Biogenesis of the bundle-forming pilus of enteropathogenic Escherichia coli: reconstitution of fimbriae in recombinant E. coli and role of DsbA in pilin stability – a review1

Gene 192 (1997) 33–38

Biogenesis of the bundle-forming pilus of enteropathogenic Escherichia coli: reconstitution of fimbriae in recombinant E. coli and role of DsbA in pilin stability – a review1 Michael S. Donnenberg *, Hong-Zhong Zhang 2, Kelly D. Stone Division of Infectious Diseases, Department of Medicine, University of Maryland School of Medicine, 10 S. Pine Street, MSTF 900, Baltimore, MD 21201, USA Received 22 March 1996; accepted 19 April 1996; Received by P.A. Manning

Abstract Enteropathogenic Escherichia coli (EPEC ) adhere to tissue culture cells in a distinct pattern known as localized adherence (LA). We have defined two loci necessary for LA. A plasmid-encoded gene cluster encodes bundlin, the major structural subunit of a type-IV fimbria called the bundle-forming pilus (BFP), a prepilin peptidase necessary for processing of pre-bundlin to its mature form, and twelve other proteins. Under the control of an exogenous promoter, these 14 genes are sufficient for the biogenesis of BFP in a heterologous E. coli host. The chromosomal gene dsbA, which encodes a periplasmic disulfide-bond oxidoreductase, is also required for LA. In the absence of DsbA protein, bundlin is made but rapidly degraded. Pre-bundlin is also rapidly degraded in the absence of DsbA, suggesting that the prepilin is a transcytoplasmic protein simultaneously accessible to enzymes on both sides of the inner membrane. These studies offer a fresh perspective on the biogenesis of type-IV pili. © 1997 Elsevier Science B.V. Keywords: Adherence; Prepilin peptidase; Disulfide bond; TnphoA; Protein stability; Operon; DNA probe

1. Introduction Enteropathogenic Escherichia coli (EPEC ) are a leading cause of severe diarrhea in infants throughout the developing world (Donnenberg, 1995). The pathogenesis of EPEC infection encompasses three distinct stages: * Corresponding author. Tel. +1 410 7067560; Fax +1 410 7068700; e-mail: [email protected] 1 Presented at the Workshop on ‘Type-4 Pili – Biogenesis, Adhesins, Protein Export, and DNA Import’, Schloss Ringberg, Germany, 26–29 November 1995. 2 Present address: Department of Growth and Development, University of California San Francisco, San Francisco, CA 94143, USA. Abbreviations: aa, amino acid(s); BFP, bundle-forming pilus; bfp, gene(s) involved in BFP biogenesis; bp, base pair(s); dsbA, gene encoding DsbA; DsbA, disulfide bond protein A; EAF, EPEC adherence factor; EPEC, enteropathogenic E. coli; IPTG, isopropyl b--thiogalactopyranoside; kb, kilobase(s) or 1000 bp; LA, localized adherence; nt, nucleotide(s); ORF, open reading frame; P., Pseudomonas; re-, recombinant; TCP, toxin co-regulated pilus; TM, transmembrane (domain); Tn, transposon; V., Vibrio; wt, wild type; b, novel junction (insertion). 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved PII S 03 7 8 -1 1 1 9 ( 9 6 ) 0 0 8 26 - 8

initial adherence, signal transduction, and intimate attachment (Donnenberg and Kaper, 1992). The second and third stages result in a dramatic interaction with host cells characterized by loss of microvilli, deposition of cytoskeletal proteins and formation of a cup-like pedestal upon which the bacteria rest ( Knutton et al., 1987). This effect, known as attaching and effacing (Moon et al., 1983), is associated with phosphorylation of host cell tyrosine kinase substrates ( Rosenshine et al., 1992) and requires the genes encoded on an EPEC chromosomal pathogenicity island known as the locus of enterocyte effacement (McDaniel et al., 1995). The first stage of the interaction between EPEC and host cells, known as localized adherence (LA) because of the tight bacterial clusters formed by EPEC on the surface of cells (Scaletsky et al., 1984), is associated with a type-IV fimbria and will be the focus of this review. Baldini et al. (1983) first reported that LA is associated with the presence of a large (ca. 95-kb) plasmid. They found that a plasmid-cured EPEC strain is incapable of LA, while a laboratory E. coli strain transformed with the plasmid acquired the ability to adhere to cells in this fashion. The term ‘EPEC adherence factor’

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( EAF ) was used to describe the hypothetical ligand responsible for LA. A link between EAF plasmids and virulence was demonstrated in a landmark volunteer trial when two of nine volunteers fed a plasmid-cured strain developed diarrhea in comparison to nine of ten fed the wt parental strain (Levine et al., 1985). Nataro et al. (1987) found that two cloned regions of the EAF plasmid, represented by plasmids pJPN14 and pJPN101, could confer the ability to perform LA upon a laboratory E. coli strain when present simultaneously, though neither plasmid had such activity alone. Giro´n et al. (1991) first reported that EPEC are capable of producing a type-IV fimbria under certain conditions. Since these fimbriae have a tendency to aggregate into rope-like bundles, they were named bundle-forming pili (BFP). BFP have been observed exclusively in EPEC that possess the EAF plasmid. Expression of BFP and of the LA phenotype is induced by growth in tissue culture medium ( Vuopio-Varkila and Schoolnik, 1991; Donnenberg et al., 1992). The propensity of the pili for aggregation led to the suggestion that BFP might tether the bacteria to each other as well as to the epithelial cell, thus promoting the LA phenotype (Giro´n et al., 1991; Vuopio-Varkila and Schoolnik, 1991). In our analysis of non-invasive EPEC TnphoA mutants, we found two categories of mutants that are deficient in LA, but capable of subsequent steps in pathogenesis (Donnenberg et al., 1990). One group is composed of seven mutants, each with a transposon insertion within a 500-bp region of the EAF plasmid. This region is included in pJPN101, one of the two plasmids that together reconstitute LA in re-strain of E. coli (Nataro et al., 1987). The other category of noninvasive mutants deficient in LA includes four mutants with insertions in the EPEC chromosome. Studies of these two categories of mutants led to the identification of the bfp gene cluster required for BFP biogenesis and to characterization of the role of DsbA in BFP biogenesis.

2. The bfpA gene encodes bundlin, a plasmid-encoded type-IV pilin The nt sequence of the TnphoA junctions cloned from the seven LA-deficient mutants with EAF plasmid insertions revealed that in each mutant the transposon had inserted into the gene encoding the major structural subunit of BFP. Thus the gene was named bfpA (Donnenberg et al., 1992). Bundlin, the product of bfpA, has the characteristic features of a type-IV pilin including a short, basic, hydrophilic leader sequence; a Gly preceding and Glu 5 aa following the leader peptide processing site; two Cys residues near the C terminus; and aa sequence similarities with other members of the type-IV fimbria family. The closest relative of bfpA is

the tcpA gene of Vibrio cholerae. The presence in prebundlin of a somewhat longer leader peptide than is typical for type-IV pilins, and the absence of a terminal Phe following the processing site define BFP as a class-B type-IV fimbria (Hobbs and Mattick, 1993). The sequence of bfpA from a second, distantly related, EPEC strain is identical to the one we reported (Sohel et al., 1993), a result that suggests very recent horizontal spread of the EAF plasmids carrying bfpA genes and one that is encouraging for future vaccine development. Hybridization studies using a bfpA DNA probe revealed that the gene is universal among EPEC strains that perform LA (Giro´n et al., 1993). The high sensitivity and specificity of the probe suggest that it may be useful for the diagnosis of EPEC infection.

3. BfpP, a prepilin peptidase Attempts to complement bfpAbTnphoA mutants to restore LA revealed that plasmids containing as much as 8.5 kb of DNA downstream from bfpA do not restore LA, while plasmids containing 9.9 kb do restore LA. Interestingly, immunoblots of bfpAbTnphoA mutants containing a non-complementing plasmid with 4.5 kb of downstream DNA revealed that bundlin, the product of the bfpA gene, migrates slower than does bundlin from wt EPEC, suggesting that it is not processed in the mutant containing the 4.5-kb plasmid. In contrast, bundlin from bfpAbTnphoA mutants containing a noncomplementing plasmid with 8.5 kb of downstream DNA runs as fast as does the protein from wt EPEC. These observations led to the hypothesis that bfpA is part of a large operon and that a prepilin peptidase gene might reside on the same operon at a distance 4.5–8.5 kb downstream from bfpA. To test the hypothesis that a prepilin peptidase is encoded downstream from bfpA, we cloned several fragments from this vicinity into an E. coli-Pseudomonas aeruginosa shuttle vector and mobilized the constructs via triparental conjugation into a P. aeruginosa strain that contains a mutation in the pilD gene encoding the prepilin peptidase. Exconjugants were tested for sensitivity to bacteriophage P04, which requires intact fimbriae for productive infection. We found that a 1.3-kb fragment located 7.7 kb downstream from bfpA complemented the pilD mutant to restore P04 sensitivity. The nt sequence of this fragment revealed a 747-bp ORF that predicts a protein with 30% aa sequence identity to the product of pilD as well as similar degrees of identity to other prepilin peptidases. We named this locus bfpP, for bundle forming prepilin peptidase (Zhang et al., 1994). To further investigate functional homology with the prepilin peptidase of P. aeruginosa, we introduced the bfpA gene on a shuttle vector into a wt P. aeruginosa strain and the pilD mutant. Immunoblots of whole cell

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lysates of these constructs with anti-bundlin antiserum revealed a protein with an M of 19 000 in the wt strain, r and one of 21 000 in the pilD mutant, consistent with bundlin and pre-bundlin, respectively. No proteins were detected in control strains containing vector alone. Thus the bfpP gene of EPEC encodes a prepilin peptidase functionally homologous to that of P. aeruginosa.

4. A type-IV fimbria gene cluster sufficient for BFP biogenesis A series of complementation studies allowed us to define the minimum DNA fragment required for biogenesis of a type-IV fimbria in an E. coli re-strain (Stone et al., 1996). A fragment of 11 565 bp confers LA and BFP production on a laboratory E. coli strain provided that it is present together with pJPN14, one of the two plasmids described by Nataro et al. (1987) as required for LA (see Section 1). An E. coli strain containing the same fragment without pJPN14 is not capable of LA or BFP production. Immunoblots using anti-bundlin antiserum revealed that the strain containing both plasmids produces much more bundlin than does the strain lacking pJPN14, suggesting that pJPN14 encodes functions that increase transcription or translation of bfpA or increase stability of bundlin. To determine whether these functions can be substituted by an exogenous promoter, the bfp cluster was cloned under the control of the IPTG-inducible trc promoter. A laboratory E. coli strain containing this construct is capable of producing BFP in the presence of IPTG, but not in its absence ( Fig. 2). Thus, for the first time, biogenesis of a type-IV fimbria has been demonstrated in an E. coli re-strain using a defined set of genes. However, the E. coli re-strain containing this fragment is not capable of LA even in the presence of IPTG, indicating that additional functions encoded by pJPN14 are also required for LA. One possibility is that an adhesin, distinct from bundlin, is encoded on pJPN14. Alternatively, the level of BFP expression directed by the trc construct may not be sufficient for adherence, or another function encoded by pJPN14 is required for localization, modification, or correct conformation of the actual adhesin. DNA sequencing and T7 promoter directed protein expression of the fragment sufficient for LA and BFP biogenesis in conjunction with pJPN14, revealed that

Fig. 1. Physical and genetic map of the bfp gene cluster. Numbers on the scale indicate kb. Selected features of each protein are described in Table 1. Only the closest relative identified in protein databases is noted ( X=any aa).

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this fragment contains 14 genes ( Fig. 1 and Table 1). These genes encode bundlin (BfpA), the prepilin peptidase (BfpP), three additional potential prepilin peptidase substrates (BfpIJK ), two potential ATP-binding proteins (BfpD and BfpF ), and a potential cytoplasmic TM protein (BfpE). All of the foregoing have homologues in other type-IV pilus biogenesis systems. The cluster also encodes BfpB, a protein related to the MshD protein from V. cholerae, which is encoded within a locus required for mannose sensitive hemagglutination ( Ha¨se et al., 1994), and BfpH, which resembles the product of the trbN gene of the broad-host-range transmissible plasmid RP4 (Lessl et al., 1992). No function has yet been assigned to trbN, but the Tra2 locus within which this gene resides is necessary for formation of mating pairs and could therefore be related to pilus biogenesis (Lessl et al., 1992). The BfpG and BfpB proteins have signal peptidase II processing sites, suggesting that they are lipoproteins, while the BfpU protein has a signal peptidase I processing site. BfpG, BfpC, BfpU and BfpL lack homologues in the databases. A plasmid containing bfpA-bfpK, but lacking bfpL does not confer the ability for LA or BFP biogenesis on a laboratory E. coli strain containing pJPN14, indicating that the BfpL protein is required for these phenotypes (Stone et al., 1996). The demonstration that 14 genes are sufficient for assembly of a type-IV pilus in an E. coli re-strain should facilitate a detailed mutational analysis of the role of each gene product in fimbria biogenesis. Furthermore, this system may be useful for assembly of heterologous pili in E. coli for the purposes of producing re-pilus proteins, for studies of protein interactions, and for vaccine development.

5. DsbA is required for stability of bundlin Analysis of the second category of non-invasive EPEC TnphoA mutants deficient in LA revealed that they all have insertions in the chromosomal dsbA gene. Furthermore, a plasmid containing the cloned dsbA gene of E. coli K-12 can complement each of the mutants, confirming that the dsbA gene is necessary for LA ( Zhang and Donnenberg, 1996). The product of this gene is a periplasmic enzyme required for efficient disulfide bond formation in numerous extracytoplasmic proteins (Bardwell et al., 1991; Kamitani et al., 1992). Not surprisingly, E. coli strains with mutations at this locus, or other species with mutations in homologues, have numerous phenotypes. Among these phenotypes are reduced activity of alkaline phosphatase (Bardwell et al., 1991; Kamitani et al., 1992), resistance to F-pilusspecific bacteriophages (Bardwell et al., 1991), defective assembly of flagellae (Dailey and Berg, 1993), reduced transcription of outer membrane proteins (Pugsley,

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Table 1 Gene product

Size (bp)

Distinguishing features

Closest relative

(% identity; No. aa)

Pre-bundlin (BfpA) BfpG BfpB BfpC BfpU BfpD BfpE BfpF BfpP BfpH BfpI BfpJ BfpK BfpL

20.3 14.8 58.3 45.3 17.7 60.4 39.9 35.1 28.0 16.4 20.1 20.1 18.1 16.8

Prepilin peptidase cleavage site Signal peptidase II cleavage site Signal peptidase II cleavage site None Signal peptidase I cleavage site Nucleotide-binding sites TM domains Nucleotide-binding sites CXXC motifs, TM domains Signal peptidase II cleavage site Prepilin peptidase cleavage site Prepilin peptidase cleavage site Prepilin peptidase cleavage site Hydrophobic N terminus

V. cholerae TcpA None V. cholerae MshD None None V. cholerae TcpT V. cholerae TcpE V. cholerae PilT V. cholerae TcpJ Plasmid RP4 TrbN None None None None

(30% 180 aa)

Fig. 2. BFP production in an E. coli re-host containing the bfp gene cluster under the control of an inducible promoter. E. coli HB101 was transformed with a plasmid containing the bfpA-L genes under the control of a trc promoter. BFP were noted only after induction with IPTG. The white bar at the bottom of the figure indicates 200 nm. Reprinted with permission from Stone et al. (1996).

1993), reduced adherence and cholera toxin secretion in V. cholerae (Peek and Taylor, 1992; Yu et al., 1992), absence of transformation in Haemophilus influenzae ( Tomb, 1992), and reduced secretion of cellulase in Erwinia spp. (Bortoli-German et al., 1994). As bundlin contains two Cys residues, it is possible

(24% 184 aa)

(26% (24% (32% (32% (33%

353 353 248 249 114

aa) aa) aa) aa) aa)

that DsbA is required for these aa to form a disulfide bond. To investigate this possibility bundlin was examined by immunoblot from wt and dsbA mutant EPEC. These studies revealed that bundlin is present at extremely low levels in dsbA mutants, but at normal levels after complementation of the mutants with a plasmid containing the intact dsbA gene, indicating that bundlin is either rapidly degraded or produced at lower levels in a dsbA mutant. b-Galactosidase activities of wt and dsbA mutants containing a bfpAblacZ fusion are equivalent, indicating that the decreased bundlin levels in dsbA mutants are not a result of decreased transcription. To determine whether failure of disulfide bond formation results in decreased bundlin levels, EPEC strains were created that have mutations in bfpA resulting in replacement of either Cys with Ser. Bundlin levels are markedly reduced in both mutants. Pulse-chase experiments confirmed that DsbA is required for pilin stability. These experiments revealed that pre-bundlin is rapidly degraded in a dsbA mutant and that in such mutants very little reduced pre-bundlin can be chased into mature oxidized bundlin. Furthermore, immunoblots of pre-bundlin from strains with or without dsbA mutations that lack the bfpP prepilin peptidase gene, reveal that pre-bundlin levels are also dependent on DsbA. This result, together with the pulse chase experiments showing degradation of pre-bundlin in the absence of DsbA, indicate that the DsbA enzyme is capable of acting on the periplasmic C terminus of the pre-pilin independently of the prepilin peptidase, which cleaves the N-terminal signal sequence in the cytoplasm. Thus during its processing pre-pilin exists as a cytoplasmic TM protein accessible to both enzymes simultaneously (Fig. 3). Our results, indicating that in the absence of DsbA the pilin protein is rapidly degraded, contrast with those of Peek and Taylor (1992), who studied the effect of a tcpG (V. cholerae dsbA homologue) mutation on the toxin co-regulated pilus ( TCP). These authors found

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Fig. 3. Model for post-translational processing of bundlin. Pre-bundlin is represented by three segments: a hydrophilic signal sequence (solid), a hydrophobic TM mature N terminus (striped), and a C terminus containing both Cys residues (cross-hatched). OM and IM represent the outer and inner (cytoplasmic) membranes. (1) Pre-bundlin is directed to the cytoplasmic membrane by its hydrophobic N terminus while the C terminus is exposed to the periplasmic space. (2) Disulfide-bond formation is catalyzed by the DsbA protein and the prepilin peptidase enzyme BfpP removes the hydrophilic signal peptide in reactions that proceed simultaneously and independently. In the absence of the DsbA protein (3), bundlin is rapidly degraded.

that TCP are still produced in a tcpG mutant, but that the tcpG mutant is deficient in hemagglutination and colonization of mice, phenotypes attributed to TCP. The authors postulated that the pilus assumes an abnormal conformation in the absence of TcpG that is responsible for the lack of hemagglutination and colonization. The different effects of DsbA on two similar type-IV pili could reflect differences in the pilins themselves, differences between EPEC and V. cholerae proteases, or differences in the kinetics of pilus biogenesis and disulfide bond formation in the two species.

6. Conclusions (1) Bundlin, the major structural subunit of a type-IV fimbria of EPEC, is encoded by the bfpA gene on the EAF plasmid and is ubiquitous among EPEC strains. (2) A prepilin peptidase encoded by the bfpP gene downstream from bfpA is functionally homologous to the PilD protein of P. aeruginosa. (3) The 14-gene bfp cluster is sufficient for the biogenesis of a type-IV pilus in an E. coli re-host and contains homologues of genes previously described in other type-IV pilus systems as well as novel genes thus far unique to BFP biogenesis. (4) The DsbA enzyme is required for stability of bundlin.

(5) Studies of the effect of a dsbA mutation in the absence of bfpP reveal that the pre-pilin molecule is a cytoplasmic TM protein accessible to processing enzymes in both the cytoplasm and periplasm.

Acknowledgement These studies were supported by the Office of Research and Development, Medical Research Service, Department of Veterans’ Affairs, by Public Health Service award AI37606 from the National Institutes of Health, and by a Designated Research Initiative Fund award from the University of Maryland.

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