyvinyltransferase prephenate pehydrogenase Hypothetical protein PA2799 Probable two-component response regulator Phosphatidylcholine synthase Probable CDP-alcohol phosphatidyltransferase Hypothetical protein PA2089 Hypothetical protein PA0224 Conserved hypothetical protein

32 32 45 31 32 31 32 31 44 32 37 30 45

54 47 58 47 48 47 54 45 63 54 53 46 54

pmtA pmtA pmtA pmtA pmtA pcs pcs pcs pcs pcs pcs pcs pptA

Identity: indicates the percentage of identical aminoacids between the two

sequences blasted. b

Positive: represents the percentage of aminoacids with similar properties, however not

necessarily identical, between the two sequences blasted. c

ChoP 25ºC: Result of the screening for the association of ChoP to EF-Tu at 25ºC in the

mutants of the PA14 transposon library for the corresponding gene. The detection of ChoP associated to EF-Tu is indicated in green. The gene of which the mutant was not available is indicated in red.

Mariette Barbier


Appendix

Arrays data

8.2.2. Arrays data

PA4175 PA0897 PA3915 PA3877 PA1914 PA0896 PA0534 PA4648 PA1894 PA0899 PA2939 PA1877 PA2134 PA3337 PA4653 PA5355 PA0535 PA1874 PA2753 PA4649 PA4610 PA1895 PA5170 PA1876 PA3006 PA0529 PA0895 PA1168 PA1818 PA4294 PA1561 PA4650 PA1875 PA3274 PA0898 PA2161 PA5212 PA2160 PA2147 PA1984 PA4171 PA1897 PA4359 PA3369 PA2405 PA0200 PA2407 PA2166 PA5232 PA3362 PA2404 PA2398 PA5026 PA2412 PA2165

piv aruG moaB1 narK1 aruF aruB rfa-D glcD acrD aruC aruD katE fpvA -

protease IV (prpL) arginine/ornithine succinyltransferase AII subunit Molybdopterin biosynthetic protein B Nitrite extrusion protein 1 Conserved hypothetical protein - halovibrin Arginine/ornithine succinyltransferase AII subunit Conserved hypothetical protein Hypothetical protein Hypothetical protein Succinylarginine dihydrolase Probable aminopeptidase Probable secretion protein Hypothetical protein ADP-L-glycero-D-mannoheptose 6-epimerase Hypothethical protein glycolate oxidase subunit GlcD Probable transcriptional regulator Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Arginine/ornithine antiporter Probable ATP-binding/permease fusion ABC transporter Transcriptional regulator PsrA, TetR familly Hypothetical protein Succinylornithine aminotransferase Hypothetical protein Orn/Arg/Lys decarboxylase LdcC Hypothetical protein Aerotaxis receptor Aer Hypothetical protein Hypothetical protein Hypothetical protein Succinylglutamate 5-semialdehyde dehydrogenase Hypothetical protein Hypothetical protein Probable glycosyl hydrolase Catalase HPII Probable aldehyde dehydrogenase Probable protease Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Ferripyoverdine receptor Hypothetical protein Hypothetical protein Hypothetical protein

Mariette Barbier

72,0848 22,5752 20,1910 20,1849 19,1777 18,1742 18,0468 16,5323 15,5628 15,0982 14,6923 14,5339 14,5149 13,6683 13,1343 11,6589 10,5572 10,2671 10,0163 9,8631 9,8559 9,5073 9,0327 8,8566 8,8486 8,7744 8,7079 8,5169 8,3712 8,2279 7,9914 7,8901 7,8102 7,7481 7,5893 7,4849 7,3930 7,3794 7,3026 6,8600 6,6356 6,6150 6,5951 6,4511 6,4164 6,3756 6,3218 6,2831 6,2655 6,2308 6,2042 6,0852 5,9235 5,8676 5,8600


ChoP 37ºCb

ChoP 25ºCa

Fold Change

Genes with increased expression levels in PAO1 grown at 25ºC versus 37ºC

Protein

Gene name

Gene locus

8.2.2.1.

Appendix

PA2397 PA1195 PA4625 PA4651 PA1896 PA0567 PA2159 PA3231 PA2158 PA2119 PA0052 PA0830 PA5208 PA2143 PA4139 PA0050 PA5481 PA4624 PA0901 PA3934 PA2386 PA0051 PA1404 PA3341 PA3289 PA2413 PA1137 PA0059 PA3371 PA2864 PA2174 PA2190 PA4310 PA5027 PA2433 PA2513 PA2178 PA5482 PA0196 PA2717 PA4876 PA2345 PA2396 PA4786 PA2153 PA3788 PA2403 PA5181 PA4877 PA2393 PA2698 PA3370 PA2183 PA3629 PA5173 PA0900 PA5171 PA0527 PA2384 PA1202 PA2442 PA3068 PA1745 PA4298 PA2394 PA2411 PA2754 PA4590 PA0158 PA0788 PA4497

Arrays data

pvdE aruE pvdA phzH pctB antB pntB pvdF glgB adhC arcC arcA pvdN pra -

Pyoverdine biosynthesis protein PvdE Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Probable alcohol dehydrogenase (Zn-dependent) Alcohol dehydrogenase (Zn-dependent) Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Succinylglutamate desuccinylase Hypothetical protein L-ornithine N5-oxygenase Potential phenazine-modifying enzyme Hypothetical protein Hypothetical protein Hypothetical protein L-2,4-diaminobutyrate:2-ketoglutarate 4-aminotransferase, PvdH Hypothetical protein Osmotically inducible protein OsmC Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Chemotactic transducer PctB Hypothetical protein Hypothetical protein Anthranilate dioxygenase small subunit Hypothetical protein Hypothetical protein Pyridine nucleotide transhydrogenase, beta subunit Chloroperoxidase precursor cpo Osmotically inducible lipoprotein OsmE Hypothetical protein Pyoverdine synthetase F Probable short-chain dehydrogenase Glycogen branching enzyme Hypothetical protein Hypothetical protein Probable oxidoreductase Hypothetical protein Hypothetical protein Probable hydrolase Hypothetical protein Hypothetical protein Alcohol dehydrogenase class III Carbamate kinase Hypothetical protein Arginine deiminase Transcriptional regulator Dnr Hypothetical protein Probable hydrolase Glycine cleavage system protein T2 NAD-dependent glutamate dehydrogenase Hypothetical protein Hypothetical protein PvdN Probable thioesterase Hypothetical protein Protein activator Hypothetical protein Hypothetical protein Probable binding protein component of ABC transporter

Mariette Barbier

5,8262 5,7943 5,7514 5,7068 5,6947 5,6110 5,5621 5,5274 5,5264 5,4282 5,4047 5,3960 5,3756 5,2901 5,1390 5,1012 5,0270 5,0117 5,0048 5,0014 4,9360 4,8185 4,7955 4,7585 4,7562 4,7425 4,6502 4,6221 4,5953 4,5657 4,5314 4,5263 4,4663 4,4252 4,4250 4,3502 4,3471 4,3463 4,3448 4,3195 4,3176 4,2521 4,1899 4,1856 4,1824 4,1457 4,1378 4,1130 4,1129 4,0705 4,0440 4,0050 3,9699 3,9512 3,9364 3,8955 3,8920 3,8513 3,8495 3,8483 3,7953 3,7614 3,6694 3,6461 3,6384 3,5980 3,5883 3,5757 3,5573 3,5535 3,5534


Appendix

PA4788 PA5172 PA0157 PA2409 PA4015 PA4348 PA2171 PA3721 PA0540 PA1080 PA2663 PA1789 PA4703 PA0314 PA2501 PA3677 PA5111 PA4735 PA1819 PA3221 PA2242 PA2485 PA1647 PA0462 PA5271 PA4311 PA3858 PA4577 PA3572 PA3905 PA5153 PA5217 PA3250 PA4571 PA4915 PA4352 PA4117 PA1289 PA2476 PA1095 PA4328 PA2026 PA5108 PA4874 PA1689 PA1078

a

Arrays data

arcB nalC flgE fliY gloA3 csaA pslL bphP dscbG -

Hypothetical protein Ornithine carbamoyltransferase Hypothetical protein Probable permease of ABC transporter Hypothetical protein Hypothetical protein Hypothetical protein Transcriptional regulator Hypothetical protein Flagellar hook protein FlgE Hypothetical protein Hypothetical protein Hypothetical protein L-cysteine transporter of ABC system FliY Hypothetical protein Resistance-Nodulation-Cell Division efflux membrane fusion precursor Lactoylglutathione lyase Hypothetical protein Probable amino acid permease CsaA PslL Hypothetical protein Probable sulfate transporter Hypothetical protein Hypothetical protein Hypothetical protein Probable amino acid-binding protein Hypothetical protein Hypothetical protein Hypothetical protein Amino acid ABC transporter periplasmic binding protein Probable binding protein component of ABC iron transporte Hypothetical protein Probable cytochrome c Probable chemotaxis transducer Hypothetical protein Bacterial phytochrome, BphP Hypothetical protein Thiol:disulfide interchange protein DsbG Hypothetical protein Hypothetical protein, cytoplasmic Hypothetical protein, cytoplasmic membrane Hypothetical protein Hypothetical protein Hypothetical protein Flagellar basal-body rod protein FlgC

3,5448 3,5415 3,5376 3,5174 3,5068 3,4690 3,4387 3,4325 3,4285 3,4203 3,4189 3,4129 3,3858 3,3815 3,3798 3,3633 3,3632 3,3574 3,3354 3,3354 3,3348 3,2765 3,2736 3,2638 3,2635 3,2441 3,2440 3,2439 3,2336 3,2284 3,2148 3,2120 3,2008 3,2007 3,1828 3,1809 3,1773 3,1757 3,1746 3,1687 3,1596 3,1342 3,1324 3,1223 3,1120 3,0869


mutants of the PA14 transposon library for the corresponding gene. The detection of ChoP associated to EF-Tu is indicated in green. The gene of which the mutant was not available is indicated in red. b


mutants of the PA14 transposon library for the corresponding gene. The absence of ChoP on EF-Tu appears in blue. The gene of which the mutant was not available is indicated in red.

Mariette Barbier


Arrays data

PA5316 PA0796 PA3113 PA4219 PA3353 PA2741 PA4484 PA4250 PA4271 PA3791 PA4142 PA1742 PA4506 PA0964 PA4242 PA3330 PA0578 PA0617 PA0482 PA1787 PA5570 PA0271 PA0621 PA2860 PA4502 PA0428 PA2262 PA0789 PA3989 PA2644 PA1714 PA0722 PA0637 PA0615 PA0626 PA0265 PA3769 PA0620 PA2641 PA4268 PA1774 PA1333 PA3431 PA0633 PA1616 PA0636 PA3664 PA1871 PA4640 PA1792 PA2263 PA1869 PA0631 PA3361 PA3190 PA2640 PA2116 PA2627 PA1248 PA5315

rpmB trpF rplT gatB rpsN rplL pmpR rpmJ glcB acnB rpnH nuoI exsD gabD nuoF rpsL lasA mqoB lecB nuoE aprF rpmG

50S ribosomal protein L Carboxyphosphonoenolpyruvate phosphonomutas N-(5'phosphoribosyl)anthranilate (PRA) isomerase Hypothetical protein Hypothetical protein 50S ribosomal protein L20 Glu-tRNA(Gln) amidotransferase subunit B 30S ribosomal protein S14 50S ribosomal protein L7 / L12 Hypothetical protein Probable secretion protein Probable aminotransferase Probable ATP-binding component of ABC dipeptide transporter pqsR-mediated PQS regulator 50S ribosomal protein L36 Probable short chain dehydrogenase Hypothetical protein Probable bacteriophage protein Malate synthase G Aconitate hydratase 2 50S ribosomal protein L34 Hypothetical protein Hypothetical protein Hypothetical protein Probable binding protein component of ABC transporter Probable ATP-dependent RNA helicase Probable 2-ketogluconate transporter Probable amino acid permease DNA polymerase III, delta subunit holA NADH Dehydrogenase I chain I ExsD Bacteriophage hypothetical protein Hypothetical protein Hypothetical protein Putative tail formation protein Succinate-semialdehyde dehydrogenase succinate-semialdehyde dehydrogenase, NADP-dependent activity probable bacteriophage protein NADH dehydrogenase I chain F 30S ribosomal protein S12 CfrX protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Protease precursor Malate:quinone oxidoreductase Hypothetical protein Probable 2-hydroxyacid dehydrogenase Probable acyl carrier protein Hypothetical protein Frucose-binding lectin PA-IIL Probable binding protein component of ABC sugar transporter NADH dehydrogenase I chain E Hypothetical protein Hypothetical protein Alkaline protease secretion outer membrane protein AprF precursor 50S ribosomal protein L33

Mariette Barbier

0,3537 0,3531 0,3530 0,3526 0,3512 0,3506 0,3500 0,3499 0,3486 0,3480 0,3474 0,3472 0,3471 0,3458 0,3457 0,3434 0,3430 0,3417 0,3414 0,3405 0,3401 0,3393 0,3383 0,3363 0,3362 0,3356 0,3346 0,3326 0,3325 0,3321 0,3319 0,3313 0,3306 0,3299 0,3291 0,3285 0,3280 0,3256 0,3256 0,3256 0,3221 0,3218 0,3215 0,3210 0,3208 0,3207 0,3199 0,3195 0,3191 0,3177 0,3174 0,3172 0,3165 0,3145 0,3136 0,3130 0,3120 0,3107 0,3102 0,3095


ChoP 37ºCb

ChoP 25ºCa

Genes with decreased expression levels in PAO1 grown at 25ºC versus 37ºC

Protein

Gene name

Gene locus

8.2.2.2.

Fold Change

Appendix

Appendix

PA4843 PA3334 PA4006 PA0432 PA2564 PA3525 PA0632 PA3344 PA3569 PA4217 PA1712 PA4223 PA2300 PA0952 PA0618 PA5285 PA2323 PA5522 PA4181 PA1192 PA2301 PA3841 PA3790 PA2261 PA0619 PA0129 PA3770 PA5523 PA0630 PA0723 PA2322 PA0641 PA5049 PA3192 PA1106 PA4563 PA0639 PA0131 PA0634 PA0713 PA0625 PA0628 PA0638 PA2274 PA1156 PA0629 PA5569 PA2624 PA2259 PA1711 PA1710 PA1246 PA4852 PA4225 PA0795 PA2302 PA2304 PA4231 PA4567 PA2776 PA1123 PA0627 PA3182 PA0266 PA3183 PA3126 PA0635 PA3479 PA4182 PA3193 PA4230

Arrays data

nadD sahH argG recQ mmsB phzS exsB chic exoS oprC gabP guaB coaB gnuB rpmE gltR rpsT nrdA rnpA Idh ptxS exsE exsC aprD pchF prpC pchA rpmA pgl gabT zwf ibpA rhlA glk pchB

Probable two-component response regulator Probable acyl carrier protein Nicotinic acid mononucleotide adenylyltransferase S-adenosyl-L-homocysteine hydrolase Hypothetical protein Argininosuccinate synthase Hypothetical protein ATP-dependent DNA helicase RecQ 3-hydroxyisobutyrate dehydrogenase Flavin-containing monooxygenase Exoenzyme S synthesis protein B Putative ATP-binding component of ABC transporter Chitinase Hypothetical protein Putative phage baseplate assembly protein Conserved hypothetical protein Putative glyceraldehyde-3-phosphate dehydrogenase Putative glutamine synthetase Conserved hypothetical protein Putative ATPase of the PP-loop superfamily Putative tRNA synthase Exoenzyme S Outer membrane copper receptor OprC Putative 2-ketogluconate kinase Putative phage tail protein Gamma-aminobutyrate permease Inosine-5'-monophosphate dehydrogenase Putative glutamate-1-semialdehyde aminotransferase Hypothetical protein Coat protein B of bacteriophage Pf1 Gluconate permease Putative phage-related protein, tail component 50S ribosomal protein L31 Two-component response regulator GltR Conserved hypothetical protein 30S ribosomal protein S20 Conserved hypothetical protein Hypothetical protein Hypothetical protein Hypothetical protein Putative tail length determinator protein Conserved hypothetical protein Putative minor tail protein L Hypothetical protein NrdA, catalytic component of class Ia ribonucleotide reductase Conserved hypothetical protein Ribonuclease P protein component Monomeric isocitrate dehydrogenase Transcriptional regulator PtxS ExsE Exoenzyme S synthesis protein C precursor Alkaline protease secretion protein AprD Putative tRNA-dihydrouridine synthase Pyochelin synthetase PchF Citrate synthase 2 Putative non-ribosomal peptide synthetase Putative regulatory protein Salicylate biosynthesis isochorismate synthase 50S ribosomal protein L27 Putative oxidoreductase Hypothetical protein Conserved hypothetical protein 6-phosphogluconolactonase 4-aminobutyrate aminotransferase Glucose-6-phosphate 1-dehydrogenase Putative small heat shock protein Hypothetical protein Rhamnosyltransferase chain A Putative transcriptional regulator Glucokinase Salicylate biosynthesis protein PchB

Mariette Barbier

0,3092 0,3050 0,3042 0,3021 0,3016 0,3011 0,3007 0,3000 0,2991 0,2896 0,2894 0,2885 0,2884 0,2849 0,2823 0,2816 0,2809 0,2797 0,2772 0,2752 0,2743 0,2715 0,2714 0,2690 0,2686 0,2665 0,2665 0,2662 0,2651 0,2611 0,2579 0,2571 0,2540 0,2534 0,2532 0,2526 0,2522 0,2506 0,2498 0,2484 0,2480 0,2469 0,2439 0,2432 0,2416 0,2271 0,2249 0,2209 0,2185 0,2160 0,2127 0,2126 0,2093 0,2090 0,2059 0,2034 0,2032 0,2026 0,2016 0,2005 0,2002 0,1988 0,1931 0,1893 0,1891 0,1861 0,1822 0,1817 0,1813 0,1807 0,1799


Appendix

PA0622 PA3191 PA4224 PA4229 PA0623 PA0624 PA4677 PA0610 PA3233 PA3478 PA2260 PA3194 PA3022 PA1155 PA4228 PA3496 PA2303 PA1247 PA2575 PA3568 PA4218 PA4226 PA2305 PA3038 PA1556 PA0807 PA2320 PA3181 PA4221 PA0612 PA1432 PA1555 PA3232 PA0613 PA2679 PA0130 PA4290 PA3195 PA3529 PA5445 PA1245 PA3662 PA2321 PA0132 PA3234 PA4220 PA3235 PA0887

a

Arrays data

pchC prtN rhlB edd nrdB pchD aprE pchE gntR eda fptA ptrB lasI gapA apta acsA

Putative phage tail sheath protein Putative two-component sensor Putative two-component sensor Pyochelin biosynthetic protein PchC Putative phage tail tube protein Conserved hypothetical protein Conserved hypothetical protein Transcriptional regulator PrtN Putative signal-transduction protein Rhamnosyltransferase chain B Hypothetical protein 6-phosphogluconate dehydratase Conserved hypothetical protein NrdB, tyrosyl radical-harboring cmpt. of ribonucleotide reductase Pyochelin biosynthesis protein PchD Hypothetical protein Putative regulatory protein Alkaline protease secretion protein AprE Putative nitroreductase Putative acetyl-CoA synthetase Probable transporter Pyochelin synthetase Putative non-ribosomal peptide synthetase Putative outer membrane porin Putative cytochrome c oxidase subunit Hypothetical protein Transcriptional regulator GntR 2-keto-3-deoxy-6-phosphogluconate aldolase Fe(III)-pyochelin outer membrane receptor precursor Repressor, PtrB Autoinducer synthesis protein LasI Putative cytochrome c oxidase, cbb3-type, subunit III Putative nuclease Hypothetical protein Conserved hypothetical protein Probable aldehyde dehydrogenase Putative chemotaxis transducer Glyceraldehyde-3-phosphate dehydrogenase Putative alkyl hydroperoxide reductase subunit Putative coenzyme A transferase Conserved hypothetical protein Hypothetical protein Gluconokinase Beta-alanine-pyruvate transaminase Putative sodium/proline:solute symporter Conserved hypothetical protein Putative membrane protein Acetyl-coenzyme A synthetase

0,1798 0,1796 0,1755 0,1754 0,1744 0,1726 0,1723 0,1713 0,1675 0,1637 0,1617 0,1590 0,1589 0,1468 0,1465 0,1447 0,1442 0,1401 0,1378 0,1300 0,1289 0,1276 0,1240 0,1226 0,1225 0,1220 0,1182 0,1146 0,1096 0,1070 0,1043 0,1030 0,0968 0,0960 0,0880 0,0865 0,0810 0,0730 0,0695 0,0694 0,0679 0,0670 0,0606 0,0519 0,0413 0,0360 0,0282 0,0059


mutants of the PA14 transposon library for the corresponding gene. The detection of ChoP associated to EF-Tu is indicated in green. The gene of which the mutant was not available is indicated in red. b


mutants of the PA14 transposon library for the corresponding gene. The absence of ChoP on EF-Tu appears in blue. The gene of which the mutant was not available is indicated in red.

Mariette Barbier
