Identification of Secretion Determinants of the Bordetella pertussis BrkA Autotransporter
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3*6')0d?, 百拇医药
Received 8 August 2002/ Accepted 28 October 2002*6')0d?, 百拇医药
ABSTRACT*6')0d?, 百拇医药
The autotransporters comprise a functionally diverse family of gram-negative proteins that mediate their own export across the bacterial outer membrane. They consist of an amino-terminal passenger region called the "-domain" and the structural hallmark of the autotransporter family, a carboxy-terminal transporter region usually referred to as the "ß-domain." The passenger region can be quite diverse and constitutes the effector functions of these proteins, whereas the C-terminal region is conserved and is responsible for translocating the passenger moiety across the outer membrane. BrkA is the 103-kDa autotransporter protein in Bordetella pertussis that is cleaved to yield a 73-kDa N-terminal -domain and a 30-kDa C-terminal ß-domain. We have previously shown that a recombinant form of the ß-domain of BrkA is capable of forming channels in artificial membranes. Here, we define two additional secretion determinants of BrkA. N-terminal sequencing of the 73-kDa BrkA passenger from B. pertussis and Escherichia coli revealed that BrkA has a 42-amino-acid signal peptide. In addition, deletion analysis of BrkA identified a 31- to 39-amino-acid region found immediately upstream of the ß-domain that was essential for surface expression. This 31- to 39-amino-acid linker region, together with the ß-domain, defines the minimal BrkA translocation unit. The linker region may also serve to anchor the BrkA passenger to the bacterial surface.
INTRODUCTION?jy, 百拇医药
Autotransporters are outer membrane proteins found in gram-negative bacteria. They are multidomain proteins composed of an N-terminal signal sequence to direct translocation across the inner membrane, a passenger region to be delivered to the cell surface, and a conserved C-terminal transporter region that is proposed to mediate secretion across the outer membrane (9). Most autotransporters are proteolytically processed close to the transporter region to yield an domain, which comprises most of the passenger region, and the remaining fragment called the ß-domain, which encompasses the transporter.?jy, 百拇医药
Several hundred autotransporters can now be identified from the growing list of completed bacterial genomes. They represent putative virulence factors (8), possible vaccine candidates (33), and an efficient way to display heterologous proteins (18), including antigenic determinants (14), important enzymes (16), heavy metal-detoxifying agents (32), and platforms for steroid biosynthesis (10). A detailed understanding of autotransporter secretion mechanisms not only will shed light on the biological problem of traversing the outer membrane, seemingly in one step, but also will contribute to the improved engineering of autotransporters for specific biotechnological purposes. At the moment, it is not known whether or what parts of the autotransporter secretion process (9, 26, 35) apply universally to all autotransporters.
Bordetella pertussis is the gram-negative mucosal pathogen that causes whooping cough (17). The autotransporter protein BrkA is a B. pertussis virulence factor that confers serum resistance and also acts as an adhesin (4). BrkA expression is controlled by a sensor kinase response regulator system called "Bvg" (37). Consistent with the model of autotransporter secretion, BrkA is expressed as a 103-kDa precursor that is processed during secretion to yield a 73-kDa N-terminal -domain and a 30-kDa C-terminal ß-domain (28). Following translocation, the cleaved -domain remains tightly associated with the bacterial surface and is not detected in B. pertussis culture supernatants (24). The processed ß-domain has been isolated from B. pertussis outer membrane fractions, and the processing site has been determined to occur between residues Asn731 and Ala732 (25).|, 百拇医药
In an effort to elucidate the mechanism(s) of autotransporter secretion, we have begun to characterize BrkA secretion. We have shown that a recombinant form of the C-terminal region of BrkA encompassing the ß-domain has the capacity to form channels with a conductivity of 3.2 nS in planar lipid bilayer experiments (28). Such a channel would be sufficient to translocate an unfolded or partially unfolded passenger past the outer membrane. Here we report the characterization of additional regions within BrkA that are necessary for secretion. We show that BrkA has a 42-amino-acid signal peptide to traverse the inner membrane, and we define the minimal translocation unit necessary to mediate secretion of the BrkA passenger to the bacterial surface.
MATERIALS AND METHODS6], http://www.100md.com
Bacterial strains and plasmids and growth conditions. The bacterial strains and plasmids used in this study are listed in E. coli strains were cultured at 37°C on Luria broth or Luria agar supplemented with the appropriate antibiotics. B. pertussis strains were maintained on Bordet-Gengou (BG) medium (BBL, Cockeysville, Md.) containing 15% sheep's blood (RA Media, Calgary, Alberta, Canada), as previously described (4). When necessary, the following antibiotics were added at the indicated concentrations to the media: nalidixic acid, 30 µg/ml; kanamycin, 50 µg/ml; ampicillin, 100 µg/ml; and gentamicin, 10 µg/ml.6], http://www.100md.com
fig.ommitted6], http://www.100md.com
Strains and plasmids used in this study6], http://www.100md.com
Recombinant DNA techniques. All DNA manipulations were carried out by standard techniques (27). Restriction enzymes were purchased from New England Biolabs (Beverly, Mass.). The primers used in this study were purchased either from the University of British Columbia (UBC) Nucleic Acid Protein Services Unit (Vancouver), Sigma-Genosys (The Woodlands, Tex.), or Alpha DNA (Montreal, Quebec, Canada) . DNA sequencing was done with an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, Calif.) at the UBC Nucleic Acid Protein Services Unit.
fig.ommitted8@($;{, http://www.100md.com
Primers used in this study8@($;{, http://www.100md.com
The B. pertussis strain BBC9DO was made by introducing a second copy of the brkA gene (on plasmid pUW2171) into the chromosome of strain BBC9, a pertactin mutant of B. pertussis, as described previously (5).8@($;{, http://www.100md.com
Construct pDO6935, which constitutively expresses low levels of BrkA in E. coli, was derived by excision of a 476-bp AatII fragment of pRF0166. Plasmid pDO6935 was used as a template in all subsequent PCRs described in this study. All PCRs were performed with Vent polymerase (New England BioLabs) with the following cycles: an initial denaturation step of 2 min at 94°C followed by 30 cycles of 45 s at 94°C, 30 s at 60°C, and 1 min/kb at 72°C. The last cycle was followed by an additional 10 min at 72°C. Amplified PCR products were separated on an agarose gel, and a band of the expected size was extracted and cloned as described below. The primers used in this study are listed in.
Construct pDO181 was made by PCR with primer pairs DO1210F and DO1614R and DO2894F and BRK-CR. The resulting products were digested with restriction enzyme pairs AscI and XbaI and XbaI and BamHI, respectively. In a triple-ligation reaction, these products were ligated into a 5-kb AscI- to BamHI-digested fragment of pDO6935 to yield pDO181. Construct pDO182 was generated via the same strategy with primer sets DO1210F and DO1893R and DO2894F and BRK-CR. Constructs pDO244 and pDO246 were made with primer pair DO1975F and BRK-CR to generate a PCR product that was subsequently digested with AscI and BamHI. The resulting 1.3-kb product was then ligated into either a 5.3-kb XbaI- to BamHI-digested fragment of pDO181 or a 5.5-kb XbaI- to BamHI-digested fragment of pDO182 to yield pDO244 and pDO246, respectively.:2{u|, http://www.100md.com
Constructs pGD1, pGD2, pGD3, pGD4, pGD5, pGD6, pGD7, pGD8, pGD9, pGD10, pGD10.5, pGD11, and pGD12 were made by PCR with forward primers BRK-2113F, BRK-2398F, BRK-2650F, BRK-2752F, BRK-2821F, BRK-2890F, BRK-3010F, BRK-3184F, BRK-3238F, BRK-3289F, BRK-3310F, BRK-3370F, and BRK-3601F, respectively. BRK-CR was used as the reverse primer in each of the reactions. The amplified products were purified, digested with XbaI and HindIII, and ligated into a 4.3-kb XbaI- to BamHI-digested fragment of pDO246.
SDS-PAGE and immunoblot analysis. For detection of expressed BrkA via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or immunoblotting, E. coli cultures were grown to an optical density at 600 nm (OD600) of 0.7 and pelleted. Trypsin accessibility experiments were performed according to a previously described protocol (18) with slight modifications. In brief, cell pellets were resuspended in 0.2 ml of phosphate-buffered saline (PBS) to an OD600 of ~ 10. To 0.1 ml of cells, 2 µl of 10-mg/ml trypsin was added to yield a final trypsin concentration of 200 µg/ml. Cells were incubated in the presence of protease for 10 min at 37°C, pelleted by centrifugation, and washed three times with PBS containing 10% fetal calf serum to stop digestion and once in PBS alone. As a control, cell pellets were simultaneously processed in the same manner in the absence of trypsin. Washed pellets were finally resuspended in sample buffer and immediately boiled for 5 min prior to SDS-PAGE.
For immunoblot analysis, samples were resolved by SDS-PAGE (4, 15) and transferred to Immobilon-P membranes (Millipore, Etobicoke, Ontario, Canada) as described previously (24). Blots were probed with heat-inactivated rabbit anti-BrkA antiserum and horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (ICN Biomedicals, Costa Mesa, Calif.) diluted 1/50,000 and 1/10,000, respectively (24). Renaissance chemiluminescent reagent (NEN Life Science Products, Boston, Mass.) was used to develop immunoblots. The rabbit anti-BrkA antiserum is specific for residues Met1 to Glu693 of BrkA (24). Molecular masses were determined with Kaleidoscope prestained markers purchased from Bio-Rad (Hercules, Calif.).-0, http://www.100md.com
N-terminal sequencing. Whole-cell lysates of strains BBC9DO (a pertactin [prn] mutant with two copies of brkA), and BBC9BrkA (a prn brkA double mutant) (4) were resolved by SDS-PAGE and transferred to an Immobilon-P membrane (Millipore). A unique band migrating at approximately 73 kDa in the BBC9DO lane was excised from the membrane and sequenced by Edman degradation by the UBC Nucleic Acid and Protein Services core facility.
Immunofluorescence analysis. E. coli cells were grown to an OD600 of 0.7, pelleted by centrifugation, and resuspended in PBS. Resuspended cells were immobilized on a glass slide that had been previously treated with 0.1% poly-L-lysine (Sigma). Slides were washed three times with PBS to remove unbound bacteria and subsequently probed with a 1/200 dilution of heat-inactivated rabbit anti-BrkA antiserum (24) and a 1/100 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, Pa.), respectively. Slides were washed three times with PBS containing 1% bovine serum albumin between each step to remove unbound material. Bacteria were visualized under epifluorescence with a Zeiss Axioscop-2 microscope. Phase-contrast and fluorescent images were captured digitally.qh), http://www.100md.com
RESULTS AND DISCUSSIONqh), http://www.100md.com
Identification of the BrkA signal peptide. It was previously reported that sequence analysis of the 1,010-amino-acid protein BrkA did not identify a conventional signal peptide (4). More recent analysis with SignalP V2.0 (22) has predicted a signal peptide of 44 amino acids by the neural network prediction method and a cleavage site at 43 amino acids by the hidden Markov model method (23). To experimentally determine the BrkA signal peptide, N-terminal sequencing was performed with the 73-kDa moiety of BrkA. The amount of BrkA seen in whole-cell lysates of B. pertussis represents a small fraction of the total amount of cellular protein. Furthermore, at 73 kDa, BrkA migrates to a similar position on SDS-PAGE gels as the 69-kDa protein pertactin, a protein with which it shares sequence identity (4). To circumvent these issues, we introduced a second copy of the brkA gene into the chromosome of strain BBC9, a pertactin mutant of B. pertussis, to create strain BBC9DO. Western blot analysis of this strain with antibodies to pertactin and BrkA confirmed the lack of expression of pertactin and the increased expression of BrkA relative to that in wild-type strains (data not shown). Whole-cell lysates of strain BBC9DO were resolved by SDS-PAGE and transferred to an Immobilon-P membrane, and a unique band migrating at approximately 73 kDa was excised and sequenced by Edman degradation. Six cycles of Edman degradation revealed an N-terminal sequence of QAPQA. This sequence corresponds to amino acids 43 through 47 of BrkA. Similar results were obtained with a recombinant brkA construct expressed in E. coli (data not shown). Thus, both in B. pertussis and in E. coli, BrkA is processed between residues Ala42 and Gln43. A signal peptide of this length is not unusual for autotransporters (9).
Expression of BrkA in E. coli. We chose to study BrkA secretion in E. coli, since it has been used as a host to study secretion of a variety of autotransporters (11-13, 18, 21, 29, 31, 34, 35), thus allowing comparisons to be made between different autotransporters and because mutational analysis of BrkA is greatly facilitated in E. coli. Plasmid pDO6935 was derived from pRF1066 (4), which carries the entire brk locus encoding the divergently transcribed brkA and brkB genes (Table 1). pDO6935 was generated by excision of a 476-bp AatII fragment from pRF1066, resulting in a deletion of the 5' region of the brkB gene. pDO6935 was transformed into E. coli strain UT5600, which is deficient for the outer membrane proteases OmpT and OmpP (7). UT5600 has been used in the past to study secretion of the Neisseria immunoglobulin A (IgA) protease (11, 34, 35), the E. coli AIDA-1 adhesin (18, 19), and the Shigella VirG (IcsA) autotransporters (31). BrkA expression was assessed by a previously described anti-BrkA polyclonal antiserum (24) that specifically recognizes both denatured and native forms of the 73-kDa BrkA -domain. Immunoblots of whole-cell lysates resolved by SDS-PAGE show that BrkA was expressed to yield two major species migrating at approximately 103 and 73 kDa. The 103-kDa product corresponds to the unprocessed full-length precursor and the species migrating at 73 kDa corresponds to the cleaved -domain lane 1). Although BrkA is Bvg regulated in B. pertussis, the promoter region responsible for driving BrkA expression from pDO6935 in E. coli is not known. We previously reported that the overexpression of full-length BrkA in E. coli is toxic (24); however, in the absence of IPTG induction, the levels of BrkA expression in E. coli with this construct are similar to those seen in B. pertussis (4, 24).
fig.ommittedrd, 百拇医药
BrkA expression in E. coli strain UT5600. (A) BrkA domain organization: signal peptide (SP [residues 1 to 42]), passenger or -domain (residues 43 to 731), and ß-domain (residues 732 to 1010). (B) Western immunoblot of E. coli UT5600 whole-cell lysates resolved by SDS-PAGE (11% polyacrylamide), probed with anti BrkA antiserum, and detected with goat anti-rabbit antiserum conjugated to horseradish peroxidase. Lanes: 1 and 2, pDO6935 (wild-type copy of brkA gene); 3 and 4, pBluescript (vector control). Specific BrkA bands are indicated. U, unprocessed 103-kDa precursor protein; *, 73-kDa processed passenger moiety. Cells were processed in the presence or absence of trypsin as described in Materials and Methods. (C) Surface expression of BrkA in E. coli UT5600 detected via indirect immunofluorescence. The top panels show phase-contrast images, and the bottom panels show epifluorescence images.rd, 百拇医药
To determine whether BrkA is translocated to the surface of E. coli, trypsin accessibility and immunofluorescence experiments were performed with whole cells. When cells were incubated with trypsin, a marked decrease in the 73-kDa moiety was observed, and two products of approximately 40 and 45 kDa were detected by Western immunoblotting. The cleavage sites producing the 40- and 45-kDa species are unknown, and over time, both species are lost. The intensity of the 103-kDa product remained constant following trypsin digestion, suggesting that the 103-kDa band represents an intracellular form of the protein inaccessible to trypsin. Concomitant with this result, BrkA was detected on the surface of E. coli (Fig. 1C) and appeared evenly distributed, as shown by indirect immunofluorescence staining. Secreted BrkA could not be detected in concentrated culture supernatants, suggesting that the cleaved passenger remains noncovalently associated with the bacterium (data not shown). Taken together, these data indicate that BrkA is exported to the surface of E. coli strain UT5600 and is processed (independently of proteases OmpT or OmpP) in a manner similar to that observed in B. pertussis (24).
Identification of the minimal BrkA translocation unit necessary for surface expression. The natural cleavage of three well-characterized autotransporters, IgA protease (11), VirG/IcsA (6), and AIDA-1 (30), results in ß-domains of 45, 37, and 48 kDa, respectively. By using a series of protease susceptibility assays and experiments with heterologous proteins fused to N-terminally-truncated ß-domains, minimal regions necessary to display passenger proteins have been identified for these autotransporters. They have in common, a membrane-embedded ß-core of 25 to 30 kDa found at the extreme C terminus, preceded by a so-called "linker" region (11, 19, 31). In these autotransporters, the linker region has been shown to be necessary for the translocation of the passenger domain to the bacterial surface. The linker region together with the outer membrane-embedded ß-core make up what has been coined the "translocation unit" (19).;p3o7d&, http://www.100md.com
Having demonstrated that BrkA is targeted to the outer membrane of E. coli, we next developed a deletion-based strategy to define the boundaries of the minimal translocation unit of BrkA. N-terminal sequencing of proteins from outer membrane preparations of B. pertussis has localized the processing of BrkA to between Asn731 and Ala732 (25), resulting in a ß-domain of 30 kDa (28). At 30 kDa, the BrkA ß-domain is smaller than the ß-domains for IgA protease, VirG/IcsA, and AIDA-1, but it approaches the size of the outer membrane-embedded ß-cores identified for these proteins (11, 19, 31). We constructed a series of brkA deletion mutants by using PCR mutagenesis. As shown in mutant proteins consisted of the first 228 amino acids of BrkA (Met1 to Gly228) fused in frame to processive deletions of the C-terminal region of the BrkA -domain leading into the BrkA ß-domain. BrkA (Met1 to Gly228) was chosen as a passenger, since heterologous passengers such as cholera toxin B subunit (12) may be inefficiently translocated due to structural limitations (i.e., disulfide bond formation). In addition, it has been suggested that the extended signal sequences observed in many autotransporters may play a role in secretion (9). Therefore, the inclusion of the native BrkA signal sequence within the passenger avoids any influence that a nonnative signal sequence may have on secretion. All deletion strains were derivatives of pDO6935, thereby ensuring a common promoter for the wild-type and mutant constructs (Table 1).
fig.ommitted$!2, 百拇医药
Expression of BrkA deletion constructs in E. coli UT5600. (A) Diagram illustrating positions of BrkA in-frame deletions. Deleted regions are indicated by dotted lines, and deletion boundaries correspond to the wild-type BrkA amino acid designation. The BrkA domain structure is described in Construction of mutations is described in the Materials and Methods. Plasmids are described in (A) pGD1. (B) pGD2. (C) pGD3. (D) pGD4. (E) pGD5. (F) pGD6. (G) pGD7. (H) pGD8. (I) pGD9. (J) pGD10. (K) pGD10.5. (L) pGD11. (M) pGD12. E. coli UT5600 bacteria were transformed with BrkA deletion constructs (plasmids A to M) and grown to an OD600 of approximately 0.7. Bacteria were harvested, and BrkA surface expression was assessed by immunoblotting or indirect immunofluorescence. (B) Immunoblotting following resolution of whole-cell lysates by SDS-PAGE. The band migrating within the region denoted as "U" in each lane corresponds to the unprocessed, precursor form of BrkA, and the band denoted with an asterisk (*) corresponds to the processed passenger domain of BrkA. Cells were processed in the presence (+) or absence (-) of trypsin as described in Materials and Methods. Molecular mass markers (in kilodaltons) are indicated to the left of the panel. (C) BrkA expression in E. coli strain UT5600 detected by indirect immunofluorescence. The top panels show phase-contrast images, and the bottom panels show epifluorescence images.
An attempt was made to target our deletions to regions that would not disrupt the core structure of the protein. Secondary structural analysis with PsiPred (20) predicts that BrkA is predominantly composed of ß-strands (data not shown). In addition, the closest relative to BrkA in the database is the B. pertussis autotransporter pertactin (4). The structure of the pertactin passenger domain has been solved and was shown to be a monomer folded into a single domain that is almost entirely made up of a right-handed cylindrical ß-helix (3). Given that BrkA and pertactin passenger domains share 27% sequence identity and 39% sequence similarity, we refined our secondary structural prediction by overlaying the pertactin structural coordinates (1DABA) onto a BrkA-pertactin primary amino acid sequence alignment. The best alignment was between Arg175 to Pro572 in pertactin and Val301 to Gln707 in BrkA. Using this analysis, we systematically targeted N-terminal deletions to intervening regions with the predicted ß-strands.
The effects of each deletion on BrkA expression and processing were assessed by immunoblotting of whole-cell lysates resolved by SDS-PAGE. As shown in , each mutant form of BrkA was expressed, indicating that the specific deletions did not render the individual mutant protein products markedly unstable. In deletion mutants A through J, products corresponding to both the unprocessed precursor (region designated as "U") and the cleaved passenger (asterisk) were detected . In contrast, only the unprocessed precursor could be detected in deletion mutants K, L, and M. Given our previous observation that the cleaved passenger domain represents a major fraction of the surface-expressed wild-type BrkA (Fig. 1), these data suggested that BrkA deletion mutants A through J were being exported to the bacterial surface, but mutants K, L, and M were not. In support of this observation, trypsin accessibility assays and indirect immunofluorescence experiments were performed. As expected, exposure of whole cells to trypsin digestion resulted in the complete absence of the band corresponding to the processed passenger domain (lanes A to J), whereas a significant fraction of the unprocessed precursor remained stable lanes A to M). Consistent with these data, surface expression of the passenger region was detected via indirect immunofluorescence in mutants A through J, but not in mutants K, L, and M . The absence of immunofluorescence in mutants K, L, and M supports the tenet that the unprocessed, trypsin-resistant fraction of BrkA represents an intracellular form of BrkA, and not simply a trypsin-resistant surface molecule. It should be noted that a deletion (Ala136-Pro255) within the BrkA passenger (Met1 to Gly228) construct used for mutants A to M did not affect surface expression of BrkA (data not shown). Collectively, these data show that the region spanning residues Ala136 to Glu693 of BrkA is not required for surface localization of passenger proteins in E. coli strain UT5600. Furthermore, since the processed form of the passenger is also evident in deletion constructs A to J as well as construct Ala136-Pro255, it argues against BrkA having autoproteolytic activity.
Our data indicate that the ß-domain of BrkA is itself insufficient to translocate a passenger to the cell surface. The minimal translocation unit for BrkA thus consists of the ß-core plus a preceding linker region, the N-terminal boundary of which maps within Glu693 to Ser701. Historically, the ß-domain has been defined as the C-terminal outer membrane resident fragment derived from proteolytic processing of the autotransporter protein. Although the ß-domains of IgA protease (11), VirG/IcsA (31), and AIDA-1 (19) are larger than the ß-domain of BrkA, the sizes of their minimal translocation units are remarkably similar. Indeed, a comparison of experimentally defined linkers in four diverse autotransporters including BrkA reveals a structurally conserved architecture that can be viewed as a signature for autotransporters. It consists of a 21- to 30-amino-acid -helical region that precedes a 255- to 294-amino-acid transporter domain, a region rich in ß structure . It has been proposed that the linker region is involved in forming a hairpin-like structure that leads secretion of the passenger domain through the channel formed by the ß-core (9). The common features of the translocation unit suggest that it, rather than the ß-domain, is a more appropriate operational definition for the transporter domain. The region upstream of the translocation unit would thus constitute the passenger moiety regardless of the positioning of the proteolytic processing sites .
fig.ommitted4kj#, 百拇医药
Comparison of the translocation units of different autotransporters. The C-terminal regions of four autotransporters are shown (not drawn to scale). See the text for explanation. The N-terminal boundaries noted for each translocation unit have been defined experimentally in references 11 (IgA protease), 31 (VirG/IcsA), and 19 (AIDA-1), as well as in this paper (BrkA).4kj#, 百拇医药
IgA protease, VirG/IcsA, and AIDA-1 are naturally cleaved well upstream of the predicted -helical region and either can be released naturally (6, 26) or can be induced to be released following heat treatment (1). Unlike these proteins, BrkA is steadfastly associated with the bacterial outer membrane both in B. pertussis and in E. coli and cannot be released by heat treatment (G. Huang and R. Fernandez, unpublished data). Cleavage of BrkA occurs within the predicted -helical region. Thus, it is possible that the linker region also acts as the anchor (11) for BrkA, since none of the deletion mutant proteins spanning Ala136 to Glu693 was detected in immunoblots of concentrated culture supernatants (data not shown).
In summary, we have shown that the B. pertussis autotransporter BrkA can be surface expressed in E. coli, enabling dissection of autotransporter secretion mechanisms in a host more amenable to genetic manipulation. Adding to our previous studies on the BrkA ß-domain (28), which we demonstrated has the capacity to form a channel, we have identified two additional secretion determinants of BrkA: a 42-amino-acid signal peptide and a 30- to 39-amino-acid region preceding the ß-domain that, together with the ß-domain, defines the BrkA translocation unit . The data presented provide further experimental support for the importance of the predicted -helical region in autotransporter secretion of both native and heterologous passengers (12, 19, 31).4me'y, 百拇医药
ACKNOWLEDGMENTS4me'y, 百拇医药
We thank V. deLorenzo and L. Fernandez for the gift of UT5600 and UT2300. D.C.O. was a recipient of a University of British Columbia graduate student fellowship.
This work was funded by a grant from the Natural Sciences and Engineering Research Council of Canada.$.\v, 百拇医药
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Received 8 August 2002/ Accepted 28 October 2002*6')0d?, 百拇医药
ABSTRACT*6')0d?, 百拇医药
The autotransporters comprise a functionally diverse family of gram-negative proteins that mediate their own export across the bacterial outer membrane. They consist of an amino-terminal passenger region called the "-domain" and the structural hallmark of the autotransporter family, a carboxy-terminal transporter region usually referred to as the "ß-domain." The passenger region can be quite diverse and constitutes the effector functions of these proteins, whereas the C-terminal region is conserved and is responsible for translocating the passenger moiety across the outer membrane. BrkA is the 103-kDa autotransporter protein in Bordetella pertussis that is cleaved to yield a 73-kDa N-terminal -domain and a 30-kDa C-terminal ß-domain. We have previously shown that a recombinant form of the ß-domain of BrkA is capable of forming channels in artificial membranes. Here, we define two additional secretion determinants of BrkA. N-terminal sequencing of the 73-kDa BrkA passenger from B. pertussis and Escherichia coli revealed that BrkA has a 42-amino-acid signal peptide. In addition, deletion analysis of BrkA identified a 31- to 39-amino-acid region found immediately upstream of the ß-domain that was essential for surface expression. This 31- to 39-amino-acid linker region, together with the ß-domain, defines the minimal BrkA translocation unit. The linker region may also serve to anchor the BrkA passenger to the bacterial surface.
INTRODUCTION?jy, 百拇医药
Autotransporters are outer membrane proteins found in gram-negative bacteria. They are multidomain proteins composed of an N-terminal signal sequence to direct translocation across the inner membrane, a passenger region to be delivered to the cell surface, and a conserved C-terminal transporter region that is proposed to mediate secretion across the outer membrane (9). Most autotransporters are proteolytically processed close to the transporter region to yield an domain, which comprises most of the passenger region, and the remaining fragment called the ß-domain, which encompasses the transporter.?jy, 百拇医药
Several hundred autotransporters can now be identified from the growing list of completed bacterial genomes. They represent putative virulence factors (8), possible vaccine candidates (33), and an efficient way to display heterologous proteins (18), including antigenic determinants (14), important enzymes (16), heavy metal-detoxifying agents (32), and platforms for steroid biosynthesis (10). A detailed understanding of autotransporter secretion mechanisms not only will shed light on the biological problem of traversing the outer membrane, seemingly in one step, but also will contribute to the improved engineering of autotransporters for specific biotechnological purposes. At the moment, it is not known whether or what parts of the autotransporter secretion process (9, 26, 35) apply universally to all autotransporters.
Bordetella pertussis is the gram-negative mucosal pathogen that causes whooping cough (17). The autotransporter protein BrkA is a B. pertussis virulence factor that confers serum resistance and also acts as an adhesin (4). BrkA expression is controlled by a sensor kinase response regulator system called "Bvg" (37). Consistent with the model of autotransporter secretion, BrkA is expressed as a 103-kDa precursor that is processed during secretion to yield a 73-kDa N-terminal -domain and a 30-kDa C-terminal ß-domain (28). Following translocation, the cleaved -domain remains tightly associated with the bacterial surface and is not detected in B. pertussis culture supernatants (24). The processed ß-domain has been isolated from B. pertussis outer membrane fractions, and the processing site has been determined to occur between residues Asn731 and Ala732 (25).|, 百拇医药
In an effort to elucidate the mechanism(s) of autotransporter secretion, we have begun to characterize BrkA secretion. We have shown that a recombinant form of the C-terminal region of BrkA encompassing the ß-domain has the capacity to form channels with a conductivity of 3.2 nS in planar lipid bilayer experiments (28). Such a channel would be sufficient to translocate an unfolded or partially unfolded passenger past the outer membrane. Here we report the characterization of additional regions within BrkA that are necessary for secretion. We show that BrkA has a 42-amino-acid signal peptide to traverse the inner membrane, and we define the minimal translocation unit necessary to mediate secretion of the BrkA passenger to the bacterial surface.
MATERIALS AND METHODS6], http://www.100md.com
Bacterial strains and plasmids and growth conditions. The bacterial strains and plasmids used in this study are listed in E. coli strains were cultured at 37°C on Luria broth or Luria agar supplemented with the appropriate antibiotics. B. pertussis strains were maintained on Bordet-Gengou (BG) medium (BBL, Cockeysville, Md.) containing 15% sheep's blood (RA Media, Calgary, Alberta, Canada), as previously described (4). When necessary, the following antibiotics were added at the indicated concentrations to the media: nalidixic acid, 30 µg/ml; kanamycin, 50 µg/ml; ampicillin, 100 µg/ml; and gentamicin, 10 µg/ml.6], http://www.100md.com
fig.ommitted6], http://www.100md.com
Strains and plasmids used in this study6], http://www.100md.com
Recombinant DNA techniques. All DNA manipulations were carried out by standard techniques (27). Restriction enzymes were purchased from New England Biolabs (Beverly, Mass.). The primers used in this study were purchased either from the University of British Columbia (UBC) Nucleic Acid Protein Services Unit (Vancouver), Sigma-Genosys (The Woodlands, Tex.), or Alpha DNA (Montreal, Quebec, Canada) . DNA sequencing was done with an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, Calif.) at the UBC Nucleic Acid Protein Services Unit.
fig.ommitted8@($;{, http://www.100md.com
Primers used in this study8@($;{, http://www.100md.com
The B. pertussis strain BBC9DO was made by introducing a second copy of the brkA gene (on plasmid pUW2171) into the chromosome of strain BBC9, a pertactin mutant of B. pertussis, as described previously (5).8@($;{, http://www.100md.com
Construct pDO6935, which constitutively expresses low levels of BrkA in E. coli, was derived by excision of a 476-bp AatII fragment of pRF0166. Plasmid pDO6935 was used as a template in all subsequent PCRs described in this study. All PCRs were performed with Vent polymerase (New England BioLabs) with the following cycles: an initial denaturation step of 2 min at 94°C followed by 30 cycles of 45 s at 94°C, 30 s at 60°C, and 1 min/kb at 72°C. The last cycle was followed by an additional 10 min at 72°C. Amplified PCR products were separated on an agarose gel, and a band of the expected size was extracted and cloned as described below. The primers used in this study are listed in.
Construct pDO181 was made by PCR with primer pairs DO1210F and DO1614R and DO2894F and BRK-CR. The resulting products were digested with restriction enzyme pairs AscI and XbaI and XbaI and BamHI, respectively. In a triple-ligation reaction, these products were ligated into a 5-kb AscI- to BamHI-digested fragment of pDO6935 to yield pDO181. Construct pDO182 was generated via the same strategy with primer sets DO1210F and DO1893R and DO2894F and BRK-CR. Constructs pDO244 and pDO246 were made with primer pair DO1975F and BRK-CR to generate a PCR product that was subsequently digested with AscI and BamHI. The resulting 1.3-kb product was then ligated into either a 5.3-kb XbaI- to BamHI-digested fragment of pDO181 or a 5.5-kb XbaI- to BamHI-digested fragment of pDO182 to yield pDO244 and pDO246, respectively.:2{u|, http://www.100md.com
Constructs pGD1, pGD2, pGD3, pGD4, pGD5, pGD6, pGD7, pGD8, pGD9, pGD10, pGD10.5, pGD11, and pGD12 were made by PCR with forward primers BRK-2113F, BRK-2398F, BRK-2650F, BRK-2752F, BRK-2821F, BRK-2890F, BRK-3010F, BRK-3184F, BRK-3238F, BRK-3289F, BRK-3310F, BRK-3370F, and BRK-3601F, respectively. BRK-CR was used as the reverse primer in each of the reactions. The amplified products were purified, digested with XbaI and HindIII, and ligated into a 4.3-kb XbaI- to BamHI-digested fragment of pDO246.
SDS-PAGE and immunoblot analysis. For detection of expressed BrkA via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or immunoblotting, E. coli cultures were grown to an optical density at 600 nm (OD600) of 0.7 and pelleted. Trypsin accessibility experiments were performed according to a previously described protocol (18) with slight modifications. In brief, cell pellets were resuspended in 0.2 ml of phosphate-buffered saline (PBS) to an OD600 of ~ 10. To 0.1 ml of cells, 2 µl of 10-mg/ml trypsin was added to yield a final trypsin concentration of 200 µg/ml. Cells were incubated in the presence of protease for 10 min at 37°C, pelleted by centrifugation, and washed three times with PBS containing 10% fetal calf serum to stop digestion and once in PBS alone. As a control, cell pellets were simultaneously processed in the same manner in the absence of trypsin. Washed pellets were finally resuspended in sample buffer and immediately boiled for 5 min prior to SDS-PAGE.
For immunoblot analysis, samples were resolved by SDS-PAGE (4, 15) and transferred to Immobilon-P membranes (Millipore, Etobicoke, Ontario, Canada) as described previously (24). Blots were probed with heat-inactivated rabbit anti-BrkA antiserum and horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (ICN Biomedicals, Costa Mesa, Calif.) diluted 1/50,000 and 1/10,000, respectively (24). Renaissance chemiluminescent reagent (NEN Life Science Products, Boston, Mass.) was used to develop immunoblots. The rabbit anti-BrkA antiserum is specific for residues Met1 to Glu693 of BrkA (24). Molecular masses were determined with Kaleidoscope prestained markers purchased from Bio-Rad (Hercules, Calif.).-0, http://www.100md.com
N-terminal sequencing. Whole-cell lysates of strains BBC9DO (a pertactin [prn] mutant with two copies of brkA), and BBC9BrkA (a prn brkA double mutant) (4) were resolved by SDS-PAGE and transferred to an Immobilon-P membrane (Millipore). A unique band migrating at approximately 73 kDa in the BBC9DO lane was excised from the membrane and sequenced by Edman degradation by the UBC Nucleic Acid and Protein Services core facility.
Immunofluorescence analysis. E. coli cells were grown to an OD600 of 0.7, pelleted by centrifugation, and resuspended in PBS. Resuspended cells were immobilized on a glass slide that had been previously treated with 0.1% poly-L-lysine (Sigma). Slides were washed three times with PBS to remove unbound bacteria and subsequently probed with a 1/200 dilution of heat-inactivated rabbit anti-BrkA antiserum (24) and a 1/100 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, Pa.), respectively. Slides were washed three times with PBS containing 1% bovine serum albumin between each step to remove unbound material. Bacteria were visualized under epifluorescence with a Zeiss Axioscop-2 microscope. Phase-contrast and fluorescent images were captured digitally.qh), http://www.100md.com
RESULTS AND DISCUSSIONqh), http://www.100md.com
Identification of the BrkA signal peptide. It was previously reported that sequence analysis of the 1,010-amino-acid protein BrkA did not identify a conventional signal peptide (4). More recent analysis with SignalP V2.0 (22) has predicted a signal peptide of 44 amino acids by the neural network prediction method and a cleavage site at 43 amino acids by the hidden Markov model method (23). To experimentally determine the BrkA signal peptide, N-terminal sequencing was performed with the 73-kDa moiety of BrkA. The amount of BrkA seen in whole-cell lysates of B. pertussis represents a small fraction of the total amount of cellular protein. Furthermore, at 73 kDa, BrkA migrates to a similar position on SDS-PAGE gels as the 69-kDa protein pertactin, a protein with which it shares sequence identity (4). To circumvent these issues, we introduced a second copy of the brkA gene into the chromosome of strain BBC9, a pertactin mutant of B. pertussis, to create strain BBC9DO. Western blot analysis of this strain with antibodies to pertactin and BrkA confirmed the lack of expression of pertactin and the increased expression of BrkA relative to that in wild-type strains (data not shown). Whole-cell lysates of strain BBC9DO were resolved by SDS-PAGE and transferred to an Immobilon-P membrane, and a unique band migrating at approximately 73 kDa was excised and sequenced by Edman degradation. Six cycles of Edman degradation revealed an N-terminal sequence of QAPQA. This sequence corresponds to amino acids 43 through 47 of BrkA. Similar results were obtained with a recombinant brkA construct expressed in E. coli (data not shown). Thus, both in B. pertussis and in E. coli, BrkA is processed between residues Ala42 and Gln43. A signal peptide of this length is not unusual for autotransporters (9).
Expression of BrkA in E. coli. We chose to study BrkA secretion in E. coli, since it has been used as a host to study secretion of a variety of autotransporters (11-13, 18, 21, 29, 31, 34, 35), thus allowing comparisons to be made between different autotransporters and because mutational analysis of BrkA is greatly facilitated in E. coli. Plasmid pDO6935 was derived from pRF1066 (4), which carries the entire brk locus encoding the divergently transcribed brkA and brkB genes (Table 1). pDO6935 was generated by excision of a 476-bp AatII fragment from pRF1066, resulting in a deletion of the 5' region of the brkB gene. pDO6935 was transformed into E. coli strain UT5600, which is deficient for the outer membrane proteases OmpT and OmpP (7). UT5600 has been used in the past to study secretion of the Neisseria immunoglobulin A (IgA) protease (11, 34, 35), the E. coli AIDA-1 adhesin (18, 19), and the Shigella VirG (IcsA) autotransporters (31). BrkA expression was assessed by a previously described anti-BrkA polyclonal antiserum (24) that specifically recognizes both denatured and native forms of the 73-kDa BrkA -domain. Immunoblots of whole-cell lysates resolved by SDS-PAGE show that BrkA was expressed to yield two major species migrating at approximately 103 and 73 kDa. The 103-kDa product corresponds to the unprocessed full-length precursor and the species migrating at 73 kDa corresponds to the cleaved -domain lane 1). Although BrkA is Bvg regulated in B. pertussis, the promoter region responsible for driving BrkA expression from pDO6935 in E. coli is not known. We previously reported that the overexpression of full-length BrkA in E. coli is toxic (24); however, in the absence of IPTG induction, the levels of BrkA expression in E. coli with this construct are similar to those seen in B. pertussis (4, 24).
fig.ommittedrd, 百拇医药
BrkA expression in E. coli strain UT5600. (A) BrkA domain organization: signal peptide (SP [residues 1 to 42]), passenger or -domain (residues 43 to 731), and ß-domain (residues 732 to 1010). (B) Western immunoblot of E. coli UT5600 whole-cell lysates resolved by SDS-PAGE (11% polyacrylamide), probed with anti BrkA antiserum, and detected with goat anti-rabbit antiserum conjugated to horseradish peroxidase. Lanes: 1 and 2, pDO6935 (wild-type copy of brkA gene); 3 and 4, pBluescript (vector control). Specific BrkA bands are indicated. U, unprocessed 103-kDa precursor protein; *, 73-kDa processed passenger moiety. Cells were processed in the presence or absence of trypsin as described in Materials and Methods. (C) Surface expression of BrkA in E. coli UT5600 detected via indirect immunofluorescence. The top panels show phase-contrast images, and the bottom panels show epifluorescence images.rd, 百拇医药
To determine whether BrkA is translocated to the surface of E. coli, trypsin accessibility and immunofluorescence experiments were performed with whole cells. When cells were incubated with trypsin, a marked decrease in the 73-kDa moiety was observed, and two products of approximately 40 and 45 kDa were detected by Western immunoblotting. The cleavage sites producing the 40- and 45-kDa species are unknown, and over time, both species are lost. The intensity of the 103-kDa product remained constant following trypsin digestion, suggesting that the 103-kDa band represents an intracellular form of the protein inaccessible to trypsin. Concomitant with this result, BrkA was detected on the surface of E. coli (Fig. 1C) and appeared evenly distributed, as shown by indirect immunofluorescence staining. Secreted BrkA could not be detected in concentrated culture supernatants, suggesting that the cleaved passenger remains noncovalently associated with the bacterium (data not shown). Taken together, these data indicate that BrkA is exported to the surface of E. coli strain UT5600 and is processed (independently of proteases OmpT or OmpP) in a manner similar to that observed in B. pertussis (24).
Identification of the minimal BrkA translocation unit necessary for surface expression. The natural cleavage of three well-characterized autotransporters, IgA protease (11), VirG/IcsA (6), and AIDA-1 (30), results in ß-domains of 45, 37, and 48 kDa, respectively. By using a series of protease susceptibility assays and experiments with heterologous proteins fused to N-terminally-truncated ß-domains, minimal regions necessary to display passenger proteins have been identified for these autotransporters. They have in common, a membrane-embedded ß-core of 25 to 30 kDa found at the extreme C terminus, preceded by a so-called "linker" region (11, 19, 31). In these autotransporters, the linker region has been shown to be necessary for the translocation of the passenger domain to the bacterial surface. The linker region together with the outer membrane-embedded ß-core make up what has been coined the "translocation unit" (19).;p3o7d&, http://www.100md.com
Having demonstrated that BrkA is targeted to the outer membrane of E. coli, we next developed a deletion-based strategy to define the boundaries of the minimal translocation unit of BrkA. N-terminal sequencing of proteins from outer membrane preparations of B. pertussis has localized the processing of BrkA to between Asn731 and Ala732 (25), resulting in a ß-domain of 30 kDa (28). At 30 kDa, the BrkA ß-domain is smaller than the ß-domains for IgA protease, VirG/IcsA, and AIDA-1, but it approaches the size of the outer membrane-embedded ß-cores identified for these proteins (11, 19, 31). We constructed a series of brkA deletion mutants by using PCR mutagenesis. As shown in mutant proteins consisted of the first 228 amino acids of BrkA (Met1 to Gly228) fused in frame to processive deletions of the C-terminal region of the BrkA -domain leading into the BrkA ß-domain. BrkA (Met1 to Gly228) was chosen as a passenger, since heterologous passengers such as cholera toxin B subunit (12) may be inefficiently translocated due to structural limitations (i.e., disulfide bond formation). In addition, it has been suggested that the extended signal sequences observed in many autotransporters may play a role in secretion (9). Therefore, the inclusion of the native BrkA signal sequence within the passenger avoids any influence that a nonnative signal sequence may have on secretion. All deletion strains were derivatives of pDO6935, thereby ensuring a common promoter for the wild-type and mutant constructs (Table 1).
fig.ommitted$!2, 百拇医药
Expression of BrkA deletion constructs in E. coli UT5600. (A) Diagram illustrating positions of BrkA in-frame deletions. Deleted regions are indicated by dotted lines, and deletion boundaries correspond to the wild-type BrkA amino acid designation. The BrkA domain structure is described in Construction of mutations is described in the Materials and Methods. Plasmids are described in (A) pGD1. (B) pGD2. (C) pGD3. (D) pGD4. (E) pGD5. (F) pGD6. (G) pGD7. (H) pGD8. (I) pGD9. (J) pGD10. (K) pGD10.5. (L) pGD11. (M) pGD12. E. coli UT5600 bacteria were transformed with BrkA deletion constructs (plasmids A to M) and grown to an OD600 of approximately 0.7. Bacteria were harvested, and BrkA surface expression was assessed by immunoblotting or indirect immunofluorescence. (B) Immunoblotting following resolution of whole-cell lysates by SDS-PAGE. The band migrating within the region denoted as "U" in each lane corresponds to the unprocessed, precursor form of BrkA, and the band denoted with an asterisk (*) corresponds to the processed passenger domain of BrkA. Cells were processed in the presence (+) or absence (-) of trypsin as described in Materials and Methods. Molecular mass markers (in kilodaltons) are indicated to the left of the panel. (C) BrkA expression in E. coli strain UT5600 detected by indirect immunofluorescence. The top panels show phase-contrast images, and the bottom panels show epifluorescence images.
An attempt was made to target our deletions to regions that would not disrupt the core structure of the protein. Secondary structural analysis with PsiPred (20) predicts that BrkA is predominantly composed of ß-strands (data not shown). In addition, the closest relative to BrkA in the database is the B. pertussis autotransporter pertactin (4). The structure of the pertactin passenger domain has been solved and was shown to be a monomer folded into a single domain that is almost entirely made up of a right-handed cylindrical ß-helix (3). Given that BrkA and pertactin passenger domains share 27% sequence identity and 39% sequence similarity, we refined our secondary structural prediction by overlaying the pertactin structural coordinates (1DABA) onto a BrkA-pertactin primary amino acid sequence alignment. The best alignment was between Arg175 to Pro572 in pertactin and Val301 to Gln707 in BrkA. Using this analysis, we systematically targeted N-terminal deletions to intervening regions with the predicted ß-strands.
The effects of each deletion on BrkA expression and processing were assessed by immunoblotting of whole-cell lysates resolved by SDS-PAGE. As shown in , each mutant form of BrkA was expressed, indicating that the specific deletions did not render the individual mutant protein products markedly unstable. In deletion mutants A through J, products corresponding to both the unprocessed precursor (region designated as "U") and the cleaved passenger (asterisk) were detected . In contrast, only the unprocessed precursor could be detected in deletion mutants K, L, and M. Given our previous observation that the cleaved passenger domain represents a major fraction of the surface-expressed wild-type BrkA (Fig. 1), these data suggested that BrkA deletion mutants A through J were being exported to the bacterial surface, but mutants K, L, and M were not. In support of this observation, trypsin accessibility assays and indirect immunofluorescence experiments were performed. As expected, exposure of whole cells to trypsin digestion resulted in the complete absence of the band corresponding to the processed passenger domain (lanes A to J), whereas a significant fraction of the unprocessed precursor remained stable lanes A to M). Consistent with these data, surface expression of the passenger region was detected via indirect immunofluorescence in mutants A through J, but not in mutants K, L, and M . The absence of immunofluorescence in mutants K, L, and M supports the tenet that the unprocessed, trypsin-resistant fraction of BrkA represents an intracellular form of BrkA, and not simply a trypsin-resistant surface molecule. It should be noted that a deletion (Ala136-Pro255) within the BrkA passenger (Met1 to Gly228) construct used for mutants A to M did not affect surface expression of BrkA (data not shown). Collectively, these data show that the region spanning residues Ala136 to Glu693 of BrkA is not required for surface localization of passenger proteins in E. coli strain UT5600. Furthermore, since the processed form of the passenger is also evident in deletion constructs A to J as well as construct Ala136-Pro255, it argues against BrkA having autoproteolytic activity.
Our data indicate that the ß-domain of BrkA is itself insufficient to translocate a passenger to the cell surface. The minimal translocation unit for BrkA thus consists of the ß-core plus a preceding linker region, the N-terminal boundary of which maps within Glu693 to Ser701. Historically, the ß-domain has been defined as the C-terminal outer membrane resident fragment derived from proteolytic processing of the autotransporter protein. Although the ß-domains of IgA protease (11), VirG/IcsA (31), and AIDA-1 (19) are larger than the ß-domain of BrkA, the sizes of their minimal translocation units are remarkably similar. Indeed, a comparison of experimentally defined linkers in four diverse autotransporters including BrkA reveals a structurally conserved architecture that can be viewed as a signature for autotransporters. It consists of a 21- to 30-amino-acid -helical region that precedes a 255- to 294-amino-acid transporter domain, a region rich in ß structure . It has been proposed that the linker region is involved in forming a hairpin-like structure that leads secretion of the passenger domain through the channel formed by the ß-core (9). The common features of the translocation unit suggest that it, rather than the ß-domain, is a more appropriate operational definition for the transporter domain. The region upstream of the translocation unit would thus constitute the passenger moiety regardless of the positioning of the proteolytic processing sites .
fig.ommitted4kj#, 百拇医药
Comparison of the translocation units of different autotransporters. The C-terminal regions of four autotransporters are shown (not drawn to scale). See the text for explanation. The N-terminal boundaries noted for each translocation unit have been defined experimentally in references 11 (IgA protease), 31 (VirG/IcsA), and 19 (AIDA-1), as well as in this paper (BrkA).4kj#, 百拇医药
IgA protease, VirG/IcsA, and AIDA-1 are naturally cleaved well upstream of the predicted -helical region and either can be released naturally (6, 26) or can be induced to be released following heat treatment (1). Unlike these proteins, BrkA is steadfastly associated with the bacterial outer membrane both in B. pertussis and in E. coli and cannot be released by heat treatment (G. Huang and R. Fernandez, unpublished data). Cleavage of BrkA occurs within the predicted -helical region. Thus, it is possible that the linker region also acts as the anchor (11) for BrkA, since none of the deletion mutant proteins spanning Ala136 to Glu693 was detected in immunoblots of concentrated culture supernatants (data not shown).
In summary, we have shown that the B. pertussis autotransporter BrkA can be surface expressed in E. coli, enabling dissection of autotransporter secretion mechanisms in a host more amenable to genetic manipulation. Adding to our previous studies on the BrkA ß-domain (28), which we demonstrated has the capacity to form a channel, we have identified two additional secretion determinants of BrkA: a 42-amino-acid signal peptide and a 30- to 39-amino-acid region preceding the ß-domain that, together with the ß-domain, defines the BrkA translocation unit . The data presented provide further experimental support for the importance of the predicted -helical region in autotransporter secretion of both native and heterologous passengers (12, 19, 31).4me'y, 百拇医药
ACKNOWLEDGMENTS4me'y, 百拇医药
We thank V. deLorenzo and L. Fernandez for the gift of UT5600 and UT2300. D.C.O. was a recipient of a University of British Columbia graduate student fellowship.
This work was funded by a grant from the Natural Sciences and Engineering Research Council of Canada.$.\v, 百拇医药
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