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UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New series No. 1019
ISSN 0346-6612
Porins of Borrelia burgdorferi
Marija Pinne
Department of Molecular Biology Umeå University Sweden, 2006
Cover: Electron micrograph and a density map of 2-D crystals formed by the P66 porin in an outer-membrane protein fraction of B. burgdorferi
Photo: Akemi Takade Image processing: Ignas Bunikis
Copyright © 2006 by Marija Pinne ISBN 91-7264-059-6 Printed by Solfjädern Offset AB Umeå, Sweden, 2006
To my mother, in deepest memory…
Kaut tā mana māmuliņa Jo ilgāki dzīvājuse, Es būt liepas kuplumiņu, Ievas ziedu baltumiņu.
Latviešu tautasdziesma (Latvian Folksong)
TABLE OF CONTENTS ABSTRACT
7
ABBREVIATIONS
8
PAPERS IN THIS THESIS
9
INTRODUCTION
11
1. Lyme disease Borrelia
11
1.1. Classification
11
1.2. General features
11
1.3. Transmission and adaptation to different environments
12
1.4. Clinical manifestations of Lyme disease
13
2. Genome structure of B. burgdorferi
14
2.1. The role of plasmids
14
2.2. Paralogous gene families
15
2.3. Genetic tools
16
3. Borrelia membrane structure 3.1. Biogenesis and translocation of spirochetal OMPs 3.1.1. C-terminal processing 3.2. Outer surface lipoproteins of B. burgdorferi
17 19 22 23
3.2.1. Lipoproteins with roles in transmission and adaptation
24
3.2.2. Adhesins
25
3.2.3. Lipoproteins with roles in persistence and diagnosis
26
3.2.4. Lipoproteins conferring serum resistance
27
3.3. Integral outer membrane proteins in Borrelia
28
3.4. General features of outer membrane channels (Porins)
30
3.5. Functional reconstitution of porins in artificial lipid bilayer membranes 3.6. Porins in spirochetes 3.6.1. Porins in B. burgdorferi
32 33 34
3.6.2. Channel-forming proteins of relapsing fever Borrelia
34
3.6.3. Porins in Treponema sp. and Leptospira
35
3.7. Active transport systems in B. burgdorferi
36
3.8. Structural characterization of membrane proteins
38
AIMS OF THIS THESIS
41
RESULTS AND DISCUSSION
42
4. Molecular analysis of P13 and its paralogue family 48 (Paper I)
42
5. P13, a channel-forming protein of B. burgdorferi (Paper II)
44
6. C-terminal processing of P13 by carboxyl-terminal processing enzyme A, CtpA (Paper III)
47
7. Characterization of the BBA01 protein, the paralog of P13 (Paper IV) 8. The function and oligomeric status of P66 (paper V)
50 53
9. Structural characterization of P13 and P66 by electron crystallography (Paper VI)
56
CONCLUSIONS
60
ACKNOWLEDGMENTS
61
REFERENCES
63
PAPERS I-VI
85
ABSTRACT Borrelia burgdorferi is a pathogenic spirochete which cycles between its arthropod vector and vertebrate host. If transmitted to humans, B. burgdorferi causes Lyme disease, an infection which can impair different organs, such as the skin, joints, nervous system and heart. Alterations in protein expression due to the different environments Borrelia encounters during its complicated life cycle require advanced adaptation mechanisms. The outer surfaceexposed proteins play a critical role in survival and pathogenesis of Borrelia in different hosts and tissues, being involved in avoiding the host immune response, adhesion to different tissues and nutrient acquisition. This thesis aimed to characterize integral outer membrane proteins which play a role in solute and nutrient uptake, and provides support for their role in the environmental adaptation of Borrelia. In this thesis, three B. burgdorferi proteins, P13, BBA01 and P66, were shown to be porins, and characterized structurally and functionally using a combination of biochemical, biophysical and genetic methods. The channel-forming function of the 13 kDa protein, P13, was elucidated by a lipid bilayer assay. Post-translational processing of P13 occurred at the C-terminus by C-terminal processing protease (CtpA)-dependent cleavage. The membrane-spanning architecture of P13 was determined by epitope mapping and computer-based structural predictions which revealed that P13 is an unusual porin, not possessing the structural properties of conventional porins: rather than forming β-barrels, it is predicted to span the membrane with hydrophobic α-helices. p13 belongs to a paralogous gene family. The transcription of p13 and other gene family members during in vitro growth and in a mouse infection model was therefore investigated. The paralog BBA01, which has the highest sequence homology to P13, is expressed during in vitro growth in all three Lyme disease causing species, although at very low levels. Like P13, BBA01 is also processed by CtpA and exhibits very similar channelforming activity. Furthermore, in the absence of P13, a proportion of total BBA01 protein is relocated to the bacterial surface with strong indications that BBA01 and P13 are functionally interchangeable. P66, an integrin binding protein, was also determined to be a porin. The oligomeric state of native P66, elucidated by chemical cross-linking, indicated that P66 forms trimers, as do the majority of conventional porins. Electron crystallography and a projection map of P66 crystals at 2.2 nm resolution revealed tetragonal unit cell symmetry with the area intercalated between the assembled protein structures consistent with the approximate expected size of the channel formed by P66. Finally, the biological relevance of two porins, P13 and P66, was demonstrated in a double mutant displaying a stress response as revealed by increased sensitivity to high osmolarity and elevated expression of the B. burgdorferi heatshock protein HtrA homolog.
7
ABBREVIATIONS 2-D 3-D aa ABC AFM ATPase bp cp CtpA Da EM FPLC G+C GAG HtrA kb Lep lp LPS Lsp MAbs NMR nS OGP OM OMPs Oms ORF Osps PTS RT-PCR SDS-PAGE Sec SRP
two-dimensional three-dimensional amino acid(s) ATP binding cassette atomic force microscopy adenosine triphosphate hydrolyse base pair circular plasmid carboxyl-terminal protease Daltons electron microscopy fast performance liquid chromatography guanosine + cytosine glycosaminoglycan high-temperature requirement protease (DegP) kilobases leader peptidase (signal peptidase I) linear plasmid lipopolysaccharide lipoprotein signal peptidase (signal peptidase II) monoclonal antibodies nuclear magnetic resonance nano siemens octyl-glucopyranoside outer membrane outer membrane proteins Outer membrane spanning protein open reading frame outer surface proteins phosphorylation transport system reverse transcription-polymerase chain reaction Sodium dodecyl sulphate polyacrylamide gel electrophoresis preprotein translocase complex signal recognition particle
8
PAPERS IN THIS THESIS This thesis is based on the following articles and manuscripts, which will be referred to by their Roman numerals (I-VI) I
M. Pinne, Y. Östberg, P. Comstedt, and S. Bergström. 2004. Molecular analysis of the channel-forming protein P13 and its paralogue family 48 from different Lyme disease Borrelia species. Microbiology. 150(3): 549-559.
II
Östberg Y, Pinne M, Benz R, Rosa P, Bergström S. 2002. Elimination of channel-forming activity by insertional inactivation of the p13 gene in Borrelia burgdorferi. J Bacteriol. 184(24):6811-9.
III
Y. Östberg, J. A. Caroll, M. Pinne, J.G. Krum, P. Rosa, and S. Bergström. 2004. Pleiotropic Effects of Inactivating a CarboxylTerminal Protease, CtpA, in Borrelia burgdorferi. J Bacteriol. 186 (7):2074-2084.
IV
M. Pinne, K. Denker, E. Nilsson, R. Benz, and S. Bergström. 2006. The BBA01 protein, a member of paralog family 48 from Borrelia burgdorferi, is potentially interchangeable with the channel-forming protein P13. Submitted to J Bacteriol.
V
M. Pinne, M. Thein, K. Denker, J. Coburn, R. Benz, and S. Bergström. 2006. The P66 protein of Borrelia burgdorferi demonstrates dual functions of channel-forming and integrinbinding activity. Manuscript.
VI
M. Pinne, I. Bunikis, A. Takade, Y. Mizunoe, S. Nyunt Wai, and S. Bergström. 2006. Structural characterization of Borrelia burgdorferi porins by electron crystallography. Manuscript
9
Contributions to the following work, not included in the thesis, were also made: ¤
L. Noppa, Y. Östberg, M. Lavrinovicha, and S. Bergström. 2001. P13, an Integral Membrane Protein of Borrelia burgdorferi, Is Cterminally Processed and Contains Surface- Exposed Domains. Infection and Immunity. 69- 5: 3323- 3334.
¤
Nilsson, C, L., H. J. Cooper, K. Håkansson, A. G. Marshall, Y. Östberg, M. Lavrinovicha, and S. Bergström. 2002. Characterization of the P13 membrane protein of Borrelia burgdorferi by mass spectrometry. J Am Soc Mass Spectrom. Apr;13(4):295-9.
10
INTRODUCTION
INTRODUCTION 1.
Lyme disease Borrelia
The causative agent of Lyme disease is the tick-transmitted spirochete, Borrelia burgdorferi. The disease is named after the town of Old Lyme in Connecticut, USA, from where the first reports of Lyme arthritis originated (Steere et al., 1977; Steere et al., 1977). The causative agent was discovered in 1982 by Willy Burgdorfer and co-workers (Benach et al., 1983; Burgdorfer et al., 1982; Steere et al., 1983) and named Borrelia burgdorferi in honour of its discoverer (Johnson et al., 1984).
1.1
Classification
Lyme disease Borrelia belongs to the spirochete group, an ancient evolutionary branch of bacteria only remotely related to Gram-negative and Gram-positive bacteria (Woese, 1987). The family Spirochaetaceae also includes other important pathogenic bacteria such as: Treponema denticola, causing periodontal disease, Treponema pallidum, causing syphilis, Leptospira interrogans, causing leptospirosis, and Brachyspira hyodysenteriae, causing swine dysentery. The genus Borrelia contains different pathogenic groups: the relapsing fever Borrelia, Lyme disease Borrelia, and the aetiological agents of avian and bovine spirochaetosis, Borrelia anserina, and Borrelia coriaceae, respectively. The Lyme disease Borrelia burgdorferi complex contains at least 12 different species, including the well studied human pathogens, Borrelia burgdorferi, Borrelia afzelii, Borrelia garinii (Baranton et al., 1992; Canica et al., 1993; Marconi & Garon, 1992) and the newly discovered Borrelia spielmani (Richter et al., 2004).
1.2
General features
Borrelia are elongated (10-30 μm in length), thin (~0.3 μm in width), motile and helically curved (Fig. 1) bacteria (Goldstein et al., 1996). Borrelia exhibits chemotaxis (Shi et al., 1998) by virtue of 7-11 bipolar endoflagella located in the periplasmic space and attached to the poles of the bacterial cell (Barbour & Hayes, 1986; Goldstein et al., 1996; Motaleb et al., 2000). These flagella also confer the characteristic flat wave-like shape of Borrelia. Although B. burgdorferi is considered Gram-negative because they have both an outer and inner membrane, they share many features of Gram-positive bacteria. These include, the low G+C
11
INTRODUCTION content of the genome (28.6%) and the close association of the inner membrane with a peptidoglycan cell wall. Unlike Gram-negative bacteria, the Borrelia outer membrane is also fluid and labile consisting of 45-62% protein, 23-50% lipid and 3-4% carbohydrate (Barbour & Hayes, 1986) and lacking lipopolysaccharides (LPS) (Takayama et al., 1987). This labile outer membrane also spontaneously generates extracellular vesicles or blebs during in vitro growth (Barbour & Hayes, 1986; Radolf et al., 1994). The unusual genome and membrane composition of B. burgdorferi will be discussed in later sections of this thesis. B. burgdorferi is an obligate parasite, not able to survive outside its arthropod or vertebrate host. This natural life cycle of Borrelia has led to relatively small genome with only limited biosynthetic pathways. Thus, Borrelia has fastidious growth requirements for in vitro cultivation. In laboratory, Borrelia can be cultivated only in rich growth medium, Barbour-Stoenner-Kelly II (BSK II) supplemented with the rabbit serum and is based on the eukaryotic cell-line cultivation medium CMRL (Barbour, 1984). Their generation time is about 10 hours at optimal temperature of about 35-37oC.
Figure 1. B. burgdorferi B31 visualized under phase contrast microscopy. (Photo: Marija Pinne)
1.3
Transmission and adaptation to different environments
Lyme disease is the most common arthropod-borne human disease in the northern hemisphere- the incidence increases yearly and reached 40,800 cases in the USA alone in 2001-2002 (CDC, 2004). Lyme disease spirochetes are transmitted to humans via the bite of a tick from the Ixodidae family that was previously infected during a blood meal taken from a Borrelia-contaminated vertebrate. Reservoir hosts for Borrelia usually are small rodents, although an importance of birds in enzootic lifecycle of Borrelia is emerging (Anderson, 1988; Burgdorfer, 1989; Gylfe et al., 2001; Humair, 2002; Kurtenbach et al., 1998; Olsen et al., 1995). The tick has three developmental stages: larva, nymph and adult. The tick
12
INTRODUCTION feeds only once before it molts into the next stage and if larval ticks acquire Borrelia, the spirochetes survive through the molts and are present in subsequent stages. The prevalence of Borrelia in different tick populations varies between 1-70% (De Michelis et al., 2000; Gustafson, 1994; Rauter & Hartung, 2005). The arthropod vector competence for Lyme disease Borrelia species is observed leading to the preference in transmission. Thus, in North America, I. scapularis and I. pacificus transmit B. burgdorferi (Postic et al., 1998), while in Europe and Asia, I. ricinus and I. persulcatus transmit B. afzelii and B. garinii and to a lesser extent B. burgdorferi (Hubalek & Halouzka, 1997; Li et al., 1998). Lyme disease Borrelia has a complex lifecycle, cycling between their tick vector and the vertebrate reservoir. This is accompanied by many changes in environmental factors encountered by Borrelia, such as temperature, pH, osmolarity, nutrient availability, and immune system barriers, which precipitated the development of advanced strategies (see sections 2.2 and 3.2) to sense and survive in these environments (Hefty et al., 2002b; Thomas et al., 2001; Tokarz et al., 2004).
1.4
Clinical manifestations of Lyme disease
The clinical manifestations of Lyme disease (Lyme borrelisosis) involve multiple organs, including the skin, joints, heart, and nervous system. Symptoms are classified according to the infections stage- early localized, disseminated, or chronic infection. The most common and distinctive manifestation of an early localized infection is a skin-rash termed erythema migrans (EM), normally occurring approximately one week post-tick bite at the site of inoculation. At this stage of infection, Borrelia are susceptible to antibiotic treatment and the infection can be eliminated (Steere, 1989). More severe symptoms occur if left untreated, which is most probably in those ~10% of cases that do not exhibit initial EM symptoms (Nadelman et al., 1996). The next stages of Lyme disease occur after Borrelia disseminates from its initial site of infection. The most common clinical manifestations of early disseminated infection is neuroborreliosis and Lyme arthritis (Nadelman & Wormser, 1998; Steere, 1989). Manifestations of chronic (persistent) Lyme disease involve acrodermatitis chronica atrophicans (ACA), meningitis, and more rarely Lyme meningoencephalitis (Asbrink et al., 1984; Nadelman & Wormser, 1998; Steere, 1989). The specific distribution patterns of Borrelia species in North America and Europe may account for variable frequency of Lyme disease manifestations, partially depending on which species causes the infection (Wang
13
INTRODUCTION et al., 1999).
Neuroborrelisosis and ACA more commonly occurs in Europe, usually caused by B. garinii, and B. afzelii, respectively, whereas B. burgdorferi infections causing Lyme arthritis are more common in North America (Nadelman & Wormser, 1998). In this context, it is noteworthy that although a human vaccine (LYMErix) based on recombinant outer surface protein A (OspA) has been developed and used in the past (Steere et al., 1998; Wallich et al., 1996), at the moment there is no available vaccine for prevention of Lyme disease.
2.
Genome structure of B. burgdorferi
B. burgdorferi has an unusual genome structure, consisting of a 0.91 Mb linear chromosome and 21 plasmids (12 linear and 9 circular) that comprise an additional 0.61 Mb, based on the genome of the sequenced strain B31 MI (Casjens et al., 2000; Fraser et al., 1997). To date, the amount of extra chromosomal DNA elements in Borrelia is the highest of any known organism. The B. burgdorferi genome encodes only 1283 putative genes (E. coli has 4405 genes). Most notable is the absence of genes coding for proteins involved in amino acid, fatty acid, or nucleotide biosynthesis (Fraser et al., 1997). This limited metabolic capacity probably reflects convergent evolution by the loss of genes, prompted by the parasitic lifestyle of Borrelia. Most bacteria possess a circular chromosome and plasmids, but such DNA molecules are a minority in the Borrelia genera. Interestingly, the linear chromosome and linear plasmids of B. burgdorferi have covalently closed hairpin loops resembling those of eukaryotic telomeres (Casjens et al., 1997a; Hinnebusch et al., 1990; Hinnebusch & Tilly, 1993).
2.1
The role of plasmids
Probably nowhere else in the prokaryotic world have plasmids evolved to be as central to an organism’s life cycle as they have in Borrelia spirochetes. Each plasmid has a copy number of one per chromosome (Casjens & Huang, 1993; Hinnebusch & Barbour, 1992). Many plasmids contain homologous regions and the plasmid content among different B. burgdorferi isolates can vary (Casjens et al., 1997b; Palmer et al., 2000; Stewart et al., 2005). The infectivity of most pathogenic bacteria requires certain plasmids on which the genes for virulence factors are located. More than 90% of the predicted open reading frames (ORFs) carried on B. burgdorferi plasmids have no known homologues, such that very few virulence factors have been identified based on homology to ORFs in other pathogenic bacteria (Casjens et al., 2000; Fraser et al., 1997). B. burgdorferi plasmids
14
INTRODUCTION important for infectivity include the 25-kb linear plasmid (lp25), lp28-1, and lp28-4. Genes encoding many outer surface proteins are located on the plasmids and are differentially expressed during the infectious cycle (Hefty et al., 2002a; Purser & Norris, 2000; Purser et al., 2003; Thomas et al., 2001). The fact that plasmids are easily lost during in vitro cultivation of B. burgdorferi (Barbour, 1988), correlating with a subsequent loss of infectivity (Elias et al., 2002; Labandeira-Rey & Skare, 2001; Schwan et al., 1988; Xu et al., 1996), is further evidence for their important roles in maintaining the bacterial lifecycle. Although many plasmids carry genes essential for infectivity but not for survival in artificial growth media, the circular plasmid cp26 is essential for in vitro growth (Byram et al., 2004). Generally, the circular plasmids contain fewer pseudogenes and are more stable in vitro than their linear counterparts. With exception of cp26, all other circular plasmids are related, being derivatives of the cp32 (1-9) plasmid family. The cp32s are temperate bacteriophages, whose prophage forms are autonomously replicating circular plasmids acquired by Borrelia through lateral gene transfer (Eggers & Samuels, 1999).
2.2
Paralogous gene families
Although not fully understood, another striking feature of Borrelia is the amount of paralogous gene (homologous genes within the same specie) families in their genome. The B. burgdorferi B31 genome exhibits extensive genetic redundancy, with most of the 175 paralogous gene families (in the order of 70%) situated on plasmids. Many putative proteins encoded by these gene families exhibit no homology with proteins of known function (Casjens et al., 2000; Fraser et al., 1997). The biological advantage of maintaining so many paralogous genes, an energetically expensive process, is unclear. Several gene families have been investigated in B. burgdorferi, including bmp (Aron et al., 1994; Gorbacheva et al., 2000; Ramamoorthy et al., 1996; Simpson et al., 1994), erp (Stevenson et al., 1996; Stevenson et al., 1998b), vls (Wang et al., 2001; Zhang et al., 1997; Zhang & Norris, 1998a), elp (Akins et al., 1999), bdr (Carlyon et al., 2000; Zuckert et al., 1999), mlp (Porcella et al., 2000; Yang et al., 1999), and family 95 (bapA and eppA) (Champion et al., 1994; Miller & Stevenson, 2003). Our own investigation of paralogous gene family 48 will be discussed in sections 4, 7 and Papers I, IV. Perhaps the existence of multiple copies of a given gene controlled by different promoter sequences may allow differential regulation of individual gene family members, especially since specific paralogs may function only at certain stage of the Borrelia lifecycle, responding to a particular set of environmental conditions (Ojaimi et al., 2003;
15
INTRODUCTION Revel et al., 2002; Roberts et al., 2002; Tokarz et al., 2004). In this respect, temperature
shift, serum deprivation, tick feeding, and the mammalian environment influence the regulation of several paralogous gene family members, including mlp, elp, vls, erp, bdr, and family 54 genes (Brooks et al., 2003; Carroll et al., 2000; Hefty et al., 2001; Roberts et al., 2002; Stevenson et al., 1998a; Stevenson et al., 2002; Tokarz et al., 2004; Yang et al., 2000; Yang et al., 2003; Zhang & Norris, 1998b).
Interestingly, spatial control of protein expression may also be an important contributing factor since differential cellular localization of proteins from the same paralog family has been reported for the OspE/F/Elp and Bdr paralogs (Hefty et al., 2002b; Roberts et al., 2002). Our studies on P13 and its paralogs also support the hypothesis that B. burgdorferi possesses many paralogous gene families to replace or regulate certain protein functions under different conditions. This will be presented in sections 4 and 7 along with Papers I, IV.
2.3
Genetic tools
Genetic manipulation techniques are widely used to elucidate and characterize the functions of genes and their products. Genetic studies in Borrelia have been hindered because of the difficulties to grow these bacteria on solid media, the lack of exogenous selectable markers, and the extremely low DNA transformation efficiency. However, major improvements in gene inactivation and complementation have occurred during the last 6 years, although currently genetic manipulations are possible only in Lyme disease Borrelia. In B. burgdorferi, the gene of interest can be inactivated by allelic-exchange, plasmid integration or random genome-wide transposon mutagenesis. Historically, the first gene inactivated in B. burgdorferi by allelic-exchange was ospC (Tilly et al., r 1997) using the selective marker GyrB , a mutated form of the chromosomal gyrB gene conferring resistance to coumermycin A1 (Samuels et al., 1994). Nevertheless, coumermycin as a selectable marker is limited by a high frequency of recombination with the endogenous gyrB gene (Tilly et al., 1998). More recently, an efficient selectable marker was developed by linking the B. burgdorferi flagelline A (flaA) or flagelline B (flab) promoter with the kanamycin-resistance gene from Tn903 (Bono et al., 2000). This strategy of linking flaB gene promoter to the selectable markers is now an established technique to genetically manipulate B. burgdorferi. Several selectable markers have now been developed including ermC (conferring resistance to erythromycin) (Sartakova et al., 2000), aacC1 (gentamycin) (Elias et al., 2002), aadA (spectinomycin and streptomycin) (Frank et al., 2003), and some additional
16
INTRODUCTION antibiotic resistance markers are also described (Sartakova et al., 2003). Our use of the flaBP-kan and flaBP-aadA resistance cassettes to inactivate genes is discussed in Papers II to V. The next challenge is to inactivate genes in infectious B. burgdorferi. Low passage, infectious B. burgdorferi isolates grow more slowly, display lower plating efficiency and transformation frequencies are 100-fold lower than those of high passage, non-infectious isolates (Tilly et al., 2000). Interestingly, just as the loss of plasmids lp25, lp28-1, and lp28-4 correlates with loss of infectivity of B. burgdorferi, the absence of lp25 correlates with an increase in transformation efficiency. This correlation likely occurs because reduced transformation frequency is associated with restriction/modification system that is encoded on the lp25 and lp56 plasmids in infectious B. burgdorferi and is absent or nonfunctional in in vitro cultivated Borrelia (Lawrenz et al., 2002). Moreover, the nicotinamidase gene pncA (BBE02), located on lp25, also strongly effects transformation efficiency of B. burgdorferi. Inactivation of BBE02 eliminated the transformation barrier for infectious B. burgdorferi B31, providing a valuable tool for determining virulence factors contributing to Lyme disease (Kawabata et al., 2004). Genetic complementation based on the use of shuttle vectors has been developed for B. burgdorferi. The first shuttle vector introduced in B. burgdorferi was pGK12 with an ermC gene conferring resistance to erythromycin (Sartakova et al., 2000). Another vector, pBSV2 including a region of B. burgdorferi cp9 and a kanamycin resistance marker, was successfully transformed in low passage B. burgdorferi N40 (Stewart et al., 2001) and served as the basis for the most widely used shuttle vector pBSV2G, containing a gentamycin resistance (GmR) marker (Elias et al., 2003). Additional shuttle vectors and other complementation techniques for B. burgdorferi are also available (Eggers et al., 2002; Hubner et al., 2001; Sartakova et al., 2001). Our genetic complementation studies based on pBSV2G are described in sections 6-8 and Papers III-V.
3.
Borrelia membrane structure
The outer membrane of bacteria is a selective barrier between the cell and environment that excludes certain molecules and simultaneously allows entry of appropriate nutrient substances. The outer membrane of conventional Gramnegative bacteria consists of an asymmetrical bilayer with an inner leaflet composed of phospholipids and an outer leaflet containing lipopolysaccharides
17
INTRODUCTION (LPS) (Kamio & Nikaido, 1976). Interestingly, LPS and phosphatidylethanolamine are notable omissions from the outer membrane of Borrelia (Belisle et al., 1994; Takayama et al., 1987). Moreover, this outer membrane also has a very low density of integral, membrane-spanning proteins (Radolf et al., 1994; Walker et al., 1991), but maintains a extraordinary high abundance of lipoproteins (Brandt et al., 1990; Fraser et al., 1997). In fact, 8% (105 in total) of all B. burgdorferi ORFs are predicted to encode lipoproteins, the highest frequency among those bacterial genomes sequenced (Fraser et al., 1997; Haake, 2000). This number could even be higher, since a new computer algorithm, SpLip- designed to specifically identify spirochetal lipoproteins, predicted 127 lipoprotein ORFs in B. burgdorferi (Setubal et al., 2006). Integral outer membrane protein
Osps Porin C C
C
C
C
C
C
C
OM C
Flagella Lipoproteins
Peptidoglycan C
C
Integral membrane protein
IM Transporter
Membrane protein
Channel
Figure 2. Schematic illustration of the Borrelia cell envelope. OM, outer membrane. IM, inner membrane. Osps, outer surface proteins (lipoproteins).
Outer membrane proteins (OMPs) of Gram-negative bacteria are of great interest because of their location at the cell surface, which may facilitate interactions of bacterial pathogens with their host. In particular, OMPs may play a role in pathogenesis by acting as (i) adhesins, (ii) targets for bactericidal antibodies, (iii) receptors for various molecules, or (iv) porins. In addition, the selective permeability of the outer membrane is due to the physico-chemical properties of the membrane itself and to the action of specific binding and uptake molecules associated with the outer membrane. Substances can be taken into bacteria by diffusion through the outer membrane via porins (Benz, 2001) or 18
INTRODUCTION by receptor-mediated uptake (Nikaido, 2003). Clearly, OMPs allow Borrelia to adapt and respond to the different environments during its life cycle, in this way playing a critical role in survival, biology and infectivity of Lyme disease spirochetes. Since the outer membrane also makes direct contact with the host, B. burgdorferi molecules exposed on the bacterial surface must mediate the interactions with the host and contend with specific host defence mechanisms that permit colonization of various tissues. Borrelia outer membranes are composed of phospholipids and OMPs, which include outer surface (lipo)proteins (Osps), other lipoproteins, and integral (trans-membrane) outer membrane proteins. A schematic illustration of the Borrelia cell envelope is given in figure 2.
3.1
Biogenesis and translocation of spirochetal OMPs
All the spirochetal OMPs studied to date are synthesized with a signal peptide at their N-terminus, which initiates export of the protein across the inner membrane, and is subsequently cleaved to allow the translocation to the outer membrane (Cullen et al., 2004; Haake, 2000). While the mechanisms of secretion, processing and sorting are generally elucidated for bacterial lipoproteins, how non-lipidated OM proteins traffic remains unclear. In spirochetes the molecular mechanisms of protein secretion are unknown, but from analysis of the genome sequence of B. burgdorferi, homologues exist for all the essential components of Sec-dependent bacterial secretory machinery (Fraser et al., 1997). This suggests that similar secretion pathways exist in Borrelia. Biogenesis of conventional outer membrane is illustrated in figure 3. As a nascent lipoprotein emerges from the ribosome, a signalrecognition particle Ffh (BB0694) binds to the signal peptide and traffics it to the preprotein translocase complex (Sec) in the inner membrane. Sec complexes consist of a SecYE (BB0498, BB0395) transmembrane channel, a SecA (BB0154) cytoplasmic domain, and a SecDF (BB0652, BB0653) periplasmic domain. While secretion through the Sec translocase complex consumes energy, it is assumed that all proteins with an N-terminal signal peptides follow this pathway (Haake, 2000). After secretion across the inner membrane, proteins are lipidated and the signal peptide is cleaved off at the cysteine residue (Fig. 3). Lipoprotein processing takes place on the periplasmic side of the inner membrane and involves three separate enzymes located in the inner membrane (Wu, 1996). First, the diacylglyceryl transferase, Lgt (BB0362) transfers two fatty acids to the sulphur atom of cysteine. Next, the signal peptidase II, also termed lipoprotein signal peptidase, Lsp (BB0469) proteolytically removes the signal 19
INTRODUCTION peptide. The consensus ‘’lipobox’’ amino acid sequence for Lsp recognition in E. coli has formed the basis of the Psort computer prediction program for in silica lipoprotein identification (Nakai & Horton, 1999). Nevertheless, spirochetal lipoproteins possess highly variable lipoboxes indicating that spirochetal Lsp has reduced substrate specificity and/or the possibility of slow rates of lipoprotein biosynthesis, which is also supported by long generation time of spirochetes (Haake, 2000). The increased plasticity of the spirochetal lipobox has meant that 43% of experimentally verified lipoproteins are left unrecognized by the Psort program. Therefore, recently the new computer program (SpLip) has been developed to specifically identify spirochetal lipoproteins (Setubal et al., 2006). Finally, an apolipoprotein N-acyltransferase, Lnt (BB0237) transfers the third fatty acid from membrane phospholipids to the nitrogen atom of cysteine. Thus, lipoproteins have three fatty acids attached to their cysteine residue, preventing N-terminal amino acid sequencing by the Edman degradation technique.
LPS
?
p o rin
L o lB S C
In te g ra l OMP
op
ro t e in
s
S o r tin g
li p
Skp H tr A S u rA
Integral OMPs
P
Lsp
C
C
SP
Sec
Lgt
SP
IM
Lo l A
?
O m p85
S C
OM
Imp
S C
Lnt C
C
D C
S C L o lC D E
OMPs
C
Figure 3. Biogenesis of conventional outer membrane. Both lipoproteins and integral OMPs are synthesized in the cytoplasm (C) and translocated across the inner membrane (IM) by the Sec machinery. SP, signal peptide. P, periplasm, OM, outer membrane. LolCDE, an ABC transporter. The Lol-avoidance motif Asp (D) at position +2 aa results in retention of the lipoprotein in the IM. A serine (S) at position +2 aa results in OM localization. Imp, OMP required for the assembly of LPS. Omp85, OMP involved in the assembly of integral OMPs.
20
INTRODUCTION Interestingly, most bacteria analysed have lipoproteins with homologous acylation profiles (Mizuno, 1979; Zlotnick et al., 1988). In contrast, spirochetal lipoproteins appear to have heterogeneous fatty acid compositions and those from B. burgdorferi even possesses glyceryl residues acylated predominantly with oleic and linoleic acid, while the amide-linked fatty acid on the cysteine is predominantly stearic acid (Beermann et al., 2000). This unique lipid composition in B. burgdorferi might be important in pathogenesis and immune evasion by Borrelia, where the role of lipid modification is established (Erdile et al., 1993; Morr et al., 2002; Radolf et al., 1995; Sellati et al., 1996). Lipoproteins become associated with membranes via a hydrophobic interaction between the lipid moiety and the membrane phospholipids. The three hydrophobic fatty acid moieties anchor otherwise hydrophilic proteins to hydrophobic membrane bilayers. Lipoproteins can be localized to one or more of four cellular compartments: the periplasmic leaflet of the inner membrane, the periplasmic or outer leaflets of the outer membrane, or beyond the outer membrane (Cullen et al., 2004; Haake, 2000). In E. coli, two proteins are involved in localization of lipoproteins to the OM (Fig. 3). A periplasmic chaperone, LolA, transports the lipoproteins to the OM and LolB incorporates murein lipoprotein into the OM (Matsuyama et al., 1997). Nascent lipoproteins form a complex with LolA and on recognition of the outer membrane sorting signal, lipoproteins are released in an ATP-dependent manner from the inner membrane by LolCDE (Narita et al., 2002; Yakushi et al., 2000). The resultant LolA-lipoprotein complex is hydrophilic and can traverse the periplasm and interact with an outer membrane lipoprotein receptor LolB, which mediates the anchoring into the OM (Matsuyama et al., 1997). It is assumed that +2 aa (relative to the N-terminus) determines whether the lipoprotein interacts with LolA and therefore if it remains in the inner membrane or is transported to the outer membrane (Matsuyama et al., 1995). The importance of the environment around +2 sorting signal has been also implicated in this cellular localization (Robichon et al., 2003). The factors that determine the cellular localization of spirochetal lipoproteins are still largely unknown, but presumably the sequence or physico-chemical properties of the signal peptide or mature protein are critical. Thus, acidic and/or basic residues following the N-terminal Cys may have relevance in sorting spirochetal lipoproteins (Haake, 2000), although conformation might also play a significant role (Cullen et al., 2004). Homologues to the E. coli lipoprotein sorting can be found only in Leptospira interrogans, suggesting that lipoprotein sorting may even differ between spirochete species. Perhaps unique pathways for transportation of spirochetal lipoproteins to the OM exist. A recent analysis of the lipoprotein-sorting signals of B. burgdorferi indicated a mechanism distinct
21
INTRODUCTION of the ‘’+2 rule’’ and that secretion to the OM occurs independent of any obvious N-terminal sequence or overall lipoprotein secondary structure (Schulze & Zuckert, 2006). Moreover, negatively charged residues within the first nine Nterminal amino acids of a mature lipoprotein can cause its retention in Borrelia periplasm. This lead to the proposal that Borrelia lipoproteins are targeted to the cell surface by default, but can be retained in the periplasm or inner membrane by sequence-specific signals (Schulze & Zuckert, 2006). In contrast to lipoproteins, trafficking of nonlipoprotein OMPs beyond the inner membrane and their subsequent integration into the outer membrane is poorly understood. The Sec machinery is involved also in translocation of integral OMPs across the inner membrane (Fig. 3). In case of integral OM proteins, the recognition and cleavage of N-terminal signal sequence is achieved by signal peptidase I, also called leader peptidase (Lep). Further protein sorting and trafficking mechanisms to the outer membrane are only now being unraveled even in E.coli. LPS is implicated in porin assembly and insertion into the bacterial OM (Laird et al., 1994; Ried et al., 1990). Thus, one reason for the low abundance of transmembrane OMPs in spirochetes such as B. burgdorferi or T. pallidum, is the absence of LPS, which may hamper assembly and insertion of transmembrane OMPs into these unusual outer membranes (Cullen et al., 2004). Several proteins involved in OMP assembly in various Gramnegative bacteria are now being identified including HtrA (DegP), Skp, Imp, SurA, and Omp85 (Braun & Silhavy, 2002; De Cock et al., 1999; Lazar & Kolter, 1996; Rouviere & Gross, 1996; Spiess et al., 1999; Voulhoux et al., 2003). Yet, only HtrA and Omp85 homologues are present in all the published spirochetal genome sequences (Fraser et al., 1997; Fraser et al., 1998; Ren et al., 2003; Seshadri et al., 2004; Voulhoux et al., 2003). Thus, at least in some aspects, translocation and insertion of OMPs into the outer membranes of Borrelia must be unique (Cullen et al., 2004). In fact, the possibility of a novel OMP translocation pathway involving Cterminal processing became evident only after our studies on the integral outer membrane protein P13 (Noppa et al., 2001) and in Papers II and III with further discussions in sections 5 and 6. 3.1.1 C-terminal processing Many proteins of prokaryotic and eukaryotic origins are synthesized as precursors and subsequently converted to the mature forms by proteolytic cleavage of signal peptides. As discussed above, the N-terminal processing is a universal translocation signal for proteins being transported across inner membranes. On the other hand, the C-terminal processing is a much less
22
INTRODUCTION common phenomenon, although a couple of bacterial proteins are known to contain cleavable C-terminal peptides (Nagasawa et al., 1989; Navarre & Schneewind, 1994; Silber et al., 1992; Zhang et al., 2003). Several surface proteins of Grampositive bacteria possess C-terminal sorting signals that are cleaved off by sortase (Mazmanian et al., 2001). In E. coli, the C-terminal processing is required for the maturation of [NiFe]-hydrogenase (Menon et al., 1993; Zhang et al., 2003). Furthermore, Tail specific protease (Tsp) is a periplasmic protease of E. coli, which is involved in the C-terminal processing of penicillin binding protein 3 (Hara et al., 1991; Silber et al., 1992). Inactivation of tsp gene leads to an increased sensitivity of E.coli to heat and osmotic stress (Hara et al., 1991). The phenomenon of C-terminal processing is best studied in photosynthetic organisms where the cleavage of a C-terminal precursor plays a crucial role in translocation and insertion of photosystem II component (the D1 protein) in thylakoid membranes of chloroplasts (Hankamer et al., 2001; Ivleva et al., 2000; Karnauchov et al., 1997; Nixon et al., 1992). The carboxyl-terminal processing protease A (CtpA) is involved in this cleavage and may also be involved in maturation of proteins destined to the cell envelope. CtpA and Tsp belong to an unusual class of serine endoproteases that utilize a Ser/Lys catalytic dyad instead of the ordinary Ser/His/Asp catalytic triad (Paetzel & Dalbey, 1997). While CtpA was initially characterized in cyanobacterium Synechocystis sp. PCC 6803 (Anbudurai et al., 1994; Shestakov et al., 1994), Bartonella baciliformis was the first eubacterium in which CtpA was described, although no target was identified (Mitchell & Minnick, 1997). In our study (Paper III), we identified CtpA and its substrates in B. burgdorferi and elucidated that CtpA homologues actually exist in the vast majority of bacteria. Recently, CtpA from Brucella suis was implicated in cell morphology and persistence in macrophages (Bandara et al., 2005). CtpA, Tsp, as well as HtrA (high-temperature requirement) protease (Pallen & Wren, 1997) all exhibit common PDZ-domains that in HtrA and Tsp facilitate binding to their substrates (Spiers et al., 2002). Homologues of PDZdomains are present in many mammalian, yeast, plant and bacteria proteins (Ponting, 1997; Ponting et al., 1997).
3.2
Outer surface lipoproteins of B. burgdorferi
The unusual membrane architecture of spirochetes and their ancient phylogeny suggest that features of the export, structure and function of spirochetal lipoproteins are unique to these organisms. As an indication of their importance in Borrelia, lipoproteins are the most abundant proteins (Haake, 2000). This,
23
INTRODUCTION together with the general importance of outer surface proteins in adaptation and infection has led many researchers to focus their investigations on the surface exposed lipoproteins of B. burgdorferi. While there is little immune pressure on spirochetes in the tick environment, survival in mammalian hosts demands that B. burgdorferi must evade both the innate immune response and the adaptive immune response throughout an infection (Sigal, 1997). Borrelial lipoproteins play a major role in host inflammatory response activation and inflammation of tissues at sites of infection is a unifying feature in the manifestation of Lyme disease. While activation of the innate immune response by lipoproteins is crucial for defence against B. burgdorferi infection, surface exposed lipoproteins play an important role in adaptive responses and pathogenicity of Borrelia spirochetes. Lipoproteins dominate pathogenic mechanisms exploited by B. burgdorferi, including those of antigenic variation (Zhang et al., 1997; Zhang & Norris, 1998a), evasion of complement killing (Alitalo et al., 2002; Kraiczy et al., 2001; Stevenson et al., 2002) and adherence mechanisms (Coburn et al., 2005). Furthermore, a dynamic interplay between lipoprotein expression and life cycle transitions of B. burgdorferi is envisioned (Revel et al., 2002). 3.2.1 Lipoproteins with roles in transmission and adaptation The most abundant and best studied outer membrane proteins in Borrelia spirochetes are lipoproteins called outer surface proteins (Osps). Noteworthy, Osps have very little sequence or structural homology to any other known protein. The most abundant and best studied is OspA, encoded on the lp54, in an operon with another highly related lipoprotein OspB (Barbour, 1988; Bergström et al., 1989). Immunization with OspA protects against subsequent B. burgdorferi infection (Wallich et al., 1996), but is unsuitable as a vaccine because of adverse side effects (Lathrop et al., 2002). OspC is encoded on cp26 (Fraser et al., 1997) and is homologous to variable small proteins (Vsps) of relapsing fever Borrelia (Carter et al., 1994). The expression of OspA/B and OspC are reciprocally regulated, undergoing a switch during Borrelia transition from its tick vector to the mammalian host. In unfed ticks, Borrelia express high amounts of OspA and OspB and almost no OspC, whereas during the blood meal and upon mammalian infection, OspA/B synthesis is down-regulated and OspC production is up-regulated (de Silva et al., 1996; Schwan et al., 1995). OspA may function as an adhesin in the tick midgut epithelium (Pal et al., 2000) and also provide protection for other surface proteins, such as P66, against protease attack (Bunikis & Barbour, 1999). OspC might facilitate the invasion of Borrelia in
24
INTRODUCTION tick salivary glands that is initiated by a blood meal promoting the transmition of spirochetes to the mammalian host (Gilmore & Mbow, 1999; Pal et al., 2004). Indeed, in B. burgdorferi, OspC was strictly required to infect mice, but did not seem necessary to correctly localize or migrate in the tick (Grimm et al., 2004). The structures of OspA, OspC and C-terminal part of OspB have been solved by X-ray crystallography. OspA consists of four β-sheets, formed by 21 antiparallel β-strands and a C-terminal α-helix (Li & Lawson, 1995). The crystal structure of the OspB C-terminus (residues 152-296) was identical to that of OspA (Becker et al., 2005). Strikingly, three-dimensional structure analysis of OspC revealed a totally different structural organization. OspC predominantly consists of α-helices, organized as dimer of five parallel α-helices and two short β-strands (Eicken et al., 2001; Kumaran et al., 2001). The Mlp (multicopy lipoproteins) family is one of many paralog protein families in B. burgdorferi, and up to ten mlp genes are encoded on cp32 and cp18 (Caimano et al., 2000; Porcella et al., 1996). Mlps are differentially expressed in response to various in vitro culture conditions as well as in the host-adapted environment. Thus, Mlp expression is up-regulated coordinately by either elevated temperature (37oC versus 23oC), reduced culture pH (6.8 versus 8.0), increased spirochete cell density, and by some mammalian host factors (Ojaimi et al., 2003; Ojaimi et al., 2005; Porcella et al., 2000; Yang et al., 1999; Yang et al., 2003). These data indirectly suggest that Mlp proteins are also involved in B.
burgdorferi infections of the mammalian host. Lp6.6, a small lipoprotein, is significantly down-regulated during mammalian infection, such that its function might be associated with Borrelia encountering the tick environment (Lahdenne et al., 1997). The RevA protein is synthesized during mammalian infection, and transcription of revA is upregulated shortly after the Borrelia-infected tick feeds on the mammal (Gilmore & Mbow, 1998; Gilmore et al., 2001). Recently, several new B. burgdorferi outer surface exposed lipoproteins have been identified (BB0689, BBA36, BBA66, BBA69, and BBI42) that are actively expressed and immunogenic during Borrelia infections and are targets for bactericidal antibody action, making them attractive vaccine candidates for Lyme disease (Brooks et al., 2006). 3.2.2 Adhesins Attachment to tissues and cellular matrix is an important prerequisite of pathogenic bacteria to colonize the host. B. burgdorferi colonize collagenous tissues by the binding of surface adhesins to collagen-associated proteoglycan decorin. Two ~20 kDa lipoproteins called decorin-binding proteins A and B
25
INTRODUCTION (DbpA/B) are known adhesins (Guo et al., 1995; Guo et al., 1998; Hanson et al., 1998). As decorin is found in various tissues and presumably in skin and joints, the sites of high prevalence during infection by B. burgdorferi, it is tempting to hypothesize that the decorin binding proteins might be involved in tissue tropism of Lyme disease spirochetes. Moreover, expression of both dbpA and dbpB are drastically up-regulated at 35oC versus 23oC (Ojaimi et al., 2003) and antibodies against DbpA can prevent an infection (Hagman et al., 1998; Hanson et al., 1998), illustrating the importance of adhesion in the pathogenesis of Lyme disease. Bgp (Borrelia glycosaminoglycan-binding protein) is B. burgdorferi hemagglutinin with the capacity to bind heparan sulphate and dermatan sulphate. Bgp is surface exposed, but its classification as a lipoprotein is still unresolved (Parveen & Leong, 2000). Since the adaptation of spirochetes to the mammalian host environment leads to enhanced glycosaminoglycan (GAG) binding, Bgp may play a significant role during mammalian infection (Parveen et al., 2003). Moreover, Bgp production was up-regulated during conditions that mimicked tick feeding (Ramamoorthy & Philipp, 1998), while secretion of Bgp into extracellular environment has even been observed indicating a possible role as a immune response decoy (Cluss et al., 2004). P35 incorporates two immunogenic lipoproteins, BBA64 and BBK32. Not only do these serve as important diagnostic tools for early Lyme disease, being recognized by sera from B. burgdorferi infection, but they are also expressed in vivo and up-regulated at 35oC versus 23oC (Fikrig et al., 2000; Indest et al., 1997; Indest & Philipp, 2000; Ojaimi et al., 2003). BBK32 binds fibronectin (Probert & Johnson, 1998) and promotes Borrelia attachment to GAGs (Fischer et al., 2006). Furthermore, inactivation of bbk32 in low-passage, infectious B. burgdorferi leads to significantly attenuated phenotype in the mouse model of infection (Seshu et al., 2006). Thus, the borrelial fibronectin-BBK32 interaction appears to be an important pathogenic mechanism of B. burgdorferi, particularly for dissemination and persistence (Seshu et al., 2006). 3.2.3 Lipoproteins with role in persistence and diagnosis B. burgdorferi is capable of persisting in a mammalian host for long periods. Persistence can be achieved by antigenic variation of Vls (Vmp-like sequence). Vmp stands for variable major proteins of relapsing fever Borrelia and are the best studied antigenically variable proteins in spirochetes (Barbour, 1990; Barbour, 1993; Burman et al., 1990). VlsE is encoded on lp28-1 of B. burgdorferi, and this lipoprotein undergoes antigenic variation through an elaborate gene conversion
26
INTRODUCTION mechanism (Zhang et al., 1997; Zhang & Norris, 1998a). The solved 3-D structure of VlsE is unlike OspA-C, consisting of eleven α-helices and four short β-strands (Eicken et al., 2002). This is despite VlsE and OspC sharing general structural features, including the close proximity of their N and C termini and a hypervariable sequence corresponding to a surface exposed region (Cullen et al., 2004). The Bmp lipoprotein family has four members, BmpA-D and are encoded by bmp genes of paralogous chromosomal gene family 36 (Aron et al., 1994; Gorbacheva et al., 2000; Ramamoorthy et al., 1996). Bmp lipoproteins are immunogenic and BmpA (P39) is widely used in Lyme disease diagnosis assays (Simpson et al., 1990; Simpson et al., 1991). These lipoproteins may be necessary for in vitro and in vivo growth and they may have a role in virulence (Dobrikova et al., 2001; Gorbacheva et al., 2000; Ramamoorthy et al., 1996). Yet, another lipoprotein important for serodiagnosis of Lyme disease is P22 (Lam et al., 1994). 3.2.4 Lipoproteins conferring serum resistance Human and animal sera exhibit bactericidal activity through the action of the complement system (Medzhitov & Janeway, 2000). Complement is a system of plasma proteins that can be activated by antibody or lectins bound to the bacterial surface or by the pathogen surface itself. Gram-negative bacteria have developed multiple strategies to avoid the bactericidal effects of complement by utilizing LPS, capsule or OMPs (Rautemaa & Meri, 1999). Several borrelial lipoproteins promote resistance to complement-mediated killing by preventing complement activation or membrane attack complex formation. OspE and OspF are the main members of OspE/F related proteins (Erp) encoded on both cp32 and cp18 plasmids. B. burgdorferi can carry as many as nine different erp loci per cell and all can be expressed simultaneously (Akins et al., 1999; Casjens et al., 1997b; Casjens et al., 2000; El Hage et al., 1999; Stevenson et al., 1996; Stevenson et al., 1998a). Many Erps can bind complement
inhibitory factor H enabling B. burgdorferi to avoid host-mediated serum killing (Hellwage et al., 2001; Stevenson et al., 2002). Interestingly, another group of lipoproteins contain OspE/F-like leader peptides (Elp). Although they are otherwise unrelated to OspE/F, some of their genes are transcriptionally linked (Akins et al., 1999; Hefty et al., 2001). Gene rearrangements within the Erp and Elp families has generated sequence diversity fostering the parasitic strategy and immuno-evasiveness of Lyme disease Borrelia (Akins et al., 1999; Caimano et al., 2000). Members of Erp and Elp families are differentially expressed during B. burgdorferi migration in tick and transmission to mammalian host (Akins et al.,
27
INTRODUCTION 1995; Hefty et al., 2001; Stevenson et al., 1998) and host-specific signals were found
to alter the expression patterns and final cellular localization of these lipoproteins (Hefty et al., 2002b). Complement regulator acquiring surface proteins (CRASPs) of B. afzelii also bind complement inhibitory factor H and factor H-like protein to provide complement resistance (Kraiczy et al., 2001). Borrelia can be classified as resistant, intermediate-sensitive or sensitive to the bactericidal effects of human serum (Kraiczy et al., 2001), which might correlate with possession of one or more of the CRASPs and OspE. B. burgdorferi OspE and B. afzelii CRASP-II might even be homologous proteins (Cullen et al., 2004).
3.3
Integral outer membrane proteins in Borrelia
In contrast to E.coli, the fluid and fragile outer membrane of B. burgdorferi contains very low amounts of integral membrane proteins (Brandt et al., 1990; Radolf et al., 1994; Walker et al., 1991). The integral outer membrane proteins partly maintain bacterial cell structure, bind to different substances and transport nutrients, bactericidal and toxic agents (Achouak et al., 2001). Protein secondary structure is characterized by the presence of αhelices, β-strands and loop regions. Integral membrane proteins span the hydrophobic lipid bilayer membrane, which requires hydrophobic stretches of at least 20 amino acids. Depending on their size, structure and oligomeric properties, proteins can span the membrane one to several times. Hydrophobicity scales have been developed by Kyte and Doolittle on the basis of solubility measurements of the amino acids in different solvents (Kyte & Doolittle, 1982), which are used in hydropathy plots to identify transmembrane helices by computer prediction programs. Noteworthy, of all secondary structures, only transmembrane helices are predicted from novel amino acid sequences with any reasonable degree of confidence. However, bioinformatic approaches to identify β-barrels are now available (Martelli et al., 2002; Zhai & Saier, 2002). From accumulated knowledge based on crystallization experiments and secondary structure predictions, membrane spanning proteins consists of αhelices. Nevertheless, Gram-negative bacteria have two membranes with no known pathway for the translocation of hydrophobic, α-helical proteins to the outer membrane. Moreover, the majority of all crystallized integral outer membrane proteins (mostly porins) span the membrane with β-strands (Koebnik et al., 2000; Schulz, 2004). Herein, contradictory, or as I would like to say, breakthrough data, shows that an integral outer membrane protein (porin) spanning
28
INTRODUCTION the outer membrane can do so with predicted α-helices. This has also been reported for two integral outer membrane proteins of T. pallidum. For example, Tromp1 (Treponemal rare outer membrane protein) spans the outer membrane with hydrophobic α-helices (Lee et al., 1999; Zhang et al., 1999). Amphipathic αhelices are an additional structure element characteristic of proteins which function in signal transduction in cellular membranes. Their function is regulated by amphitropism, the capacity of a protein to exist in both soluble and membrane-integrated forms (Johnson & Cornell, 1999). Although such protein architecture lacks precedent among integral outer membrane proteins, OMP (Tp0453) of T. pallidum may insert in the outer membrane by amphipathic αhelices and induce membrane permeability (Hazlett et al., 2005). To date, no Borrelia transmembrane OMPs have been crystallizedthe mention of protein structural characteristics is solely based on computer predictions and need to be evaluated with caution. Only a few outer membranespanning proteins of B. burgdorferi are characterized, all having channelforming activity and are therefore considered to be porins. Oms28 (outer membrane spanning, 28 kDa protein) and Oms66 (P66) were the first integral OMPs characterized in B. burgdorferi (Skare et al., 1996; Skare et al., 1997). Noteworthy, membrane-spanning α-helices are not predicted for these proteins by computer analysis using the web server SOSUI and the web server TMpred (http://sosui.proteome.bio.tuat.ac.jp) (http://www.ch.embnet.org/software/TMPRED_form.html). Further descriptions of P66 will be presented in sections 8 and 9 and Papers V, VI. A substantial part of my thesis will focus on the integral outer membrane protein P13, which spans the outer membrane by α-helices. Characterization of this unusual protein will be presented in sections 4 to 6 and Papers I to III. EppA (Exported plasmid protein A) has a signal peptidase I cleavage site and is most likely an integral outer membrane protein (Champion et al., 1994). Encoded on either cp9-1 or cp9-2, eppA is expressed only in the mammalian host and therefore may play a role in survival of B. burgdorferi in the mammalian host and possibly in the pathogenesis of Lyme disease (Champion et al., 1994; Miller et al., 2000). Recently, one additional integral outer membrane protein, BB0405 of B. burgdorferi was elucidated (Brooks et al., 2006). Since porins are the signature of integral OMPs in Gram-negative bacteria and all of the B. burgdorferi integral outer membrane proteins are shown to have porin function, the following three sections will be devoted to this class of proteins.
29
INTRODUCTION
3.4
General features of outer membrane channels (Porins)
Outer membrane proteins fulfill a number of tasks that are crucial for bacterial cells, such as solute and protein transport, as well as signal transduction, and interaction with other cells (Achouak et al., 2001). Uptake of nutrients or secretion through the membranes is accomplished by several pore-forming proteins (Benz, 1994; Nakae, 1976). These proteins can be divided into three different classes: (i) general diffusion pores, (ii) specific channels containing a binding site for a certain solute and (iii) actively transporting, energy consuming channels. Detailed structural and functional characterization of outer membrane proteins, has resulted in further subdivision into five groups (Schulz, 2004). These include general porins, such as E. coli OmpF (Cowan et al., 1992), which allow the unspecific diffusion of hydrophilic molecules and may show a slight selectivity towards ions, and the structurally related specific porins, such as E. coli LamB (Schirmer et al., 1995) with their β-barrels having two more strands. Generally, porins are trimers formed by monomers consisting of 16- or 18-stranded βbarrels in their functional state (Fig. 4). Another structural feature of porins is the inward folded loop attached to the inner side of the barrel wall, which constricts the internal diameter and stabilizes the porin β-barrel. Residues located in this loop determine the channel properties, like the diameter, ionselectivity or substrate affinity (Bauer et al., 1989; Jordy et al., 1996). Interestingly, these loops show very high sequence variation among homologous porins that might aid in evasion of serum antibodies, bacteriophages or proteases. Porins show very low sequence homology, however, the similarity in topology and charge distribution is striking (Schirmer, 1998). For instance, the C-terminal transmembrane segment contains an aromatic amino acid (usually phenylalanine), which is absolutely conserved in classical porins of Enterobacteriaceae (Struyve et al., 1991). Yet, this is not the case for the spirochetal porins characterized to date (Nikaido, 2003). A third, distinct group consists of composed pores, where the β-barrel itself is a homo-oligomer. For example, the β-barrel forms homo-dimer in gramidicin A (Ketchem et al., 1993), a heptamer in α-hemolysin (Song et al., 1996) and a trimer, assembled together with α-helical trans-periplasmic tunnel in TolC (see section 3.7) (Koronakis et al., 2000). The fourth group of actively transporting outer membrane transport proteins has a much larger β-barrel and additional internal domains. They do not form a pore, but open and close for the passage through the outer membrane of relative large molecules such as iron-containing siderophores, in case of FhuA (Locher et al., 1998) and cobalamins, in the case of BtuB (Chimento et al., 2003). The final group
30
INTRODUCTION of outer membrane proteins (poreless Omps) contain small β-barrels that are not likely to form a pore. Rather they function as membrane anchors, such is the case for E. coli OmpA (Pautsch & Schulz, 1998). The focus of this thesis lies with general and specific porins. A
B
Figure 4.* The structure of general porin. The OmpF porin from E. coli. (A) General fold of porin monomer. The hollow β-barrel structure is formed by antiparallel arrangement of 16 β-strands. The strands are connected by turns or short loops on the periplasmic side (bottom), whereas long, irregular loops face the cell exterior (top). The internal loop, which extends inside the barrel, is highlighted in dark. (B) Cross-section through the center of a modeled OmpF trimer structure. The three pores have size of 7 x 11 Å. *Reprinted from Journal of Structural Biology, Vol. 121, T. Schirmer, General and specific porins, pp. 101-109, Copyright (1998), with permission from Elsevier.
Primary sequences of the vast majority of outer membrane porins do not show any indication of α-helical structures, although it represents the typical structure for membrane proteins by Kyte and Doolittle (Kyte & Doolittle, 1982). As a general rule, the outer membrane porins are arranged in amphipathic β-strands and form β-barrel cylinder, whereas the inner membrane proteins consists of mostly α-helices. The cylindrical structure implies that on average every second amino acid in outer membrane spanning β-sheets is hydrophobic because it faces the lipid membrane or is hydrophilic and points towards the channel interior (Vogel & Jähnig, 1986). Besides the nutrient uptake, some porins have additional functions. Thus, porins can insert into eukaryotic cell membranes (Rudel et al., 1996), activate complement (Alberti et al., 1996; Merino et al., 1998), act as receptors for bacteriophages (Yu et al., 1998) and be targets of bactericidal compounds (Sallmann et al., 1999). Moreover, surface exposed regions of porins can adhere to cellular receptors, a feature observed for Campylobacter jejuni major outer membrane protein (MOMP) (Moser et al., 1997), Shigella flexeneri OmpC (Bernardini et al.,
31
INTRODUCTION 1993), Treponema denticola Msp (Fenno et al., 1996; Haapasalo et al., 1992; Weinberg & Holt, 1991), and B. burgdorferi P66 (Coburn & Cugini, 2003).
3.5
Functional reconstitution of porins in artificial lipid bilayer membranes
The function and properties of the porin proteins can be studied in intact cells or in vitro reconstitution systems such as the liposome-swelling assay (Nikaido & Rosenberg, 1981), measurement of exclusion limits (Nakae, 1975) or the planar lipid bilayer technique (Benz et al., 1978; Schindler & Rosenbusch, 1978). Reconstitution experiments aim to re-establish the function of purified porins, demonstrating the integrity of the pore-forming complex after the isolation process.
Addition of porin
Teflon cell filled with electrolyte
Porin trimer inserted into membrane
Figure 5*. The experimental set up of porin reconstitution in artificial lipid membranes. Magnified view on coloured lamella which is turning black is shown in the inset. Electrolyte conventionally consists of 1M KCl. *Adopted from R. Benz, Würzburg University homepage.
Many in vitro studies, including those described in this thesis, have been performed using the planar lipid bilayer assay. From a thermodynamic point of view, the bilayer is the most stable energetic form between lipids and water. The bilayers can be built as planar membranes or vesicles. Black lipid membranes separating two aqueous phases are particularly stable for electrical measurements (Benz & Janko, 1976). The cell apparatus (Fig. 5) used for membrane formation consists of a Teflon chamber with a thin wall containing a hole with diameter of 0.2-1 mm separating the two aqueous compartments. For 32
INTRODUCTION membrane formation, the lipid is dissolved mainly in n- decane in a concentration of 1% (w/v). The lipid solution is painted over the whole to form a thick, colored lamella (Fig. 5). The experiments can be started after the lamella thins out and turns optically black in reflected light, because the lipid bilayer is much thinner than the wavelength of the visible light (Dilger & Benz, 1985). The lipid bilayer technique allows very sensitive detection of current through the membrane down to 1 pA, which means that the conductance of the membrane channel can be measured with high accuracy because the bilayer itself has a very low conductance below 10 pS (corresponding to a resistance of more than 100 MΩ). The membrane current is measured with an electrode pair switched in series with a voltage source and a highly sensitive current amplifier. The addition of a channel-forming protein to the aqueous phase results in a stepwise increase of the membrane conductance as a result of reconstitution of protein in the lipid bilayer membrane. Channel-formation results in a step-wise single channel conductance (membrane current per unit voltage) increase recorded on a strip chart. The single channel conductance for an individual porin is usually homogenous and correlates with the size of the channel formed (Trias & Benz, 1994). Although the artificial lipid bilayer measurements give only a rough estimate of the channel size, it does provide additional insight into the electrostatic properties of the pore upon determination of the ratio of cation over anion permeability, i.e., the ion selectivity (Schirmer, 1998).
3.6
Porins in spirochetes
The low incidence of transmembrane spanning proteins in the spirochetal outer membrane is striking, seemingly imposing a highly restrictive diffusion barrier between the cell and its environment. How this barrier becomes permeable for ions, nutrients and metabolites, is therefore an important question, especially given the limited biosynthetic capacity and absolute dependence of Borrelia to host-supplied nutrients. The permeability of the outer membrane is achieved through channel proteins (porins) and then, once in the periplasm, nutrients are actively transported across the inner membrane to be used in different metabolic processes of the bacterium (Achouak et al., 2001). The major outer membrane protein from Spirochaeta aurantia was the first porin characterized in spirochetes. This 36.5 kDa protein forms trimers in its native state and exhibited a 7.7 nS channel-forming activity in planar lipid bilayer assay (Kropinski et al., 1987).
33
INTRODUCTION 3.6.1 Porins in B. burgdorferi The first indication of porins in B. burgdorferi came through investigation of channel-forming activities in the planar lipid bilayer assay of outer membrane vesicles (OMV). Two porin activities of 0.6 nS and 12.6 nS were found (Skare et al., 1995). Subsequent work has so far characterized three porins in Borrelia spirochetes: Oms28 (Skare et al., 1996), P66 (Skare et al., 1997), discussed in sections 8, 9 and Papers V, VI, and P13, discussed in section 5 and Paper II. Moreover, there are indications of several uncharacterized channel-forming activities present in B. burgdorferi (Paper II and section 5). Oms28 is a 28 kDa protein having 0.6 nS channel forming activity in the planar lipid bilayer assay. Recombinant Oms28 forms a ~75kDa oligomer following the paradigm of conventional porins (Skare et al., 1996). Interestingly, Oms28 is up-regulated in a CtpA deficient strain (Paper III), while mammalian factors present in the incoming blood during tick feeding may drive the oms28 repression (Brooks et al., 2003; Tokarz et al., 2004). Without these factors, temperature alone seems to increase oms28 transcription (Ojaimi et al., 2003; Revel et al., 2002). Perhaps the preferential expression of Oms28 in unfed ticks may enable acquisition of small molecules while Borrelia resides in the nutrient-poor midgut environment (Tokarz et al., 2004). This indirectly correlates with the up-regulation of Oms28 and P66 in the non-disseminating B. burgdorferi clinical isolate (B356), but not the readily disseminating isolate (BL206) (Ojaimi et al., 2005). Interestingly, the oms28 knock-out strain was impaired for enhanced nutrient uptake that might be required for entry of Borrelia into log-phase growth (Ojaimi et al., 2005). Therefore, Oms28 might be required for growth adjustment during transition to different environments such as during natural transmission from tick to mammalian host. 3.6.2 Channel-forming proteins of relapsing fever Borrelia In Borrelia hermsii, the causative agent of relapsing fever, different porin activities ranging from 0.2 nS to 7.2 nS are present in outer membrane vesicle (OMV) preparations (Shang et al., 1998), although no porin has been identified so far. In the outer membrane (OM) fractions of another relapsing fever spirochete, B. duttonii, porin activities range from 0.25 nS to 11 nS. Attempts to purify porins from the OM of B. duttonii has so far identified a 80 pS channel-forming activity, but not the responsible protein (K. Denker, C. Larsson, unpublished results).
34
INTRODUCTION 3.6.3 Porins in Treponema sp. and Leptospira The T. denticola major surface protein (Msp) is either 53 or 64 kDa (depending on the strain) and forms 116-200 kDa oligomers (Haapasalo et al., 1992). Interestingly, it forms hexagonal arrays covering the surface of T. denticola as visualized by electron microscopy (Masuda & Kawata, 1982). Similar to P66 from B. burgdorferi (section 8 and Paper V), Msp may have a dual adhesin-porin function and exhibits very large channel-forming conductance (10.9 nS) in the planar lipid bilayer assay (Egli et al., 1993). Msp permits adherence of T. denticola to host cells, such as gingival fibroblasts (Weinberg & Holt, 1991) as well as fibronectin and laminin (Fenno et al., 1996; Haapasalo et al., 1992). In Treponema pallidum, the channel-forming properties (average single-channel conductance of 0.7 nS) and trimeric conformation of the 31 kDa rare OM protein (Tromp1) has been shown by the planar lipid bilayer assay (Blanco et al., 1995; Zhang et al., 1999). However, whether Tromp1 is a porin is debated because this protein fails to permeabilize upon reconstitution into liposomes and might not possess a cleavable signal sequence (Akins et al., 1997), although N-terminal signal sequence cleavage has since been proved (Blanco et al., 1999). Another unusual feature of Tromp1 is that it lacks the extensive βbarrel structure; instead it is formed by α-helical architecture (Lee et al., 1999). Although it can be argued against the porin function of Tromp1 because of its unconventional porin properties, the possibility of novel structures for porin proteins should not be ruled out, as will be discussed in this thesis with respect to P13 (sections 5, 6 and Papers II, III). Recently, another unusual OM protein, TP0453 of T. pallidum, was shown to enhance membrane permeability (Hazlett et al., 2005). TP0453 is a ~30 kDa lipidated protein which lacks extensive β-sheet structure and contains multiple membrane-inserting, amphipathic α-helices. All known spirochetal lipoproteins are hydrophilic and become amphiphilic (membrane-associated) after modification with lipids. This is not the case for TP0453, as recombinant, non-lipidated protein is amphiphilic and inserts into liposomes. TP0453 remains as a monomer and is not surface exposed so that the peptide moiety of this lipoprotein inserts into the periplasmic leaflet of the OM, but does not traverse the membrane. This concealed TP0453 topology is consistent with the parasitic strategy of T. pallidum that focuses on resistance to specific antibody binding as a primary mechanism for immune evasion. Thus, TP0453 appears to be a novel type of OMP which promotes transient, localized lipid bilayer discontinuities allowing non-selective nutrient uptake (Hazlett et al., 2005).
35
INTRODUCTION Pathogenic Leptospira species produce a rare 31-kDa surface protein, OmpL1, which has 10 predicted amphipathic trans-membrane β-strands (Haake et al., 1993). OmpLl is a porin which forms trimers causing a 1.1 nS average channel-forming conductance in the planar lipid bilayer assay (Shang et al., 1995). The up-regulation of OmpL1 porin expression after excretion of virulent Leptospira from the host and following culture-attenuation, suggests that OmpL1 plays an important role in the adaptive response (Haake et al., 1993; Shang et al., 1995). Moreover, OmpL1 elicits protective immunity in the hamster model of leptospirosis, especially when administered in combination with lipoprotein LipL41 (Haake et al., 1999).
3.7
Active transport systems in B. burgdorferi
Although the main focus of this thesis is on outer membrane properties of Borrelia, discussion of transportation systems through both membranes is necessary to understand the whole picture of nutrient uptake, secretion and translocation. While nutrients and essential cofactors must be actively transported into the cells, end products of metabolism, toxic substances and secreted macromolecules must be actively extruded. Some characteristics of specific transport systems in B. burgdorferi will be now presented. Transport proteins catalyze the translocation of solutes by various systems differing in respect to energy coupling, substrate specificity and transmembrane topology. There are four distinct types of functionally characterized transport systems; (i) bacterial channel proteins, (ii) secondary transporters which utilizes chemiosmotic energy (proton gradient), (iii) primary transporters utilizing chemical energy, typically in the form of ATP, and (iv) group translocators that phosphorylate their substrates during transport (PTS). From a comparative genome analysis of transport capabilities from 18 different prokaryotes conducted by Paulsen and co-workers (Paulsen et al., 2000), it appeared that relative to genome size spirochetes (B. burgdorferi and T. pallidum) have comparatively few transporters, particularly secondary transporters. Moreover, both spirochetes contained the lowest numbers of transmembranal segments in their predicted integral membrane protein topologies (Paulsen et al., 2000). In another comparative study of 215 proteintranslocating outer membrane porins of Gram-negative bacteria, only TolC homologue (BB0142) belonging to the outer membrane factor (OMF) family was identified in B. burgdorferi (Yen et al., 2002). In contrast, among bacteria compared, B. burgdorferi has the largest percentage of phosphotransferase
36
INTRODUCTION system (PTS) permeases. Noticeably, B. burgdorferi also encodes a large number of peptide transporters (Paulsen et al., 2000). A short description of these translocation systems in B. burgdorferi is given below. The oligo-peptide permease (Opp) belongs to the family of ATPbinding cassette (ABC) transporters used for transportation of peptides into bacteria to be subsequently used as carbon and nitrogen sources (Lazazzera, 2001). Although the major role of bacterial Opp is in nutrient uptake, they may also be involved in sensing environmental signals in the form of peptides leading to diverse responses, including expression of virulence determinants, quorum sensing, chemotaxis, sporulation, conjugation, and competence (Leonard et al., 1996; Perego et al., 1991; Rudner et al., 1991). The Opp complex in B. burgdorferi consists of peptide-binding lipoprotein OppA, trans-membrane proteins OppB and OppC, and the ATPases OppD and OppF (Bono et al., 1998; Das et al., 1996; Kornacki & Oliver, 1998). Interestingly, B. burgdorferi has five oppA genes each controlled by promoters which respond differently to various environmental signals (Ojaimi et al., 2003; Wang et al., 2002). However, oppA genes can also be cocoordinately up-regulated in B. burgdorferi clinical isolates with different capacities for hematogenous dissemination (Ojaimi et al., 2005). Perhaps each of OppA possesses a different substrate binding affinity that is active during different stages of B. burgdorferi life cycle (Wang et al., 2004), which would attribute to the targeting and penetration of specific tissues in invertebrate and vertebrate hosts (Ojaimi et al., 2005). An intriguing group of OMF family is channel-tunnels which are engaged in exporting substances across the two membranes of Gram-negative bacterial cell envelopes. This family incorporates the efflux pumps which confer resistance to various harmful substances like antibiotics, heavy metals, dyes, bile salts and detergents. Efflux pumps consist of an inner membrane transporter, a periplasmic accessory protein and an outer membrane channel-tunnel. The well studied E. coli channel-tunnel, TolC is involved in protein secretion and can also be part of diverse efflux pumps (Andersen et al., 2000). The TolC crystal structure is determined and consists of a channel domain, a equatorial domain and a tunnel domain (Koronakis et al., 2000). The channel domain is embedded in the outer membrane and consists of nonpolar residues directed towards lipids and polar residues that locate to the interior resulting in typical amphipathic βstrands. The β-barrel of channel-tunnel is different from those of typical porins formed by single peptide chain. To form a TolC channel domain, three protomers are necessary to form a single β-barrel consisting of four β-strands. The tunnel domain of TolC consists entirely of α-helices assembled in an α-
37
INTRODUCTION barrel, representing an utterly unique structure. A TolC homologue (BB0142) is encoded in the B. burgdorferi genome and is currently being investigated in our laboratory (I. Bunikis, K. Denker, unpublished results). The genome of B. burgdorferi encodes a large amount of homologous carbohydrate transporters belonging to the PTS group (Fraser et al., 1997). Several studies have attempted to characterize some of these transporters. Chitobiose is a component of the tick cuticle and peritrophic matrix. The products of chbA, chbB, and chbC enable Borrelia to transport and utilize chitobiose when in the tick environment (Tilly et al., 2001; Tilly et al., 2004). Different chb genes may respond differently to various temperatures- chbB transcription is up-regulated at 23oC, whereas chbC transcription increases at 35oC (Ojaimi et al., 2003). Glucose is the major energy source for B. burgdorferi involving several PTS glucose transporters (von Lackum et al., 2005). BBB29 is one of the components of a glucose transporter system of B. burgdorferi (Byram et al., 2004). BBB29 expression is elevated in a B. burgdorferi non-disseminating clinical isolate compared to a disseminating isolate (Ojaimi et al., 2005) and at 23oC versus 35oC (Ojaimi et al., 2003). Mannose-modified host proteins are also a likely carbohydrate source for B. burgdorferi, with the BB0408 and BB0629 proteins suggested to be mannose transporters (von Lackum et al., 2005). The members of major intrinsic protein (MIP) group consists of essential channel-forming proteins that maintain an essential in osmotic cell equilibrium and include aquaporins and glycerol facilitators (GlpF) (Froger et al., 2001; Paulsen et al., 2000). The GlpF homologue in B. burgdorferi (BB0240) is involved in glycerol uptake, which can either be used as an energy source or for phospholipid and lipoprotein biosynthesis (Schwan et al., 2003). The process of glycerol uptake (glpF) and utilization (glpK; bb0241 and glpA; bb0243) might be required when Borrelia resides in its arthropod host since these glp genes are significantly up-regulated in B. burgdorferi grown at 23oC versus 35oC (Ojaimi et al., 2003).
3.8
Structural characterization of membrane proteins
The structural analysis plays a key role in the final characterization of proteins. The most powerful tools in three-dimensional (3-D) structure determination of proteins to atomic resolution are X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. NMR spectroscopy is based on certain atomic nuclei, such as from 1H, 13C, 15N, and 31P, having a magnetic moment or spin. For NMR analysis of proteins, usually 1H atoms are used because those are the
38
INTRODUCTION most abundant. When protein molecules are placed in a strong magnetic field, the spin of 1H atoms are recorded and used to calculate the distances between atoms in a molecule that are then used to derive a 3-D model. NMR can be applied only for small and soluble proteins, but it can also give information about various dynamic processes, such as folding (Wuthrich, 1989). However, membrane proteins are often large, oligomeric, insoluble in aqueous solutions and denatured in organic solvents, making them unsuitable for NMR analysis. X-ray crystallography can resolve individual atoms in a molecule by exposure to electromagnetic radiation (X-rays). The wavelength of X-rays is comparable to the atomic bond distances (around 1Å). However, the amount of X-ray scattering information obtained from an individual molecule is not sufficient for structural analysis. Signal amplification is done by crystallizing individual molecules into highly ordered 3-D arrays before analysis of their X-ray diffraction. Thus, obtaining well-ordered crystals that are built by numerous unit cells corresponding to an individual diffraction spot in an X-ray diffraction pattern is the absolute prerequisite for this technique. In case of hydrophobic membrane proteins, obtaining of good quality crystals (diffracting X-rays to high resolution) is particularly difficult because of their insolubility and the presence of hydrophobic surfaces that restrict crystallization. Therefore, it is no coincidence that most crystallized membrane proteins are bacterial OM porins (Schulz, 2004), not possessing prominent stretches of hydrophobic amino acids (see section 3.4). Despite the challenge, generating crystals of hydrophobic membrane proteins is possible through complex-formation with small amphiphilic molecules (detergents), such as octylglucosides (OG). Proteins can also be crystallized together with a corresponding antibody (Fc fragment). The natural epitope of a membrane protein is surface exposed and therefore less hydrophobic, which when bound by the Fc fragment increases the solubility of the complex. Given the difficulty of obtaining 3-D crystals of membrane proteins, two-dimensional (2-D) crystallization and subsequent electron microscope analysis (electron crystallography) is a useful technique for structural characterization of membrane proteins (Hasler et al., 1998a; Henderson et al., 1990). Not only are 2-D crystals, or ordered sheets of membrane proteins, easier to obtain, but in some cases they actually occur in vivo, in the plane of the membrane (Egli et al., 1993; Henderson & Unwin, 1975; Masuda & Kawata, 1982; Pajatsch et al., 1999). The amphiphilic character of integral membrane proteins promotes their integration into artificial lipid bilayers. Protein-protein interactions may lead to ordering of the proteins within the lipid bilayer into 2-D crystals that are
39
INTRODUCTION amenable to structural studies by electron microscopy (EM), or even atomicforce microscopy (AFM). AFM is a suitable tool for determining the surface topography of the native protein complexes and to monitor conformational changes directly in 2-D crystals (Muller et al., 1997; Scheuring et al., 2002). It is well documented that lipid and detergent properties, as well as temperature and pH influence the crystal type and quality. Subsequent image processing by correlation averaging projection (Saxton & Baumeister, 1982) provides sufficient resolution for structural characterization of membrane proteins. Electron crystallography has revealed 3-D structures of different eukaryotic membrane proteins, including aquaporin 1 (Aqp1), major intrinsic protein (MIP) (Hasler et al., 1998b; Walz et al., 1997) and bacterial membrane proteins like E. coli aquaporin Z (AqpZ) to high resolution (Ringler et al., 1999; Scheuring et al., 1999). The surface topography of AqpZ at 7Å resolution was also determined by AFM and image processing (Scheuring et al., 1999). Furthermore, the structure of E. coli lactose permease, a highly hydrophobic cytoplasmic membrane transporter (Zhuang et al., 1999) and certain porins such as the Campylobacter jejuni major outer membrane protein, MOMP (Amako et al., 1997; Zhuang et al., 1997), E. coli OmpF (Engel et al., 1992) and PhoE (Jap et al., 1991) was revealed by electron crystallography.
40
AIMS
AIMS OF THIS THESIS This thesis was aimed at investigating the molecular and biochemical properties of integral outer membrane proteins of B. burgdorferi. Such proteins might allow this spirochete to adapt to different environments as it cycles between invertebrate and vertebrate hosts and to function as virulence factors during the establishment of Lyme disease. Emphasis was placed on the channel-forming OM proteins of B. burgdorferi, particularly to determine the structure and function of the P13 protein and its paralogs and the integrin binding protein P66. Specific aims: Analyse the conserved and variable features of p13 and its paralogous genes from different Borrelia species. Investigate patterns of transcription within the p13 gene family during murine infection. Determine the surface exposed epitope of P13. Elucidate the function of P13. Study the possible involvement of carboxyl-terminal processing protease A (CtpA) in the maturation P13 and its paralog BBA01. Demonstrate the cellular localization and function of BBA01. Reveal the channel-forming protein, P66.
properties
of
integrin
binding
Perform a structural characterization of the B. burgdorferi porins, P13 and P66.
41
RESULTS AND DISCUSSION
RESULTS AND DISCUSSION 4.
Molecular analysis of P13 and its paralogue family 48 (Paper I)
Borrelia spirochetes exhibit a very high level of genomic redundancy, resulting in large numbers of plasmids and paralogous gene families. The biological importance of this feature remains unresolved. In the absence of major Osps (OspA-D and DbpA/B) in B. burgdorferi strain B313, a new 13kDa surface antigen (P13) was discovered (Sadziene et al., 1995). Mice infected with B313 were used to generate MAbs against P13, which in turn inhibited the growth of the Osp-deficient strain but not the wild-type strain (Sadziene et al., 1995). This implied that surface exposed lipoproteins must hide the P13-epitope, in a similar manner proposed for another integral outer membrane protein, P66 (Bunikis & Barbour, 1999). Surface exposure of P13 was later confirmed by a variety of techniques, while the presence of a N-terminal signal sequence, signal peptidase I cleavage site and three trans-membrane spanning α-helices were predicted by computer analysis (Noppa et al., 2001). Furthermore, C-terminal sequencing revealed that P13 is processed at the C-terminus, but N-terminal sequencing was blocked by a possible modification at the N-terminus (Noppa et al., 2001), which by combined mass spectrometry was confirmed to be pyroglutamylation (pyroglutamate formation at a N-terminal located glutamine) (Nilsson et al., 2002). Pyroglutamate modification is either catalyzed by the enzyme glutamine cyclotransferase or can occur spontaneously (Awade et al., 1994; Busby et al., 1987; Khandke et al., 1989). The latter might occur in P13 since no glutamine cyclotransferase homologue was found in the B. burgdorferi genome. Although pyroglutamate formation at N-terminal glutamine residues has been suggested to protect some proteins from proteolytic degradation (Strobl et al., 1997), the role of P13 pyroglutamylation remains to be elucidated. While P13 is chromosomally encoded, another 8 additional members of this paralogous gene family 48 are plasmid encoded (Fraser et al., 1997; Noppa et al., 2001). To investigate the role of the p13 paralogues we choose few other genes with highest sequence homology to p13 (i.e. bba01, bbi31, bbh41, and bbq06). The presence of these 5 genes in different Lyme disease Borrelia species was analysed by PCR. p13 and bba01 were present in the vast majority of species investigated, while other paralogues were restricted to B. burgdorferi strains. Because of wide distribution of the p13 and bba01 alleles within Lyme 42
RESULTS AND DISCUSSION disease Borrelia species, the sequence heterogeneity of P13 and BBA01 was investigated. P13 was generally well conserved, although some sequence heterogeneity occurred outside the predicted trans-membrane regions (Noppa et al., 2001). The bba01 gene sequence comparison revealed only some minor heterogeneity between Lyme disease Borrelia species. BBA01 production by in vitro cultivated Borrelia was detected in all three Lyme disease Borrelia species, being most prominent in B. afzelii ACAI. Production was also up-regulated in the p13 knock-out strain, so BBA01 might be able to substitute for the function of P13 (see Paper IV for further information). Given that the most heterogeneous region of protein is often the surface exposed epitope, we tested this theory for P13 by epitope mapping. Three fragments of mature P13, corresponding to the predicted trans-membrane regions (A and C) and the surface exposed region (B) (Fig. 6) were expressed in E. coli and analysed with immunoblot using the MAb 15G6 (described above), which recognizes the natural epitope of P13 (Sadziene et al., 1995). As only fragment B was recognized and by in silico analysis fragments A and C were predicted to contain transmembrane spanning regions (Noppa et al., 2001), we proposed a model of P13 location in the outer membrane (Fig. 6). α-P13 MAb
H2N B
OM
A C
COOH
Figure 6. Model of the P13 architecture in the outer membrane (OM) of Borrelia. Three fragments used as targets for epitope mapping are shown by dashed, coloured lines as A, B and C. The bold line shows the OM distribution of mature P13 after N- and C- terminal processing. MAb 15G6 is schematically shown to recognize the surface exposed epitope of P13. Reprinted with modification from Microbiology (Paper I), with permission of the publisher (Society for General Microbiology, SGM). Plasmids loss during in vitro cultivation of Borrelia is a common
phenomenon, but restricted to plasmids harbouring genes whose products are 43
RESULTS AND DISCUSSION not needed for in vitro growth but might be important for infectivity (Hefty et al., 2002b; Labandeira-Rey & Skare, 2001; Purser & Norris, 2000; Thomas et al., 2001). We observed herein that plasmids lp28-3 (harbouring bbh41) and lp28-4 (harbouring bbi31) are lost during in vitro cultivation, implicating that the bbh41 & bbi31 paralogues were not required. To investigate what role p13 paralogues play in Borrelia pathogenicity, we used RT-PCR to compare the transcription of p13, bba01, bbi31 and bbh41 in laboratory culture and in murine infections. p13 and bbi31 genes were transcribed in both conditions, whereas bbh41 and bba01 were transcribed only in vitro. Thus, transcription of p13 and bbi31 in mice indicate that these paralogues could be involved in adaptation of Borrelia to different environments and/or in the infection process. Although investigations of differentially expressed genes of Borrelia in response to various environments did not highlight p13 or bbi31, these studies differed in experimental set up and were not conducted in a murine infection model (Brooks et al., 2003; Ojaimi et al., 2003; Ojaimi et al., 2005; Revel et al., 2002; Tokarz et al., 2004). Nevertheless, upregulation of another p13 paralogue, bbq06 solely in ‘’mammalian hostadapted’’ B. burgdorferi (Brooks et al., 2003) and the clinical isolate of B. burgdorferi with hematogenous dissemination capacity (Ojaimi et al., 2005), indicates that there might be certain environments in which p13 paralogues could be critical, but are difficult to mimic in experimental models.
5.
P13, a channel-forming protein of B. burgdorferi (Paper II)
The host-pathogen interactions are facilitated by surface-exposed proteins. In B. burgdorferi, the scarcity of integral outer membrane proteins and extreme abundance of lipoproteins has prompted researchers to focus on surface exposed lipoproteins. This has often been at the expense of understanding how Borrelia overcomes the impermeable hydrophobic lipid membrane barrier and its low metabolic capacity. Since this implies a specific requirement of efficient nutrient uptake mechanisms, it is highly feasible that these are established by integral outer membrane channels (porins). Therefore, we aimed to elucidate the function of P13. The surface exposure and predicted trans-membrane spanning domains of P13 led us to investigate its channel-forming properties using both genetic and biophysical experiments. We purified native P13 from an outer membrane protein preparation (B-fraction) (Magnarelli et al., 1989) of B. burgdorferi B313 by FPLC, then used this in a planar lipid bilayer assay. The purity of B-fraction was verified by the absence of contaminating periplasmic FlaB protein (M. Pinne, et al., unpublished results) and is our standard outer
44
RESULTS AND DISCUSSION
75 -
B
P1 3
O M
W M
A
-a
membrane protein fraction used in the laboratory. Purified P13 had a channelforming activity with an average single-channel conductance of 3.5 nS. Moreover, this channel-forming activity could be significantly reduced by preincubation with MAbs or polyclonal rabbit serum against P13 (preliminary results). The ion selectivity measurements revealed that cations are preferentially transported through the P13 channel (Pcation/Panion=2.1), although anions can also penetrate the channel. This might indicate that positively charged molecules are actually transported through the P13 pore. Most porins exist in either open or closed states depending on the transmembrane potential, a phenomenon known as voltage gating. Whether this is physiologically significant is questionable since porin voltage dependence measurements in planar lipid bilayer assay suggest that the applied critical voltage (Vc), above which general porins close, far exceeds the naturally occurring Donnan potential across the outer membrane (Koebnik et al., 2000). However, this strategy is still widely used to characterize porins. The channel formed by P13 was not influenced by voltage increases, reflecting a stably incorporated pore in the lipid bilayer. Since substrate-specific channels tend to be voltage independent (Koebnik et al., 2000), perhaps P13 is a specific porin. While numerous substrates were tested, we have yet to demonstrate substrate specificity for the P13 channel.
P13 ’’5-mer’’
50 -
W
75 50 37 -
30 -
25 20 -
25 15 -
M
15 -
P13
10 -
C na -na 3 3 P1 P1
P13 ’’4-mer’’ P13 ’’3-mer’’ P13 ’’2-mer’’ P13
10 -
Figure 7. Oligomer formation of P13. Immunoblot analysis of (A) the outer membrane protein fraction (OM) of B. burgdorferi B313 or FPLC purified, functional P13 (P13-a), (B) non-functional P13 (P13-na), and non-functional, cross-linked P13 (P13-naC) probed with MAb 15G6 recognizing P13. Different oligomeric states of P13 are indicated by arrows and probable numbers of monomeric subunits per oligomer is indicated between inverted commas. Molecular mass markers (in kDa) are indicated on the left. (A) Reprinted with modifications from Journal of Bacteriology (Paper II), with permission from the publisher (American Society for Microbiology, ASM). 45
RESULTS AND DISCUSSION
Insertional events (%)
Interestingly, a 66 kDa oligomer was observed by western blotting of functionally active and pure P13 (Figure 7A). All conventional porins form trimers in their functional state (Koebnik et al., 2000; Schulz, 2004), and two other B. burgdorferi porins, Oms28 (Skare et al., 1996) and P66 (Paper V) have a capacity to form trimers. To achieve the final oligomeric state required for functional P13 has been met with limited success, most purification attempts result in monomeric, non-functional forms of P13 (data not shown). To investigate oligomer formation, FPLC-purified P13 was chemically cross-linked with 1mM glutaraldehyde in the presence of 0.5% OGP. Various oligomers up to 50 kDa in size were detectable by non-reducing SDS-PAGE and immunoblotting representing oligomeric intermediates up to 4 subunits, but no oligomer of 66 kDa, a probable pentamer was observed (Fig. 7). While this suggests that P13 forms oligomers, the exact number of subunits in a P13 oligomer remains to be elucidated. A (wt)
12
*
10 8 6 4 2 0 1
2
3
4
5
6
7
8
9 10 11 12
Insertional events (%)
Channel conductance (nS)
35
B (Δp13)
30 25 20 15 10 5 0 1
2
3
4
5
6
7
8
9 10 11 12
Channel conductance (nS)
Figure 8. Channel-forming activities of OM proteins. (A) wild type, B31-A (wt) and (B) p13 knock-out (Δp13) strains of B. burgdorferi in PC-n-decane membranes. The asterisk (*) indicates the average (3.5 nS) channel conductance due to P13 activity. The total numbers of insertional events, i.e. increase of the membrane conductance were 143 for wt and 139 for Δp13. Reprinted with modifications from Journal of Bacteriology (Paper II) with permission from the publisher (ASM).
The channel-forming activity of P13 was also confirmed by genetic experiments. The p13 gene was inactivated by allelic-exchange mutagenesis, 46
RESULTS AND DISCUSSION this being the first reported example of disruption of a gene encoding an integral outer membrane protein of B. burgdorferi. The porin activities of OM protein preparations derived from knock-out and wild-type strains were compared using the planar lipid bilayer assay. The 3.5 nS activity corresponding to P13 was eliminated in the p13 knock-out strain (Fig. 8). Moreover, it revealed evidence of additional porin activities present in OM of B. burgdorferi, distinct from the small 0.6 nS porin activity of Oms28 (Skare et al., 1996) and the large 11 nS porin activity of P66 (Skare et al., 1997), and the 3.5 nS channel-forming activity corresponding to P13. We predict that uncharacterized porins with channelforming activities in the range of 1-2 nS, ~6 nS and ~8 nS are present in the OM of Borrelia (Fig 8). Attempts to identify and characterize the proteins responsible for these additional porin activities are still ongoing. Several interesting characteristics of P13, such as its small size, unusual amount of subunits in an oligomer, hydrophobicity and predicted membrane-spanning α-helices are all in disagreement with common features of conventional porins (see sections 3.3 and 3.4). Thus, our study adds further the impetus to the growing notion that novel channel-forming proteins reside in outer membranes of spirochetes. It is already established that several porins of Gram-positive bacteria are formed by rather small (5 to 23 kDa) proteins (Lichtinger et al., 2001; Riess et al., 1998; Riess et al., 2003), while the crystal structure of T. pallidum channel-forming protein, Tromp1, revealed that it spans the outer membrane with α-helices (Blanco et al., 1995; Blanco et al., 1999; Lee et al., 1999; Zhang et al., 1999). Furthermore, a recently discovered T. pallidum outer membrane protein, TP0453, may enhance membrane permeability by inserting into the inner leaflet of OM by amphipathic α-helices (Hazlett et al., 2005). Yet another unusual structure element, the α-barrel, is described for the channeltunnel, TolC, of E. coli (Koronakis et al., 2000). Along with these examples, we suggest that P13 also represents another distinctive class of channel-forming proteins.
6. C-terminal processing of P13 by carboxyl-terminal processing enzyme A, CtpA (Paper III) We believe that an unusual C-terminal cleavage of P13 also plays a role in translocation and/or functionality of the protein in vivo. To investigate this notion, an attempt to identify the protease responsible for C-terminal processing of P13 was performed. Using the amino acid sequence of the known carboxylterminal protease A (CtpA) from Bartonella bacilliformis (Mitchell & Minnick,
47
RESULTS AND DISCUSSION 1997) to ‘’blast’’ the B. burgdorferi B31 MI genome (Casjens et al., 2000), a
pA
Δc t
B3
MW
1-
A
(w t)
homologous gene (bb0359) was found. Although we could also detect ctpA homologues in numerous bacteria, a detailed characterization of this protease and its substrates has been performed only in green algae and higher plants, where it plays a crucial role in the photosystem II (Anbudurai et al., 1994; Fabbri et al., 2005; Inagaki et al., 1996; Shestakov et al., 1994; Trost et al., 1997). G G V2 A V2 S S B tp pB /p +c / A A tp tp Δc Δc
50
α- CtpA
25
α- Oms28
α- P13
14.1
Figure 9. Effect of genetically manipulating ctpA. Immunoblot analysis of CtpA, P13, and Oms28 from B31-A (wild-type), ΔctpA (ctpA knock-out), ΔctpA/pBSV2G (ctpA knock-out with empty shuttle vector), and ΔctpA/pBSV2G+ctpA (complemented ctpA knock-out). The positions of molecular mass markers (in kDa) are indicated on the left. Reprinted from Journal of Bacteriology (Paper III) with permission from the publisher (ASM).
To elucidate if the activity of P13 and/or other Borrelia proteins are influenced by CtpA, we inactivated the ctpA gene by allelic exchange mutagenesis. Immunoblot analysis revealed that P13 was larger (Fig. 9) and had a more acidic pI in the ctpA knock-out, consistent with the theoretical size and pI of P13 if it was not processed at the C-terminus. The 2-D gel electrophoresis of B. burgdorferi total proteins showed that nine proteins were only present in the ctpA mutant, six proteins were observed only in wild-type (B31-A). Mass spectroscopy analysis of these potential CtpA substrates revealed that P13 and BB0323 are processed by CtpA, while Oms28 is up-regulated and produced in multiple isoforms in the absence of CtpA. These effects were due to loss of CtpA because complementation with a wild type copy of ctpA restored wild type-like protein expression profiles (Fig. 9). However, Oms28 is not a substrate for CtpA, so that its up-regulation is probably a secondary effect of ctpA inactivation. Interestingly, the expression level of CtpA in the complemented
48
RESULTS AND DISCUSSION strain is higher than in wild-type, reducing the levels of Oms28 to beyond its detection limit (Fig. 9). Thus, CtpA has pleiotropic effects, processing P13 and influencing the appearance of several other proteins.
Figure 10. Proposed model for the processing and translocation of functional P13 porin. Translated, unprocessed P13 has predicted molecular weight of 19.1 kDa and four transmembrane spanning regions (indicated in green), including a signal peptide. IM, inner membrane, P, periplasmic space, OM, outer membrane. pGlu, pyroglutamate modification (see section 4).
Being extremely hydrophobic (hydropathicity index, 0.47) and spanning the membrane with α-helices, one would have assumed that P13 would stack in inner membrane by the conventional SRP-dependent protein secretion machinery. Nevertheless, the unusual feature of C-terminal cleavage by the C-terminal processing enzyme A (CtpA) led to our hypothesis that this event initiates translocation to the outer membrane. Yet, P13 is present in OM protein preparations (B-fractions) from the ctpA knock-out mutant. Therefore, it might be the 28-amino acid C-terminal extension of P13 that is required for transportation to the outer membrane, but cleavage is necessary for correct P13 assembly. This mechanism is supported by the function of CtpA in photosynthetic organisms. A substrate for CtpA in photosystem II, the hydrophobic peptide D1 that spans the thylakoid membranes of chloroplasts (Hankamer et al., 2001), must be cleaved at its C-terminus for correct assembly and
49
RESULTS AND DISCUSSION translocation and to ensure a functional photosystem II (Ivleva et al., 2000; Nixon et al., 1992). The C-terminus of P13 is processed immediately after the alanine residue removing the extreme C-terminal 28 aa (Noppa et al., 2001). Interestingly, the C-terminal extension removed in D1 from different organisms varies from between 8 to 16 aa, but it is always cleaved after an alanine (Ivleva et al., 2000). Clearly, similarities between P13 and D1 exist and imply that CtpA might play a critical role in the translocation and assembly of functional P13. Moreover, CtpA of B. burgdorferi contains a predicted N-terminal signal sequence reminiscent of the need to transport CtpA across the thylakoid membrane in chloroplasts (Karnauchov et al., 1997). By analogy, we interpret that CtpA is transported through the Borrelia inner membrane and that C-terminal processing of P13 occurs in the periplasmic space. Based on our work, we propose a model for the processing and translocation of P13 to the OM of Borrelia (Fig. 10).
7.
Characterization of the BBA01 protein, the paralog of P13 (Paper IV)
There is great interest in understanding the biological advantage of maintaining the extraordinary amount of paralogues in the B. burgdorferi genome, especially given that this is an energetically expensive process. As Borrelia encounter different environments during their life cycle, perhaps there are circumstances in which one protein function needs to be substituted by another paralog, more suited to the ‘’new’’ task (Roberts et al., 2000; Yang et al., 2003). While initial studies on p13 paralogues, the members of gene family 48, were described in Paper I, herein we more closely analysed the interplay between P13 and its paralog BBA01. The bba01 gene was inactivated by allelic-exchange mutagenesis and subsequently complemented using the bba01 gene cloned into the pBSV2G shuttle vector. No difference in cell morphology or in vitro growth rate was observed. However, lack of a discernible phenotype may be explained by the presence of P13, which is highly expressed in all B. burgdorferi strains. In Paper I, we observed that BBA01 production was very low, being insufficient for further characterization. To overcome this obstacle, we increased the number of bba01 gene copies into B. burgdorferi B313 (Sadziene et al., 1995) via the shuttle vector pBSV2G. Strain B313 was used so that BBA01 function could be assessed in the absence of most outer membrane Osps as well as the entire plasmid lp54 on which bba01 is located. Similarly, multiple copies of bba01 was also introduced into a p13 knock-out strain (Paper II) so that BBA01 could be
50
RESULTS AND DISCUSSION analysed in the absence of P13, a protein of very similar features, and into a p13/p66 double knock-out strain (Paper V) devoid of two B. burgdorferi porins (unpublished results). On the basis of that BBA01 shows a 51.4% similarity to P13 and is smaller (~13.5 kDa) than theoretically predicted (17.8 kDa) when analysed by SDS-PAGE (Paper I), implicated that BBA01 might also be processed similarly to P13. Analysis of our CtpA knock-out strain (Paper III) revealed that BBA01 is also a substrate for CtpA. Furthermore, as BBA01 is up-regulated in the absence of P13 (Paper I), maybe these proteins are functionally redundant. Therefore, we wanted to elucidate if BBA01 is also surface-exposed and/or exhibits a channelforming activity. We have experimentally verified that the outer membrane proteins, including OspA-D, P13 and P66 are enriched in the outer membrane protein fraction (B-fraction). We therefore attempted to elucidate the localization of BBA01 by immunoblot analysis of the B-fraction from our different strains harbouring multiple copies of bba01. BBA01 was present in the B-fraction, but unlike P13, was not enriched there. Interestingly, the largest amount of BBA01 was observed in strain Δp13+bba01. Paralogous proteins need not occupy the same cellular localization. This has been already illustrated for the OspE/F/Elp and Bdr paralogs (Hefty et al., 2002b; Roberts et al., 2000). However, it is possible that in the absence of P13, a greater proportion of the cellular BBA01 protein can be transported to the outer membrane. To investigate this, we proteinase K treated intact Borrelia. BBA01 was not sensitive to proteinase K when produced in strain B313+bba01, despite the absence of major Osps, which is known to increase proteolytic sensitivity of another integral outer membrane protein, P66 (Bunikis & Barbour, 1999). In contrast however, a substantial amount of BBA01 was cleaved in strain Δp13+bba01. This supports our hypothesis that an increased proportion of total BBA01 becomes localized to the outer membrane in the absence of P13, which would be consistent with the ability of these two proteins to perform similar functions. To investigate the functional similarity between P13 and its paralog BBA01 we analysed the channel-forming ability in a planar lipid bilayer assay of native BBA01 contained in the B-fraction of B. burgdorferi carrying multiple copies of bba01, as well as recombinant proteins rBBA01 and rP13. We know that the 3.5 nS channel-forming activity is abolished in the p13 knock-out strain, Δp13 (Paper I). Therefore, by comparing this strain with strain Δp13+bba01, we were able to demonstrate that the 3.5 nS channel-forming activity was restored. Since both proteins form channels with similar size (according to the same
51
RESULTS AND DISCUSSION
A
Insertional events (%)
channel-forming conductance of 3.5 nS), we propose that BBA01 might serve as a functional substitute for P13 in its absence. We also took advantage of our p13/p66 double knock-out strain, since the P13 and P66 porins contribute to most of the channel-forming activities in the Borrelia B-fraction (compare Fig. 8A with Fig. 11A, see Paper V for additional information). bba01 introduced into this background enabled us to confirm that the 3.5 nS channel-forming activity is indeed due to BBA01 (preliminary results). Channel-forming activities in the range of 1nS to 4.5 nS were observed, but the main single channel-forming conductance measured 3 nS in strain Δp13/Δp66+bba01 (Fig. 11B). 50
Δp13/Δp66 40
30
20
10
.5 11
11
1 1.
9
1
7
8
0.
0.
0.
6
0.
0.
5
4
3
0.
2
0.
0.
0.
1
0
50
*
Δp13/Δp66+bba01
40
30
20
0 0.
5
1.
5
2.
5
3.
5
4.
5
5.
5
6.
5
7.
5
8.
5
9.
5
10
.5
.5
10
11
B
Insertional events (%)
Channel conductance (nS)
Channel conductance (nS)
Figure 11. Porin activities of OM proteins of B. burgdorferi strains. (A) p13 and p66 double knock-out (Δp13/Δp66) and (B) p13 and p66 double knock-out with introduced bba01 gene (Δp13/Δp66+bba01) in PC-n-decane membranes. In panel (A) no channel-forming activities above 1.1 nS were observed, for illustration of that the x-axis is shown as disrupted line. The asterisk (*) indicates the average (3 nS) channel conductance due to BBA01. The reference depicts the channel conductance (~1 nS) of the same porin, to better illustrate the scale differences of the two panels. The total numbers of insertional events were 39 for Δp13/Δp66 and 103 for Δp13/Δp66+bba01. 52
RESULTS AND DISCUSSION We also examined the purified, recombinant proteins rBBA01 and rP13 isolated from E. coli, for channel-forming activity. Both proteins exhibited average channel-forming activity of 4 nS, which is consistent with our findings using native proteins. The same conductance steps of 4 nS for both rP13 and rBBA01 indicate that both proteins may have very similar structures, which is supported by a common in silico structural prediction, where both proteins are composed of hydrophobic α-helices spanning the membrane (Noppa et al., 2001). This indicates that the BBA01 paralog is also a member of an unusual porin group and that BBA01 and P13 could be functionally interchangeable. We interpret these data to imply that B. burgdorferi possesses many paralogous gene families in order to fine-tune protein function under different environmental or physiological conditions. Global gene regulation in B. burgdorferi using microarrays has received much recent attention (Brooks et al., 2003; Ojaimi et al., 2003; Revel et al., 2002; Tokarz et al., 2004), where conditions mimicking the tick or host-adapted stages using temperature, pH and cell density are widely used. These conditions only approximate natural environments are regulated only by in vivo conditions. Interestingly, the up-regulation of one p13 paralogue, bbq06 occurred following Borrelia growth in dialysis membrane chambers (DMC) implanted in rats, i.e. ‘’mammalian-host adapted’’ stage (Brooks et al., 2003) and in the clinical isolate of B. burgdorferi with capacity of hematogenous dissemination compared to the clinical isolate with no such capacity of dissemination (Ojaimi et al., 2005). Thus, paralogues of the same gene family can be differentially regulated, responding to specific sets of arthropod and/pr mammalian host factors/conditions. Another B. burgdorferi porin, Om28, is also transcriptionally regulated in response to different environmental signals (Brooks et al., 2003; Ojaimi et al., 2003; Revel et al., 2002; Tokarz et al., 2004) and is inversely correlated to the levels of CtpA (Paper III). This evokes a sophisticated interplay between various regulatory mechanisms that is necessary for porins to function in nutrient acquisition from different surroundings.
8.
The function and oligomeric status of P66 (paper V)
To better understand the role of integral outer membrane proteins in biology and pathogenicity of Borrelia, we further characterized P66, a surface exposed channel-forming protein with an unusually large conductance (Bunikis et al., 1995; Skare et al., 1997). P66 can also bind to β3–chain integrins implicating a role also in attachment to host tissues (Coburn et al., 1999; Coburn & Cugini, 2003). Given the
53
RESULTS AND DISCUSSION large channel-forming conductance, the absence of predicted β-sheets, a typical porin secondary structure, and a clear ligand for αvβ3 integrins, it is now uncertain if P66 functions as a porin. Nevertheless, porins can have additional functions besides solute and nutrient uptake, including acting as adhesins (Bernardini et al., 1993; Fenno et al., 1996; Haapasalo et al., 1992; Moser et al., 1997; Weinberg & Holt, 1991). Therefore, we investigated the possible dual function of
P66 by both genetic and biophysical methods. Purified native P66 from an outer membrane protein fraction (B-fraction) of B. burgdorferi possessed channelforming properties in a planar lipid bilayer assay with an average single channel conductance of 11.5 nS. The channel is also slightly anion selective and exhibited a voltage induced closure, i.e. it was voltage dependent. These results mirrored an earlier study, although no ion selectivity and the voltage dependence was observed at lower voltages (Skare et al., 1997). Additionally, we showed that pre-incubation of purified P66 with polyclonal anti-P66 serum completely abolished channel-forming ability. Together, these data verify that P66 is a porin. Conventional porins form trimers in their functional state (Benz, 2001; Koebnik et al., 2000; Schulz, 2004). When we investigated the oligomeric status of native P66 using different concentrations of the glutaraldehyde cross-linker (Fig. 12), dimeric and trimeric structures formed in a concentration-dependent manner, implicating that P66 may function as a trimer. Glutaraldehyde (mM)
0 0.5 1 2.5 5 250 150
P66 trimer P66 dimer
100 75
P66
50 Figure 12. Determination of the oligomeric status of P66. Native P66 was chemically cross-linked with different concentrations of glutaraldehyde. Proteins were separated on SDSPAGE (Tris-Acetate 3-8% NuPage gel), blotted on PVDF membrane and probed with MAb H914 recognizing P66. The positions of molecular mass markers are indicated on the left.
To further confirm P66 porin function, while also investigating the effects of simultaneous loss of multiple porins, we inactivated p66 in the strains B31-A (Bono et al., 2000), Δp13 (Paper II) and Δbba01 (Paper IV). Furthermore, we 54
RESULTS AND DISCUSSION used also the strains HB19/K02 and HB19/K04 lacking p66 that were originally used to confirm the integrin αvβ3 binding ability of P66 (Coburn & Cugini, 2003). Moreover, we establish a second set of isogenic strains by re-introducing wild type p66 on the pBSV2G shuttle vector. We examined the channel-forming abilities in the planar lipid bilayer assay of outer membrane protein preparations from all these strains. Clearly, the large ~11 nS channel-forming conductance does correspond to P66 (Fig. 13 and Paper V), confirming by genetic methods that P66 is a B. burgdorferi porin. In addition, the 11 nS channel-forming activity due to complemented P66 was abolished after pre-incubation with polyclonal anti-P66 serum (data not shown).
Insertional events (%)
A
50
Δp66
40 30
20 10
.5
11
11
5 4.
5
4
3
3.
5
2
2.
5 1.
5 0.
1
0
Channel conductance (nS)
Insertional events (%)
B
45
*
40
Δp66+p66
35 30 25 20 15 10 5 0
1 2 3 5 6 7 8 10 11 12 13 15 16 17
Channel conductance (nS)
Figure 13. Complementation of P66 porin activity. (A) Channel-forming activities of OM proteins from strain B31-A in which p66 has been inactivated (Δp66) and (B) channelforming activities of P66 containing FPLC fraction of OM proteins from the same strain complemented with the p66 gene (strain Δp66+p66) strain in PC-n-decane membranes. In panel (A), no channel-forming activities above 4.5 nS were observed, for illustration of that the X axis is shown as disrupted line. The asterisk (*) indicates the average (11 nS) channel conductance due to complemented P66. The reference depicts the channel conductance (~1.5 nS) of the same porin to better indicate the differences in scale of both panels. The total numbers of insertional events were 120 for Δp66 and 117 for Δp66+bba01. 55
RESULTS AND DISCUSSION Depletion of the ~11 nS channel-conductance corresponding to P66 allowed us to elucidate the previously hindered smaller channel-forming activities present in OM protein preparations of B. burgdorferi. For example, a measurable 0.3 nS channel-forming activity could be attributed to the TolC homologue of B. burgdorferi, BB0142 (I. Bunikis, K. Denker, unpublished results). Moreover, the analysis of channel-forming activities of OMPs from the Δp13/Δp66 double knock-out revealed that most of the previously suggested porin activities are absent, particularly those with a conductance above 1 nS (compare Fig. 8 and Fig 11A). This could mean that P13 and P66 are needed for reconstitution of other porins or that the channel-forming activities observed are actually insertions of more than one channel simultaneously. P66 is expressed in the mammalian host as evidenced by immune responses in Lyme disease patients, being widely used in disease diagnosis (Dressler et al., 1993). Interestingly, P66 production is undetectable in unfed ticks (Cugini et al., 2003). Microarray analysis also suggest that p66 is up-regulated during the tick feeding and ‘’mammalian host-adapted’’ stage (Brooks et al., 2003; Narasimhan et al., 2002). These data collectively indicate that P66 plays a specific role in adhesion to mammalian tissues and/or nutrient acquisition in mammalian hosts. Since the adhesion process is a likely prerequisite for dissemination to various tissues, it would have been expected that B. burgdorferi isolates with an enhanced capacity to disseminate also up-regulate P66 expression. However, this is inconsistent with data observed from a clinical isolate of B. burgdorferi with no capacity for hematogenous dissemination, because transcription of p66 is also up-regulated in this strain (Ojaimi et al., 2005). Nevertheless, P66 is the first B. burgdorferi protein with probable dual functions (porin and adhesin). Further studies are required to determine in which stages of the Borrelia life cycle these two functions are most needed. In addition, a structural characterization of P66 would be valuable, with our initial structural studies presented in Paper VI.
9.
Structural characterization of P13 and P66 by electron crystallography (Paper VI).
The borrelial outer membrane localization and porin function of two integral membrane proteins of B. burgdorferi, P13 and P66, has led to a quest to resolve the structure of these proteins. Crystallization of membrane proteins is considered to be particularly difficult (section 3.8). We performed numerous empirical experimental trials to crystallize P13 and P66 porins, with most progress made with P13 (preliminary data). Good quality crystals were obtained
56
RESULTS AND DISCUSSION (Fig. 14A), giving an X-ray diffraction with high resolution of 1.4Å (Fig. 14B). However, our attempts to obtain crystals with selenium-methionine (Se-Met) labelled P13 were not successful, preventing further determination of its 3-D structure. The electron crystallography technique in connection with image processing is used for determination of many porin structures (Amako et al., 1997; Engel et al., 1992; Jap et al., 1991; Zhuang et al., 1997). Additionally, the formation of well organized 2-D structures of porins is observed in bacterial outer membranes, particularly from spirochetes (Egli et al., 1993; Henderson & Unwin, 1975; Masuda & Kawata, 1982). Therefore, we employed 2-D crystallization of two porins, P13 and P66 in B. burgdorferi OM protein preparations. The strains singularly deficient in P13 (Δp13) and P66 (Δp66) as well as a double knock-out (Δp13/Δp66) (Papers II, and V) were used in this study. Electron-microscopy and subsequent image processing was also used to determine the structure of P66 crystal.
B A
Resolution 1.4Å
Figure 14. Crystallization of P13. (A) P13 crystals grown on sitting drop in 18% PEG6000, 1M NaCl for 3 weeks. Protein concentration 1.1 mg/ml, drop size 4μl. (B) X-ray diffraction pattern of P13 crystal.
Outer membrane protein fractions of various B. burgdorferi strains were investigated by electron microscopy and revealed regularly arranged tetragonal 2-D crystal arrays. The presence of 2-D crystal sheets in strain B313 (Sadziene et al., 1995), which is deficient in the major outer membrane Osps, as well as porin Oms28, led us to hypothesize that these structures are due to other outer membrane proteins, most likely porins. To enable the characterization of 57
RESULTS AND DISCUSSION one particular porin, we compared the EM images of 2-D crystal structures from our different porin knock-outs with those taken of WT. It became evident that 2D crystals were absent only in the double knock out strain. Thus, we proposed that in the absence of P13, the 2-D crystal arrays are formed by P66 and vice versa or that it might be formed by a yet undefined outer membrane protein. Since the organization and quality of 2-D crystals in planar lattice form was better in strain Δp13 (i.e. P66-dependent), these were further subjected to structural characterization by image processing. B
D
98 Å
A
C
b
a
111 Å
Figure 15. Analysis of the 2-D crystals of P66 by EM and image processing. (A) Selected area of an electron micrograph of a negatively stained 2-D crystal array in the outer membrane protein preparation of B. burgdorferi Δp13. (B) Fourier transform of the P66 crystal. The strongest spots are observed around 1/45-1/52 Å-1. The circled spot corresponds to 1/22 Å-1. (C) Density map of the processed image after imposing a p22121 symmetry. (D) Projection map of P66 at 22 Å resolution. A one unit cell with a = 111 Å and b = 98 Å is outlined.
Electron micrographs of 2-D crystal arrays formed by P66 were scanned and subsequently selected areas (Fig. 15A) were used for image analysis. Fourier transform was computed, revealing well-defined reflections of up to 30 Å and reliable reflections to 22 Å resolution (Fig. 15B). Further examination of the phase relationships (Valpuesta et al., 1994) indicated a p22121 plane group. After imposing the p22121 symmetry, the density map of a processed image was obtained revealing the tetragonal arrangement of P66 subunits (Fig. 15C). A fully symmetrized projection map at 22 Å resolution was generated and clearly indicated a rectangular unit cell with nine protein
58
RESULTS AND DISCUSSION monomers and dimensions of a = 111 Å and b = 98 Å (Fig. 15D). The tetragonal symmetry of 2-D crystals formed by P66 in a four subunit protein complex is somewhat unusual, as conventional porins tend to have a trimeric structure. Interestingly, the area in between the protein subunits has a diameter of ~3.9 nm, which is consistent with the proposed large diameter of the channel formed by P66 (Cullen et al., 2004; Skare et al., 1997). However, further studies are required to elucidate the structural organization of functional P66 in lipid membranes. This is the first report using electron crystallography to study a B. burgdorferi protein, highlighting its usefulness for characterization the unusual structure of the borrelial outer membrane and the role of integral outer membrane proteins. Since the formation of well organized 2D plane lattices was lost in B. burgdorferi devoid of the two porins, P13 and P66, the integrity and structure of the outer membrane must be impaired. The absence of one or several components of the outer membrane of bacteria is a potential stress factor. To elucidate what phenotypic alterations the elimination of P13 and P66 porins has on B. burgdorferi cells, we investigated the stress response and osmotic sensitivity of p13, p66 and p13/p66 knock-out strains. HtrA (high temperature requirement) of E. coli is a periplasmic protease known to play a role in degradation of misfolded proteins at increased temperatures (above 42oC) (Clausen et al., 2002; Skorko-Glonek et al., 1999; Strauch et al., 1989) and in heat resistance (Clausen et al., 2002; Pallen & Wren, 1997). HtrA has also a membrane protein chaperone function at low temperatures (Spiess et al., 1999). To estimate if the absence of one or more porins leads to the alterations in this stress response marker, the levels of B. burgdorferi HtrA homologue (BB0104) was compared by western blot. Substantial up-regulation of HtrA homologue was observed in Δp66 and double knock-out strains compared to that of WT. Indeed, the absence of P13 and P66 in outer membranes of Borrelia is a substantial stress-inducing factor for the cell. This may also highlight the HtrA chaperone function as a potential player in the folding and translocation of these proteins to the B. burgdorferi outer membrane (see Fig. 3). We also wanted to investigate if the absence of P13 and P66 porins increases the sensitivity of Borrelia to high osmolarity. There was an obvious growth advantage when grown in the presence of 5% glucose for various parental strains compared to strains lacking one or both of P13 and P66. These experiments indicate that P13 and P66 are important components of the borrelial outer membrane, as their absence induces a stress response and increases sensitivity to high osmolarity in B. burgdorferi.
59
CONCLUSIONS
CONCLUSIONS The p13 gene and its paralogue bba01 are present in the vast majority of Lyme disease Borrelia strains, whereas the paralogues bbi31 and bbh41 are restricted to B. burgdorferi. p13 and its paralogue bbi31 are transcribed in vitro and also during mouse infections, whereas the paralogues bba01 and bbh41 are transcribed only in vitro. The most heterogeneous region of P13 is the surface exposed, natural epitope of the protein. The integral outer membrane protein P13 is a porin. The C-terminus of P13 and its paralog BBA01 are cleaved by the carboxyl-terminal processing protease A, CtpA. BBA01 is a channel-forming protein re-located to the borrelial outer membrane in the absence of P13. BBA01 and P13 are functionally interchangeable. P66 possesses both channel-forming and integrin-binding activities. P13 and P66 porins crystallize in two-dimensional arrays and the crystal structure of P66 at 2.2 nm resolution reveals an unusual four-fold symmetry.
60
ACKNOWLEDGMNETS
ACKNOWLEDGEMENTS During my many years in Umeå there are people who have made this whole thing possible, enjoyable and meaningful. I would like to show my appreciation and say thanks to them. My biggest gratitude goes to my supervisor Sven Bergström. I’m grateful for the chance to do my PhD in your lab, for your support, encouraging and never ending optimism! Thanks for believing in me more than I did it myself! Your great enthusiasm is the best contagious agent I know! Many thanks to former and present members of ‘’Group SB’’! It has been great to do ‘’business’’ with you! My special thanks go to Yngve Östberg, for introducing me in Borrelia and tricky P13 world as well as for good and useful discussions! Åsa Gylfe, for keeping up the group spirit and motivation, and for caring and understanding. Tina Broman, for good talks and sharing emotions. Pierre Martin, for help with computer tricks and nice holidays in Latvia. Laila Noppa, for your energy and support. Darius Straševičius for making me feel comfortable during my first days in Umeå. Ingela Nilsson, for all help in the lab and organization efforts. Elin Nilsson, for good work and calmness. Marie Andersson, for bringing new streams in the group. Pär Comstedt, for nice cooperation. Christer Larsson, for sense of humour and wish to make fun out of almost everything. Betty Guo, for valuable help with correcting my manuscript and for bringing new ideas in our group. May Ali, for all the good laughs. Leslie Baily, for being always so cool and optimistic. Ignas Bunikis, the saviour in those many cases when I don’t agree what my computer is doing, for nice work, all help and good ideas. My appreciation goes also to Alireza Shamaei-Tousi, Xihua Lu ‘’Andy’’ and Annika Nordstrand, for making a great working atmosphere. I would like to acknowledge all people at Department of Molecular Biology for creating perfect and efficient working environment! My special gratitude goes to Marita Waern, for all help in bureaucracy stuff. Berit Nordh, Anitha Bergdahl, Tord Hagervall, for keeping track of my various PhD responsibilities, Johnny Stenman, for getting all those orders reaching the destination, Siv Sääf, for sequencing assistance, Matthew Francis, for linguistic help for my thesis. Present and past members of ‘’Media’’ and ‘’Disken’’, for facilitating the practical issues for our lab-work! Bernt Eric Uhlin, for help whenever it was needed. Sun Nyunt Wai, for enthusiasm and fresh, valuable ideas. Carlos Balsalobre-Parra, for showing that success is achieved by hard work and for unforgettable skiing trips☺! Jurate Straševičiene, for caring, supporting and all that friendly teasing; I appreciate the time we were spending together! Big thanks goes also to other members of Groups ‘’BEU’’, ‘’SNW’’, and ‘’CB’’; Stina, Annika, Monica, Anna, Karolis, and Barbro, for good company in journeys and all laughs. Olena Rzhepishevska, the party queen! Thanks for believing in me, for your optimism and that amazing ability to improve my sometimes disappointing mood, you are just great! Many people from other Departments at Umeå University have helped me to try out the possibilities and facilities around. Karina Persson, for cooperation and supporting my wish to try to crystallize protein which theoretically is impossible to crystallize. Thord Johansson, for help with chromatography of Fab fragments. Lenore Johansson, for help with electron microscopy. Per-Ingvar Ohlsson, for mass-spec analyses. I’m very grateful to all present and former members of ‘’AG Benz’’ in Wüzburg, Department of Biotechnology for fruitful collaboration. Roland Benz, for leading me into a fascinating world of porins and planar lipid bilayer assay. I appreciate a lot your hospitality and great, educative, and joyful trips in Bavaria-area. Elke Maier and Bettina Schiffler, for all that great help. Katrin Denker, for cooperation, help and constant drive for learning and improving; I’m also thankful to you and your family for great time we had
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ACKNOWLEDGMENTS outside the lab. I want to thank for all help, discussions and for making my stay in Wüzburg so enjoyable also to Christian, Marcus, Frank, Noelia, Georg, Peter, Oliver, and Ursula. A lot of appreciation goes to Akemi Takade, the master of electron microscopy, and to Yoshimitsu Mizunoe from Kyushu University, Japan. My appretiation goes to Patricia Rosa and all present and past members of her group from Rocky Mountain Laboratories, Hamilton, for successful collaboration and making also us the ‘’guru of genetics’’ in Borrelia! Thanks to Jennifer Coburn from Tufts University School of Medicine, Boston, for a nice coloboration! My great appreciation goes to present and former members of Biomedical Research and Study Centre, University of Latvia for making me fit for phD studies. Especially, Valdis Bērziņš, for taking the best care of me as young student not knowing anything about complex world of research, you always will be in our memory! Viesturs Baumanis, for opening my eyes towards biochemistry and molecular biology and keeping me in touch with situation in our Institute. Inta Jansone, for tough but useful start into my scientific carrier. Jānis Kloviņš, for raising the interest towards science in me and cool Midsummer celebrations. Normunds Līcis, for being serious but warm-hearted. Elita Avota, for empathy and nice time in Wüzburg. Renāte Ranka, for improving the Borrelia research in Latvia. My course mates Aija Linē and Una Riekstiņa (Lazdiņa), I really admire you! I owe a lot of appreciation to my friends who have made my time in far north of Sweden enjoyable, fun and successful! Zita Balklava-Gross, for your friendship in all these years despite the distance between us. Kristina Wilhelm, for our talks, activities and your attitude towards life, it’s always nice to be around you! Lorenzo Giacani, for your passion! Ewa Wandzioch, for caring and for guiding me through amazing trip in Poland! Fabienne Deleuil, for taking care of my ‘’going out’’ activities! The former and present members of ‘’Lithuanian gang’’, I guess I have a big attraction towards Baltic people and since Lithuanians have been the majority of those and because they are so warm and open-hearted, I’ve spent a lot of enjoyable time with you and even learned the language, thanks for that! My Latvian friends, because of you I felt as at home in our small, cosy community! Ingvars and Vita Birznieki, it is not the same here anymore after you have left! Thanks for all good meals, activities, skiing trips and just great atmosphere you always manage to create around you! Ainārs and Ieva Reiņi, for all those nice celebrations of important dates and always keeping the spirit up! Solvita Ore, for never giving up keeping in touch. Solvita ĶieģeleKutka, thanks for a lot of fun during the time we spent both in Sweden and in Latvia! Maano Aunapuu, my Estonian outdoor and sport activities partner; thanks for your positive attitude and all great trips! Anna Zań, my polish best friend! I’m incredibly grateful for the chance to get to know you, for your understanding, for your wish to share everything with me and for teaching some good lessons of life to me! Thanks to my relatives! Dzintra, paldies par rūpēm! Rita, for having the place to stay in Riga and just for being my closest cousin! Andri, Margarita, Gita, Inga, Viestur, Lilita, Inese, Ingrīda, milzīgs paldies par atbalstu! My biggest gratitude I would like to express to my mom… Mammucīt, Tu esi un vienmēr būsi ar mani tajā podziņā, manā sirsniņā!!! I miss you so much…
This work was financially supported by grants from Swedish Medical Research Council, Swedish Council for Environment, Agricultural Sciences and Spatial Planning, Swedish Foundation for Strategic Research, Infection and Vaccinology and MicMan, the Swedish Foundation for International Cooperation in Research and Higher Education (STINT), and the J. C Kempe Memorial Foundation. 62
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