are well defined in E. coli and consist of several seo-encoded proteins (Sec), ...... Science Research Products, Cleveland, OH) were used according to vendors'.
TOXIN A SECRETION IN Pseudomonas aeruginosa by CATHERINE S. McVAY, B.S., M.S. A DISSERTATION IN MEDICAL MICROBIOLOGY Submitted to the Graduate Faculty of Texas Tech University Health Sciences Center in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY
Advisory Committee Abdul N. Hamood, Chairperson W. LaJean Chaffin Charles Faust Joe A. Fralick David J. Hentges Terence M. Joys
Accepted
Dean o^i^he' Gradiia)te School of Biomedical Sciences Texas Tech University Health Sciences Center and
Dean of the Graduate School Texas Tech University August, 1995
©1995 CATHERINE S. MCVAY All Rights Reserved
ACKNOWLEDGMENTS I owe a great debt of gratitude to my mentor Dr. Abdul Hamood for giving me the opportunity participate in this research He has been a source of leadership, instructional criticism, support, and friendship. For this I am grateful. I also give my thanks to Drs. LaJean Chaffin, Charles Faust, Joe Fralick, and David Hentges for serving on my committee. They have been generous with their support, clarifying questions and enthusiasm. My gratitude is also extended to the departmental faculty and staff and my fellow graduate students for their collegialty and support. I am especially grateful to Jane Colmer for her contagious enthusiam, support, and friendship. Finally, this project would not have been realized had it not been for the constant support of my family. To my son John and my husband Ted, I am grateful. The depth of their patience, encouragement, and love is unfathomable.
II
TABLE OF CONTENTS AKNOWLEDGEMENTS
ii
ABSTRACT
ix
LIST OF TABLES
x
LIST OF FIGURES
xi
I. INTRODUCTION
1
General Characteristics of Pseudomonas aeruginosa
1
Pseudomonas aeruginosa as an Opportunistic Pathogen of Humans
1
P. aeruginosa Infections in Cystic Fibrosis
1
P. aeruginosa Infections in Immune Dysfunction and Traumatic Injury
2
Extracellular Virulence Factors: Pathogenesis
2
Cell-associated Factors
2
Secreted Factors
3
Alginate
3
Elastases and Alkaline Protease
3
Phospholipase C
3
Exoenzyme S
4
Toxin A
4
Toxin A: Molecular Biology
5
Toxin A Gene (toxA)
5
Toxin A: Structure and Function
5
Toxin A Production
6
III
Regulation of toxA
7
Secretion in Gram-negative Bacteria
7
Single-step Pathway
7
General Secretory Pathway
8
Translocation Across the Cytoplasmic Membrane
8
Translocation Across the Outer Membrane
8
Single-step Secretion in Pseudomonas aeruginosa
9
Toxin A Secretion via the General Secretory Pathway in Pseudomonas aeruginosa
9
Translocation of Toxin A Across the Cytoplasmic Membrane
9
Extragenic Requirements
9
Intragenic Requirements: Toxin A Leader Peptide
10
Translocation of Toxin A Across the Outer Membrane
10
Extragenic Requirements
10
xcpA(pilD)
11
xcpT-W xcpR
11 12
Intragenic Requirements
12
The Purpose of the Study
13
MATERIALS AND METHODS
14
General
14
Bacterial Strains
14
Plasmid Vectors
14
Media and Reagents
17 iv
Enzyme Assays
18
ADP-ribosyl Transferase Activity
18
3-lactamase Assays
19
Glucose-6-phosphate Dehydrogenase Assays
19
Alkaline Phosphatase Assays
20
Toxin A-specific Antiserum
20
DNA Manipulation
21
Small-scale Plasmid Purification
21
Large-scale Plasmid Preparation
21
DNA Agarose Gel Electrophoresis
22
DNA Acrylamide Gel Electrophoresis
23
Elution of DNA Fragments
23
Cloning Procedures
24
Detection of toxA Subclone Products
24
Transformation of toxA Subclones into E. coli
24
Electroporation
25
Thparental Mating
25
Fractionation of E. coli
26
Fractionation of P. aeruginosa
27
Toxin A-CRM Analysis
28
SDS-PAGE Protein Analysis
28
Immunoblot Analysis
28
Pulse-labeling of Toxin A-CRMs
29
Expression of toxA Subclones from T7 Promoter
29
v
Immunoprecipitation of Toxin A-CRMs In wYro Expression of toxA Subclones Ba^l Experiments
30 31 32
ea/31 Deletions of toxA
32
Colony Blotting Assays
32
Double-stranded DNA Sequencing
33
Sequencing Reaction Mixtures
33
Electrophoresis of Sequencing Gels
34
Densitometric Analysis
34
III. RESULTS
36
Localization of Toxin A-CRMs Encoded by toxA Subclones Plasmid Constructions
36 36
Amino-terminal Subclones
36
Internal Deletion Subclones
39
Construction of Stable P. aeruginosa Replicons
39
Expression of toxA Subclones
41
Immunoblot Analysis in PA0-T1
41
Immunoblot Analysis in PA103-NT
41
Size of Toxin A-CRMs in PA103-NT
43
Toxin A Activities
43
Kinetics of Secretion
46
Cellular Localization of Toxin A-CRMs in PA103-NT
46
Processing of Toxin A-CRMs
48
Localization of toxA Subclones in XG-9 vi
48
Gene Fusion Experiments
51
toxA-regA Fusion
51
toxA-phoA fusion
54
Detection of the Product of the toxA Internal Deletion Subclone pAM30
56
Construction of toxA Subclone under T7 Promoter
57
Detection of pAM30T7-7A Product
57
Influence of the First 30 a. a. of Mature Toxin A on Toxin A Secretion by P. aeruginosa
61
Strategy for Construction and Isolation of toxA Mutants Expression of Ba/31-generated toxA Subclones Location of toxA Fusion Junctions Localization of Sa^l-generated Deletion Products in P. aeruginosa lasB-toxA Fusion
61 64 64 64 66
Construction of lasB-toxA fusion
70
Localization of pCM209 Product
70
Quantitative Analyses of Toxin A-CRMs
70
Assessment of Proteolytic Degradation of Ba/31generated Toxin A-CRMs
73
Processing of Ba^l-generated Toxin A-CRMs
73
Influence of Leader Peptide Regions of Toxin A Secretion Strategy for Construction and Isolation of toxA Leader Peptide Deletion Mutants Regions of the Leader Peptide Included in Bal3^generated Toxin A-CRMs
VII
75
75 78
Cellular Localization of Leader Peptide Mutants in E coli
78
Cellular Localization of Leader Peptide Mutants in P. aeruginosa
82
Analysis of Levels of toxin A-CRMs
88
Toxin A-CRM levels in E. coli
88
Toxin A-CRM levels in P. aeruginosa
88
Detection of Conformational Differences IV. DISCUSSION
91 93
General Secretory Pathway
93
Intragenic Secretion Signals
94
Intragenic Secretion Signals Within Mature Toxin A
95
Toxin A Leader Peptide
101
Processing of Toxin A-CRMs
103
Evidence of a Conformational Change in Toxin A
105
Model of Toxin A Secretion
106
LITERATURE CITED
108
VIII
ABSTRACT Toxin A (ToxA), an ADP-ribosyl transferase, is the most toxic among the secreted virulence factors of Pseudomonas aeruginosa. The determination of mechanisms by which ToxA reaches the extracellular space is of practical and fundamental importance. Evidence suggests that ToxA crosses the inner and outer membranes of P. aeruginosa in two separate stages according to the general secretory pathway (GSP) model of secretion. Both steps require recognition of determinants within the toxin molecule (intragenic factors) by components of the bacterial secretion machinery (extragenic factors). We have used a series of subcloning and deletion analyses to localize the intragenic signals within ToxA which are required to target the molecule to the extracellular environment. Restriction endonuclease sites within the gene for ToxA (toxA) were used to generate several toxA deletions which were expressed from the E. coli lac promoter. Bal3^ exonuclease deletion analyses were used to further examine the role of the ToxA leader peptide and the amino terminal regions in ToxA secretion. In-frame Sa/31-generated deletions were identified by colony blotting assays using ToxA-speclfIc antiserum and confirmed by DNA sequencing analyses. Localizations of the various toxA subclone products were determined by enzyme assays and Immunoblotting using ToxAspeclflc antiserum. Processing of the ToxA derivatives was examined using in wYro transcription and translation assays. Our results suggest that (1) the first 13 amino adds of the ToxA leader peptide are sufficient to direct the toxin across the cytoplasmic membrane to the periplasm in the first step of the GSP; (2) there are two secretion signals, one within the amino- and one within the carboxy-terminal regions of the mature toxin, either of which is sufficient to direct the molecule across the outer membrane in the second step of the GSP; and, (3) processing of the ToxA precursor (cleavage of the leader peptide) may not be required for release of the toxin to the periplasm or to the extracellular environment.
IX
LIST OF TABLES 2.1
Bacterial strains
15
2.2
Plasmids
16
3.1
Detection of toxin A-CRMs in PA103-NT
37
3.2
Enzymatic activities in the cellular fractions of PA103-NT containing toxA deletion subclones
45
3.3
ADP-ribosyl transferase activities In cellular fractions of the secretion mutant XG-9 containing Intact toxA and toxA subclones
52
3.4
Detection of toxA-phoA fusion product by enzymatic analysis
55
3.5
ADP-ribosyl transferase activities in the cellular fractions of PA103-NT containing fox>A subclones
69
Densitometric analysis of toxin A-CRM levels In the cellular fractions of PA103-NT containing toxA subclones
72
Toxin A activities In the cellular fractions of E. coli DH5a containing toxA leader peptide deletion subclones
83
3.6 3.7
3.8
Cellular markers in the fractions of DH5a and PA103-NT
3.9
Toxin A activities in the cellular fractions of PA103-NT containing toxA leader peptide deletion subclones
87
Densitometric analysis of toxin A leader peptide mutants In the cellular fractions of E. coli DH5a
89
Toxin A-CRM levels In fractions of PA103-NT containing toxA leader peptide deletion subclones
90
3.10
3.11 3.12
Detection of conformational differences of toxin A and toxin A-CRMs
84
92
LIST OF FIGURES 3.1
Details of plasmids carrying amino- and carboxy-terminal fox/A subclones
38
3.2
Details of plasmids carrying toxA Internal deletion subclones
40
3.3 3.4
Detection of toxA subclone products Detection of toxin A-CRMs in fractions of P. aeruginosa strain PA103-NT by immunoblot analysis
42 44
Immunblot analysis of kinetics of secretion of PA103-NT(pAM22) andPA103-NT(pAM26Q-1)
47
Localization of toxin A and toxin A-CRMs by pulse labeling/ immunoprecipitation experiments
50
3.5 3.6 3.7
Details of plasmids carrying toxA-phoA and toxA-regA fusions
3.8
Cloning strategy for the construction of plasmid pAM30T7-7A
59
3.9
Product of pAM20T7-7D expressed In E. co//K38(pGP1-2)
60
3.10
Strategy for 8a/31-generated deletions in toxA
62
3.11
Screening of Sa/31-generated toxA internal deletions for expression of stable toxin A-CRMs
63
Nucleotide and deduced a. a. sequences flanking fusion junction of Ba/31-generated deletions within the toxA region encoding the first 30 a. a. of the mature toxin
65
Localization of toxin A-CRMs In cellular fractions of P. aeruginosa
68
3.12
3.13
53
3.14
Construction of lasB-toxA gene fusion on plasmid pCM209
3.15
Immunoblot analysis of degradation of toxin A-CRMs in the supernatant of PA103-NT
74
Processing of toxin A and toxin A-CRM
76
3.16
XI
71
3.17
Nucleotide and deduced amino acid sequences flanking fusion junction of 8a/31-generated toxA leader peptide deletions
77
3.18
Toxin A leader peptide
79
3.19
Localization of toxin A-CRMs In cellular fractions of E. coli
81
3.20
Localization of toxin A-CRMs in cellular fractions of P. aerginosa.
86
4.1
Model of toxin A secretion in P. aeruginosa
XII
107
CHAPTER I INTRODUCTION General Characteristics of Pseudomonas aeruginosa Pseudomonas aeruginosa is a Gram-negative, non-fermentative, freeliving bacillus, which can be isolated from most moist environments. In nature, P. aeruginosa inhabits soils, fresh and marine waters, and other natural materials. P. aeruginosa Is also a wide-spread contaminant of fluids and moist surfaces In households and hospitals. The ecological diversity of P. aeruginosa is due In part to Its simple growth requirements, and its capacities to utilize a variety of organic compounds as carbon sources and to tolerate a wide range of temperatures (from 20-43°C) (Bergen, 1981). Although usually aerobic, P. aeruginosa can grow anaeroblcally In the presence of nitrate which serves as an electron acceptor or arglnine which serves to generate ATP by substrate phosphorylation (Bergen, 1981). Pseudomonas aeruginosa as an Opportunistic Pathogen of Humans Despite Its ubiquitous and versatile nature, P. aeruginosa is rarely the etiology of disease In healthy humans. It Is, however, the cause of serious, lifethreatening Infection In humans compromised by chronic disease, immune dysfunction, or traumatic injury (Bodey et al., 1983; Vasil, 1986). P aeruginosa Infections In Cvstic Fibrosis As an opportunist, P. aeruginosa is the predominant pathogen in cystic fibrosis (CF). CF, the most common inheritable disease among Caucasians, is characterized by abnormal secretion of electrolytes and altered properties of the mucus on epithelial surfaces (Konstan and Berger, 1993; Vasil, 1986). Eventually, almost all CF patients develop P. aeruginosa lung infections. The predisposing condition of the CF lung, the pathogenicity of P. aeruginosa products and the inherent resistance of P. aeruginosa to many antibiotics culminate In Intractable chronic lung infections (Konstan and Berger, 1993). The cycle of recrudescing P. aeruginosa infections (Davis, 1985; McGowan, 1988: Gilllgan, 1991) and the damaging host inflammatory response they elicit (Bruce et al., 1985; Konstan et al., 1990) is believed by many investigators to contribute to the progressive pulmonary deterioration, which is the ultimate
1
cause of respiratory failure and resulting demise of 95% of CF victims (Wilmott etal.. 1985). P. aeruginosa Infections in Immune Dysfunction and Traumatic Injury Immunodeficiency and traumatic injury, especially severe burns, also predispose humans to P. aeruginosa infection. Patients with leukopenia, either as a result of immunosuppressive therapy or disease, have increased risk of developing severe pulmonary infections with P. aeruginosa (Morrison and Wenzel, 1984). Recently, P. aeruginosa has been recognized as a significant cause of infections in patients with acquired immune-deficiency syndrome (AIDS) (Nichols et al., 1990; Mendelson et al., 1994). P. aeruginosa is one of the two most commonly reported bacterial isolates from severe burn patients. In contrast to the chronic, localized nature of P. aeruginosa infections associated with CF, P. aeruginosa infections in immunocompromised or burn patients are acute and can be disseminated (Konstan and Berger, 1993). Death from P. aeruginosa septicemia approaches 80% and 28% In immunocompromised and burn patients, respectively (Cryz, 1985). Extracellular Virulence Factors: Pathogenesis Cell-associated Factors P. aeruginosa elaborates a number of extracellular factors, both cellassociated and secreted, which contribute to the establishment, maintenance, and pathogenicity of infection. Colonization of the compromised host requires the function of cell-associated adhesions, which facilitate the attachment of P. aeruginosa to host cells (Woods et al., 1983). Surface components of filamentous fimbriae or pill promote attachment of P. aeruginosa to host epithelia (Woods et al., 1980; 1983). These fimbriae, which are classified as type 4 pill, bind to ganglioside receptors (Strom and Lory, 1993). There is also evidence that non-fimbrial protelnaceous components of the P. aeruginosa cell surface mediate binding to host mucin, as well as to epithelial cells (Simpson et al., 1992; Carnoyetal., 1994).
Secreted Factors Alginate Evidence suggests that the production of an extracellular polysaccharide, called alginate, correlated with the onset of chronic P. aeruginosa infection in the CF lung. Alginate, which lends a mucoid phenotype to P. aeruginosa (Evans and Linker, 1973), contributes to virulence in numerous and various ways. It is believed to act as an adhesion agent in promoting colonization of airways' epithelia (Ramphal et al., 1987). By blocking phagocytosis of P. aeruginosa cells, alginate is also a determinant in the interference with host cellular immune responses (Baltimore and Mitchell, 1980; Simpson et al., 1988). Further, the viscous nature of alginate is believed to contribute to the obstruction and clearance defect seen in the CF lung (Govan and Harris, 1986). Elastases and Alkaline Protease P. aeruginosa also secretes a number of extracellular enzymes and toxins, which potentiate virulence (Liu, 1974; Lory and Tai, 1985; Vasil, 1986). Two elastases, LasB (Schad et al., 1987; Bever and Iglewski, 1988) and LasA (Ohman et al., 1980c; Schad and Iglewski, 1987), combine to degrade elastln and other host proteins. This activity provides a means of nutrient acquisition for the organism and consequently increases the ability of the organism to Invade host tissue (Nicas and Iglewski, 1986). The degradative properties of P. aeruginosa alkaline protease also contribute to the invasiveness of the organism and Increase the organism's ability to evade host immune response by destroying host immunoglobulin, cytokines, and complement factors (Morihara and Homma, 1985; Buret and Cripps, 1993). Phospholipase C Phospholipase C, a hemolysin, degrades phospholipids which are preferentially found in eukaryotic membranes (Berka and Vasil, 1982). Evidence suggests that phospholipase C solubilizes lung tissue, and in concert with alkaline phosphatase, functions in the release of inorganic phosphate from host tissue, which is critical for P. aeruginosa growth (Liu, 1974).
Exoenzyme S P. aeruginosa secretes two ADP-ribosyl transferases, exoenzyme S (Iglewski et al., 1978) and toxin A (exotoxin A) (Iglewski and Kabat, 1975). In in vitro studies, exoenzyme S has been shown to catalytlcally transfer ADP-ribose to a small number of eukaryotic cellular proteins (Coburn et al.. 1989a). Coburn et al. (1989b) suggested that dissociated vimentin is a probable in vivo substrate for exoenzyme ADP-ribosylation. This activity is hypothesized to prevent the assembly of vimentin fibers and thus contribute to an increase in abnormal eukaryotic cellular architecture (Colburn et al., 1989b). Although the role of exoenzyme S in pathogenesis of P. aeruginosa Infections is unclear, it has been shown to be cytolytic and to cause changes In pulmonary structure (Woods, et al., 1988) Exoenzyme S has been suggested to have adhesive properties, as it has been shown to bind to lectins on bucal epithelial cells (Baker et al., 1992) Additionally, exoenzyme S has been implicated in promoting adherence of P. aeruginosa to lung tissue by causing alterations in the pulmonary epithelia (Woods and Sokol, 1985; Woods et al.. 1986). Toxin A About 90% of P. aeruginosa clinical Isolates produce another ADPribosylating enzyme (Bjorn et al., 1977; Pollack et al., 1977), toxin A, which, based on Its LD50 In mice (0.1 )Lyg/kg), Is considered the most toxic of P aeruginosa secreted products (Iglewski and Kabat, 1985). Toxin A, which is genetically distinct from exoenzyme S, enzymatlcally transfers the ADP-ribose moiety of NAD+ to eukaryotic elongation factor 2 (EF2). The addition of ADPribose to the diphthamlde residue of EF2 (Van Ness, 1980), effectively stops eukaryotic protein synthesis and leads to death of susceptible host cells (Iglewski and Kabat, 1975). In humans, antibody titers to toxin A have been correlated to recovery from P. aeruginosa Infection (Klinger et al., 1978; Cross et al., 1980; Jagger et al., 1982). High titres of neutralizing antibodies to toxin A were found in the sera of patients who had recovered from serious P. aeruginosa infections; lower titers were present In the sera of patients with fatal P. aeruginosa infections. In animal studies, the absence of toxin A was correlated with reduced virulence In the burned mouse and the mouse eye models of P. aeruginosa Infections (Ohman et al., 1980b). Evidence suggests that toxin A impedes host immune responses by killing macrophages (Pollack 4
and Anderson, 1978), Inhibiting proliferation of host progenitor cells, and activating host T-suppressor cells (Holt and Misfelt, 1984; 1986). The potential of toxin A as a major virulence factor in severe P. aeruginosa Infections has led to intense scrutiny, at the molecular level, of the structure and function of this protein. Toxin A: Molecular Biology Toxin A Gene (toxA) A single chromosomally located gene encoding toxin A (toxA) has been cloned and the nucleotide sequence determined (Gray et al., 1984). Analysis of its deduced amino acid (a. a.) sequence has revealed that toxin A is synthesized as a 638 a. a. polypeptide (Gray et al., 1984) containing eight cysteine (Cys) residues (Cys-11, Cys-15, Cys-197, Cys-214, Cys-265, Cys-287. Cys-372 and Cys 379) with the potential of forming four dissulflde bonds. The first 25 a. a. of the toxin A amino terminus are typical of those of leader peptides of other exported prokaryotic proteins (Gray et al., 1984). Toxin A: Structure and Function Studies of toxA expression In P. aeruginosa have shown that the 71 kllodalton (kDa) toxin A precursor is processed (the leader peptide cleaved) and the mature 68 kDa enzymatlcally Inactive proenzyme Is released to the extracellular medium (Lory et al., 1983). Although enzymatlcally inactive, native secreted toxin A is both toxic In animal studies and cytotoxic in cell culture (Leppla et al., 1978; Lory and Collier, 1980). Evidence, based on both in vivo and in vitro studies, suggests that the enzymatlcally active region of toxin A is sequestered within the native molecule and is accessible to substrate only after removal of certain regions of toxin A or alteration of native toxin A conformation (Lory and Collier, 1980; Wick et al., 1990a). Reduction of two of four dissulflde bonds and proteolysis, presumably by host enzymes, are both required for activity In vivo. Toxin A enzymatic activity can be achieved in vitro by reduction of dissulflde bonds and denaturation in the presence of urea (Leppla et al., 1978). Immunoblot analysis of supernatants from toxigenic P. aeruginosa strain PA103 revealed a 27kDa toxin A-cross reactive fragment with ADPribosyl transferase activity (Vasil et al., 1977). A similar 26 kDa enzymatlcally active fragment, resulting from limited proteolysis in in vitro studies (Chung and
Collier, 1977; Lory and Collier, 1980), was shown to lack toxicity both in animals and in cell culture (Lory and Tai, 1985). Toxin A is the first bacterial toxin for which a 3-dimensional crystallographic structure was reported (Allured, et al., 1986; Alouf, 1993). Using this crystalline structure, along with the deduced amino acid sequence of toxin A (Gray et al., 1984), Allured et al. (1986) proposed that the molecular structure of the inactive proenzyme form of toxin A consisted of three distinct domains. An amino-terminal domain (domain I), including amino acid residues 1-252 (domain la) and 365-404 (domain lb), is composed primarily of antiparallel p-strands. A central region (domain II) includes amino acids 253-364 in 6 consecutive a-helices. Domain III is composed of amino acids 405-613 of the carboxyl terminus of toxin A and includes an extended cleft region. Resolution of the crystalline structure of toxin A also revealed that the eight Cys residues form four sequential dissulflde bonds (Allured et al., 1986). The first three dissulflde bonds He within domain I, while the fourth is within domain II. The specific biologic functions of the structural domains of toxin A have been determined. Results from several deletion and mutation analyses (Hwang et al., 1987; Guidi-Rontani and Collier, 1987; JInno et al., 1988) have suggested that domain la of toxin A Is required for interaction with and binding to receptors on eukaryotic cells. Similar types of studies have shown that domain II is required for translocation across eukaryotic membranes and entrance into eukaryotic cells (Hwang et al., 1987; Chaudhary et al., 1988; Madshus and Collier, 1989; JInno et al., 1989). Domain III has been demonstrated to be required for enzymatic activity ( Gray et al., 1984; Siegall et al., 1989). Toxin A Production Liu (1973) has shown previously that optimal toxin A production by P. aeruginosa Is controlled by several environmental factors. Toxin A yield Is maximum when toxigenic P. aeruginosa strain PA103 is grown, with aeration, to stationary phase at 32°C in a dialysate of trypticase soy broth supplemented which glycerol and glutamic acid (Liu, 1973). Bjorn et al. (1978) demonstrated that toxin A yields are reduced when more than 5 ^M iron (Fe+3) was added to the growth medium.
Reoulation of toxA At the molecular level, two genes regA [toxR] (Hedstrom et al., 1986; Wozniak et al., 1987) and regB (Wick et al., 1990b), the expressions of which are also affected by iron concentration, have been identified as positive regulators of toxA expression. The expression of regA increases the transcription of toxA (Hindahl et al., 1987; Frank and Iglewski, 1988); however, DNA binding studies have indicated that RegA does not bind directly to toxA (Hamood and Iglewski, 1990). In a recent report. Walker et al. (1994) demonstrated, both by enzyme-linked immunoadsorbant assays and coImmunopreclpltatlon experiments, that RegA (ToxR) binds to purified P. aeruginosa RNA polymerase. Based on their findings, these investigators suggested that the product of regA (RegA) may act as an alternative sigma factor, which with RNA polymerase is required for transcription of toxA . A role for RegB Is unclear, but it is differentially expressed In the hypertoxigenic P. aeruginosa strain PA103. Therefore, re^B expression has been suggested to be required for optimal expression of toxA (Wick et al., 1990b; 1990c). Secretion in Gram-negative Bacteria P. aeruginosa is just one of several Gram-negative pathogens of plants or animals which secrete enzymes or toxins that are crucial for the pathogenesis of these organisms (Hirst and Welch, 1988). Defining the mechanisms by which these proteins reach the extracellular environment has become a focus of much research. To reach the extracellular space. Gramnegative proteins must traverse the cell envelope, which consists of the inner (cytoplasmic) and outer membranes, and the intervening periplasm. While the mechanisms by which these proteins are secreted vary, there are two secretion pathways which are widely conserved among Gram-negative bacteria (Hirst and Welch, 1988; Lory, 1992; Salmond and Reeves, 1993). Single-step Pathwav In one conserved secretion pathway, proteins are targeted to the extracellular environment by a single step mechanism (Tommassen et al., 1992; Lory, 1992; Salmond and Reeves, 1993). These proteins, the prototype of which is hemolysin (HlyA) of E. coli, lack an amino-terminal signal peptide and cross the cytoplasmic and outer membranes simultaneously, apparently
bypassing the periplasmic space (Holland et al., 1990). Secretion via the single-step pathway requires the function of accessory genes which are usually contiguous with the secreted protein's structural gene (Mackman et al., 1985; Letoffe et al., 1990; Wandersman et al., 1992). General Secretory Pathway A diverse group of secreted proteins of Gram-negative bacteria utilizes an Independent general secretory pathway (GSP), in which access to the extracellular environment is gained in two discrete stages (Lory, 1992; Reeves and Salmond, 1993; Pugsley, 1993). First, these proteins, bearing aminoterminal signal peptides, engage the translocation machinery of the cytoplasmic membrane, which facilitates their processing (cleavage of the signal peptide) and export to the periplasm. In a second step, the proteins are translocated across the outer membrane and are released to the medium. Successful negotiation of the respective membrane at each step of this pathway depends on the recognition of targeting signals within the secreted molecule (intragenic factors) by components of the bacterial secretion machinery (extragenic factors) (Lory, 1992; Reeves and Salmond, 1993; Pugsley. 1993). Translocation Across the Cytoplasmic Membrane The first step (breaching the cytoplasmic membrane) is believed to proceed similarly to the sec-dependent export of periplasmic and outer membrane proteins of E. coli. (Tommassen et al., 1992; Lory, 1992; Pugsley, 1993) The components of the cytoplasmic membrane translocation apparatus are well defined in E. coli and consist of several seo-encoded proteins (Sec), plus two cytoplasmic membrane-bound leader peptidases (LepI, Lepll) (Schatz and Beckwith, 1990). Translocation Across the Outer Membrane The terminal leg of the GSP (translocation across the outer membrane) appears to require the specific interactions between determinants within the mature protein and components of the outer membrane translocation machinery (Lory. 1992; Pugsley, 1993). This second step in the GSP has been most extensively studied in Klebsiella oxytoca, which secretes the starch degrading enzyme pullulanase. Pugsley and coworkers (Pugsley et al.. 1991; Pugsley, 8
1993) have isolated a cluster of 13 genes (pulC-pulO), the functions of which are necessary for the translocation of pullulanase across the outer membrane of K. oxytoca. Genes, which are homologous to the pul accessory genes in both gene arrangement and nucleotide sequence, have been identified as requisites of translocation of proteins across the outer membrane of a wide variety of Gram-negative bacteria (Pugsley et al, 1991; He et al., 1991: Dums et al., 1991; Jiang and Howard; 1992; Reeves et al., 1993). Despite their requirement for outer membrane translocation, the majority of the products of these genes have thus far been localized to the cytoplasmic membrane (Pugsley, 1993). Single-step Secretion in Pseudomonas aeruginosa Evidence suggests that both the single-step secretion pathway and the GSP exist In P. aeruginosa. Alkaline protease of P. aeruginosa lacks a leader peptide and appears to be translocated to the medium via a single-step pathway, which is functionally similar to that described for hemolysin of E. coli (Wretlind and Pavlovskis, 1984; Filloux et al., 1987; Guzzo et al.. 1991). The secretion of alkaline protease, like that of hemolysin, appears to require the function of four genes (apri, aprD, aprE, and aprF), three of which are located immediately upstream from the alkaline protease structural gene (Lazdunski et al., 1991; Duong etal., 1992). Toxin A Secretion via the General Secretory Pathway in Pseudomonas aeruginosa Translocation of Toxin A Across the Cytoplasmic Membrane Extragenic Requirements The extragenic factors of P. aeruginosa which facilitate the export of toxin A to the periplasm have yet to be identified. When toxin A is expressed in E. coli, however. It is processed and exported to the periplasm in a secdependent manner (Douglas et al., 1987). Thus, translocation of toxin A across the P. aeruginosa cytoplasmic membrane is believed to proceed similarly to the sec^medlated export of periplasmic and outer membrane proteins of E coli.
Intragenic Requirements: Toxin A Leader Peptide In accordance with the GSP model, the leader peptide is required for translocation of toxin A across the cytoplasmic membrane. The toxin A leader peptide Is similar to those of Gram-negative proteins exported to the periplasm and beyond (Hamood et al., 1990; Pugsley, 1989, 1993; Lory. 1992). These leader peptides, which are typically 15-30 a. a. in length, show little primary sequence homology but possess three common features: (1) a positively charged amino terminus; (2) followed by a central hydrophobic core; and (3) a consensus leader peptidase recognition site at the carboxyl terminus (Bever and Iglewski, 1988; Huang and Schell, 1990; He et al., 1991). Evidence from numerous genetic and biochemical studies of these regions, primarily in E. coli, have suggested several functions for leader peptides in the export of proteins including: (1) targeting of precursors to cytoplasmic membrane translocation machinery; (2) facilitating the translocation of proteins across cytoplasmic membrane; and (3) providing peptidase recognition and cleavage sites for processing of precursors to mature forms (von Heinje, 1985; Puziss et al., 1989; Randall and Hardy, 1989; Hikita and Mizushlma, 1992). Further, an interaction of the leader peptide with the mature region of the molecule or with another protein (chaperonin) within the cytoplasm, has been suggested to contribute to the maintenance of a translocation-competent conformation of the precursor (Randall and Hardy, 1986). Translocation of Toxin A Across the Outer Membrane Extragenic Requirements The terminal leg of the GSP (translocation across the outer membrane) appears to require specific interactions of determinants within mature toxin A with components of the outer membrane translocation machinery. Wretlind and coworkers (Wretlind et al., 1977) isolated several P. aeruginosa mutants (extracellular grotein deficient, xcp) which were pleiotropically defective in the secretion of toxin A, elastase, phospholipase C and alkaline phosphatase. The secretion of alkaline protease was not affected (Wretlind et al., 1977). These mutations, which resulted in the periplasmic accumulation of exoproteins, mapped to chromosomal locations not linked to those of the structural genes of elastase or toxin A (Wretlind and Pavlovskis, 1984). These findings led investigators to suggest that the xcp genes participate in a common secretion 10
mechanism, and their function is required for translocation of proteins across the outer membrane of P. aeruginosa (Wretlind and Pavlovskis; Filloux et al.. 1990). To date, at least 12 different xcp genes have been identified: xcpP-Zare located In the 40-min region of the chromosome (Bally et al., 1988; Filloux et al.. 1989); and, xcpA, (Ballyetal., 1991) also called pilD (Nunn and Lory, 1991), Is located at 70 min. The xcp genes are similar in nucleotide sequence, and to a lesser extent. In gene arrangement to the extragenic requirements for secretion in K. oxytoca (pul) (Pugsley et al., 1991; Pugsley, 1993), Erwinia chrysanthemi (He et al., 1991) and Erwinia carotovora (out) (Reeves et al., 1993). Aeromonas hydrophila (exe) (Jiang and Howard, 1992), and Xanthomonas campestris (xps) (Dums etal., 1991). xcpA (pilD). Thus far, of the xcp genes, only xcpA (pilD) has been definitively defined as to biochemical function (Nunn and Lory, 1991; Bally et al., 1992). The xcpA gene, which is identical to the pilD gene, is a cytoplasm leal ly located prepllin peptidase required for the processing of the type IV pilin precursors found in P. aeruginosa. The XcpA peptidase also specifically cleaves the leader peptides from the products of xcpT-W, which share significant homology to the type IV pllins (Bally et al., 1992; Nunn and Lory, 1992; 1993). None of the secreted proteins of P. aeruginosa bear leader peptides which are similar to the pilin precursor. Further, examination of the periplasmic forms of these proteins in pilD mutants revealed cleaved mature proteins (Strom et al., 1991). Based on these findings, Strom and Lory (1991) suggested that PilD acts on components of the secretion machinery of P. aeruginosa, rather than on the secreted proteins themselves. xcpT-W. As noted above, xcpT-W dse similar to type IV pllins, which are found in several Gram-negative pathogens, including Neisseria gonorrhoeae (Meyer et al., 1984) and Vibrio cholerae (Taylor et al., 1987). This homology is most apparent at the amino termini of these proteins, especially in the leader peptides and the first 25-30 amino acids of the mature molecules (Nunn and Lory, 1991). The products of xcpT-W (XcpT-W) have been localized to the cytoplasmic membrane of P. aeruginosa (Nunn and Lory, 1993); however, the function of these proteins and the significance of their similarity to type IV pilins are poorly understood. XcpT-W are homologues of PulG-J, which are required for secretion of pullulanase In K. oxytoca (Pugsley, et al., 1991). Pugsley (1993) has suggested that these proteins may participate in a pllus-IIke structure 11
(pseudopllus) which might span the periplasm between the inner and outer membranes. Such a structure is further suggested to function as a ladder to target proteins to the outer membrane (Pugsley. 1993). However, a subsequent examination, which suggests that PulG is not part of a stable multiprotein complex, does not support this theory (Pugsley and Possot, 1993). xcpR. Based on the deduced amino acid sequence, XcpR lacks an obvious leader peptide (Bally et al., 1992). It has thus been proposed to reside in the P. aeruginosa cytoplasm (Tommassen et al., 1992). XcpR possesses a highly conserved ATP-blndIng domain, "Walker box A" (Walker et al., 1982), the presence of which has been shown to be necessary for the secretion of toxin A (Turner et al., 1993). The requirement for this putative ATP-bindIng site was taken as an Indication that, by hydrolyzing ATP, XcpR functions in providing energy for the translocation of proteins across the outer membrane. Intragenic Requirements In contrast to the extensively studied extragenic factors of the GSP of P. aeruginosa, relatively little Is known of signals within extracellular proteins required to target these molecules across the outer membrane. To date, the only P. aeruginosa exoprotein examined in this regard has been toxin A. In a previous analysis of toxin A secretion, Hamood et al. (1989) examined the secretion of several toxin A deletion derivatives In P. aeruginosa. They found that a toxin A amino-terminal truncated derivative, consisting of the toxin A leader peptide and the first 223 a. a. of the mature toxin, was detected in the supernatant. A toxA Internal deletion product, consisting of the leader peptide fused with the first 30 a. a. and the final 305 a. a. of the mature toxin was also detected In the P. aeruginosa supernatant. By contrast, a carboxy-terminal derivative, consisting of only the final 305 a. a. of the mature toxin was confined to the cellular lysate. Based on their results, these authors suggested that requirements for toxin A secretion were contained within the toxin A aminoterminal region. Moreover, it was suggested that these secretion requirements may reside In a region confined by the leader peptide and the first 30 a. a. of mature toxin A. Applying the GSP model to these conclusions Implies that the signal required for translocation of toxin A across the outer membrane of P. aeruginosa resides in the first 30 a. a. of the mature protein. 12
This interpretation of these data needs to be reevaluated on two counts. First, these experiments may have been compromised by the addition of extra copies of the toxA positive regulatory gene. regA (Hedstrom et al., 1986). To achieve detectable levels of toxin A-cross reactive molecules (CRMs), Hamood et al. (1989) overexpressed the toxA subclones by the inclusion of a multi-copy plasmid containing regA. In a subsequent analysis, however, when extra copies of regA were introduced into a previously described toxin A secretion mutant (Hamood et al., 1992), toxin A was detected in the supernatant (Hamood and Iglewski, unpublished results). This suggested that regA itself may be involved, directly or Indirectly, in toxin A secretion. Second, and more importantly, the ability of the leader peptide and the first 30 a. a. of the mature toxin to be secreted was not tested: a product consisting of the toxin A leader peptide and the first 30 a. a. was not detectable In P. aeruginosa (Hamood et al., 1989). In addition, all stable internal deletion products, detected by these investigators in the P. aeruginosa supernatant. Included a substantial portion of the toxin A carboxyl terminus. Thus, a contribution of this carboxyl region to toxin A translocation across the outer membrane could not be excluded. The Purpose of the Study The goal of the research described herein has been to further define the localization of the signals within toxin A which target the molecule to the extracellular environment of P. aeruginosa. Specifically, this research addresses the following objectives: (1) To examine in P. aeruginosa the secretion of products of toxA deletion subclones expressed from the E coli lac promoter; (2) To examine in P. aeruginosa the secretion of hybrid proteins encoded by fusions of toxA and heterologous genes; (3) To generate and Isolate In-frame bi-directional deletions within the toxA region encoding the leader peptide and the first 30 a. a. of mature toxin A. using exonuclease 8 a ^ 1 ; and (4) To determine the localization of the 8aA31-generated toxA subclone products in the cellular fractions of E. coli and P aeruginosa.
13
CHAPTER II MATERIALS AND METHODS General Bacterial Strains Bacterial strains and relevant genotypes are listed In Table 2.1. Plasmid DNA was maintained and amplified in Escherichia coli DH5a (Clontech. Palo Alto, CA). DH5a competent cells were used as hosts for recombinant plasmids containing toxA deletion subclones. E. coli MM294 containing helper plasmid pRK2013 (FIgurskI and HelinskI, 1979) was used in the mobilization of recombinant plasmids to P. aeruginosa. E. coli K38 containing plasmid pG1-2 (Russell and Model, 1985; Tabor and Richardson, 1985) was used for T7 expression experiments. Pseudomonas aeruginosa wild-type (WT) strain PAK, and mutant strains PA103-NT (Kllleen and Collier, 1992) and XG9 (Nunn and Lory. 1992) were kind gifts from Stephen Lory (University of Washington, Seattle, WA). PA103-NT, derived from prototroph PA103, is a toxA insertion mutant which produces no detectable ADP-ribosyl transferase activity nor toxin A-cross reactive molecules (CRMs). XG-9, an xcpT-W deletion mutant of P. aeruginosa strain PAK, is defective In the secretion of toxin A, elastases, phopholipase C, and alkaline phosphatase (Nunn and Lory, 1992). ADD1976 is a P. aeruginosa T7 expression system strain which contains a chromosomal copy T7 RNA polymerase gene expressed from the lacR promoter (A. Darzins, Brunschwig and Darzins, 1992). Plasmid Vectors Plasmid vectors used in our study are shown In Table 2.2. Plasmids pUC19. pUC18 (Yanish-Peron et al., 1985) and pKS+ (Strategene Cloning Systems, La Jolla, CA) were used for routine cloning vectors and as vectors for double-stranded DNA sequencing experiments. Plasmid pAM41 contains intact toxA from P. aeruginosa strain PA103 on a 2.4-kilobase (kb) EcoRV-EcoRI fragment cloned Into the Smal-EcoRI sites of pUC19 (Hamood et al., 1990). Plasmid pAM21 contains intact toxA from P. aeruginosa strain PA01 similarly cloned Into pUC19 (Hamood et al., 1990). In both pAM41 and pAM21, toxA Is expressed from the E. coli lac promoter. Plasmid pMJ21 contains intact toxA from PA01 on a 2.4-kb EcoRV-EcoRI fragment cloned In to the Smal-EcoRI sites 14
Table 2.1.
Bacterial strains.
Strain or plasmid
Relevant genotype or phenotype
Source and/or reference
Strains E. coli DH5a
^ /
^
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'
VX
B. H. Iglewski; Bever and Iglewski, 1988 Hamood etal., 1990 This laboratory Hamood etal., 1990 Hamood etal., 1989 Hamood etal., 1989
of pUC18 (Hamood et al.. 1990). Plasmid pAH102 (Hamood et al.. 1989) contains a 957-base pair (bp) Sa/-EcoRI carboxy-terminal fragment of toxA cloned into the Sa/l-EcoRI sites of pUC19. The toxA subclone in pAH102 is also expressed from the lac promoter. Plasmid pRK2013 served as a helper plasmid in triparental mating experiments (FIgurski and HelinskI, 1979; Ditta et al., 1980). Plasmid pKT230, a derivative of broad host range plasmid RSF1010, was used in constructing recombinant plasmids which can replicate stably in P. aeruginosa (Bagdasarian and Timmis, 1982). Plasmid pR01614 (Olson et al., 1982) Is a recombinant derivative of plasmid pBR322 containing the origin of replication gene of the broad host range plasmid RP1 on a 1.8-kb Psfl fragment. The presence of this fragment In ColEI plasmids enables them to replicate stably in P. aeruginosa. Plasmid pMH220 (Hindahl et al., 1987) was used as a source for P. aeruginosa regA (Hedstrom et al., 1986) gene for gene fusion experiments. Plasmids pT7-7 and pGP1-2 were gifts from Stanley Tabor (Harvard University, Boston, MA) and were used In T7 expression system experiments (Tabor and Richardson, 1985). Plasmid pGP1-2 Is a derivative of pBR322 which contains the T7 RNA polymerase gene expressed from the \ Pi promoter. Plasmid T7-7 contains the T7 RNA polymerase promoter _ - ^ ,
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