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Sandra W. Ramer,
Cheng-Yen Wu, and Gary K. Schoolnik*
Departments of Medicine (Infectious Diseases and Geographic Medicine) and Microbiology & Immunology, Stanford Medical School, Stanford, California 94305
Received 29 May 2003/ Accepted 21 August 2003
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ABSTRACT |
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TEXT |
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In-frame disruption of each of the 14 bfp operon genes (designated bfpA to bfpL) (Table 1) and analysis of the resulting mutants show that with the exception of bfpH, each of the remaining 13 genes is required for normal BFP filament production and phenotype expression (1, 13). These findings for the BFP system and similar results from the study of other type IV pili have led to the idea that bfp operon-encoded proteins comprise an oligomeric structural and functional complex (4, 10, 14, 15, 22, 24). The topographical features of this complex have been explored by localizing individual proteins to compartments of the cell through the use of protein-specific antibodies and immunoblot assays to detect their presence in compartment-specific cell fractions. In this manner, BfpB and BfpG have been shown to localize exclusively to the outer membrane (12, 17); BfpU and BfpH mainly localize to the periplasmic space (see references 13 and 18 and unpublished data), and BfpA (as a pool of unassembled pilins), BfpC and BfpE, and BfpI, BfpJ, and BfpK (the last three are stoichiometrically minor pilin-like proteins of the assembly complex) localize to the inner membrane (3, 13). By contrast, BfpL (while predominantly an inner membrane protein) can be consistently detected in small amounts in outer-membrane fractions prepared from French pressure cell-disrupted bacteria and sucrose gradient centrifugation (13). BfpP, which encodes the prepilin peptidase that processes BfpA (the major repeating subunit of the pilus filament) (30) and BfpI, BfpJ, and BfpK (13), is presumed (on the basis of its functional role) to localize to the cytoplasmic face of the inner membrane, but biochemical evidence for this prediction has not been reported (21, 30). BfpD and BfpF, which contain Walker box motifs that are presumed to hydrolyze ATP and thus energize the extrusion and retraction of the pilus filament, respectively, have not been localized (13, 21).
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Construction and functional studies of a BfpB-His6 fusion protein. To determine whether proteins encoded by the BFP operon physically associate in situ as a complex, BFP-expressing cells were incubated with a membrane-permeable, disulfide-cleavable cross-linker and the cross-linked complex was isolated by affinity chromatography as described below. The cross-linked eluted complex was treated with 2-mercaptoethanol (to cleave the cross-linker), and proteins in the complex were then identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting with antisera to 10 of the 14 bfp operon-encoded proteins. This experimental strategy required that we replace one of the 14 wild-type bfp operon proteins with a hexahistidine-tagged version of the same protein by complementing the in-frame mutant of the corresponding bfp operon gene with a plasmid encoding the His-tagged variant. Proof that functional complementation had occurred was sought by determining whether the complemented strain exhibited normal LA and AA phenotypes. To efficiently express His-tagged bfp operon proteins, we constructed a plasmid containing the bfp operon promoter and the coding sequence for a His6 tag located at the C terminus of the expressed BFP protein. Inclusion of the bfp operon promoter allowed the His-tagged protein to be expressed under the control of the normal BFP transcription factor, PerABC/BfpTVW (5, 26). The multicloning site (MCS) and polyhistidine (His6) region of pBAD/Myc-HisA (Invitrogen, Carlsbad, Calif.) was obtained by PCR and used to replace the MCS of pTrc99A (Pharmacia, Peapack, N.J.) to produce pTrc99-His. Then, the upstream region of bfpA, which contains the bfp operon promoter, was obtained from the EAF plasmid by PCR and used to replace the promoter region of pTrc99-His. The resulting plasmid, pTrcAp-His, has the bfp operon promoter and C-terminal His6-tag coding sequence flanked by the MCS of pBAD/Myc-HisA. The strains and plasmids used in this study are listed in Table 2.
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B, C, D, E, F, or J, respectively) (13), only B171-8 B/pApB-His and B171-8 F/pApF-His cells retained the characteristic BFP-mediated LA and AA phenotypes (data not shown). B171-8 B/pApB-His was selected for the cross-linking studies because BfpB forms a multimer in the outer membrane, thus affording the possibility of multiplicative binding of the C-terminal His6-tagged BfpB fusion protein to the Ni affinity column (12, 17).
To prove that the BfpB-His tag fusion protein is expressed in B171-8 B/pApB-His, this recombinant strain was grown under BFP-expressing conditions and the bacteria were collected, washed, boiled in sample buffer, and analyzed by immunoblotting with a rabbit polyclonal BfpB antiserum or with a murine monoclonal anti-penta-His antibody (Qiagen, Inc., Valencia, Calif.). As the fusion protein is expressed and detectable by both antibodies (Fig. 1), it was judged suitable for Ni and/or BfpB antibody-based affinity purification of the cross-linked BFP assembly complex.
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B/pApB-His or, as a negative control, the non-BfpB-expressing recombinant B171-8B/pTrcAp-His (which lacks the BfpB-His-tagged fusion protein coding sequence but expresses each of the other Bfp operon proteins, with the exception of BfpB) and viable, exponential-phase bacteria. The negative control was used to identify proteins that interact nonspecifically with the affinity matrices and thus would be erroneously identified as being components of the BFP assembly complex. Cells grown overnight from B171-8B/pTrcAp-His or B171-8B/pApB-His in Luria-Bertani broth were diluted to an optical density at 600 nm of 0.02 in Dulbecco's modified Eagle's medium containing 0.45% glucose and 100 µg of ampicillin/ml. After incubation with shaking at 37°C for 3.5 h, at which time expression of all the BFP proteins and the AA phenotype are maximal (13), the cells were harvested by centrifugation, washed, and resuspended in phosphate-buffered saline (PBS). Disulfide-cleavable DSP (dithiobis[succinimidyl propionate]; Pierce, Rockford, Ill.) (final concentration, 0.2 mg/ml) was added for 30 min at room temperature (6). Following the addition of 0.1 M Tris-HCl (pH 7.4) to quench the reaction, the cross-linked cells were harvested, washed with PBS, and resuspended in phosphate buffer containing Triton X-100. The bacterial suspension was passed through a French press at 14,000 lb/in2. Unbroken cells were removed by centrifugation at 3,000 x g for 15 min; then, the supernatant was applied to the Ni affinity column according to the manufacturer's recommendations (Qiagen, Inc.). To promote exposure of the His6 tag at the C terminus of the BfpB fusion protein and the dissociation of non-cross-linked proteins from the assembly complex, 1% (vol/vol) Triton X-100 was added to each of the Ni affinity steps. The imidazole-eluted material from the Ni affinity column was then dialyzed against PBS and further purified by immunoaffinity chromatography using BfpB antibodies conjugated to a protein A column (8, 9).
The sequential two-step Ni and BfpB antibody affinity chromatography purification procedure began with equal cell numbers and used the same elution volumes for the His-tagged BfpB fusion protein-expressing strain (B171-8B/pApB-His) and the non-BfpB-expressing negative-control strain (B171-8B/pTrcAp-His). Figure 2A shows the SDS-PAGE protein profiles for these strains during each purification step. Bound and unbound samples from the Ni and immunoaffinity columns were boiled in sample buffer containing 5% (vol/vol) 2-mercaptoethanol to cleave the cross-linker and subjected to SDS-12% PAGE, and the proteins were detected by silver staining. Lanes 7 and 8 compare the protein profiles from the BfpB negative-control and the BfpB-His-tagged fusion protein-expressing strains, respectively, after combined, sequential Ni and immunoaffinity chromatography. A prominent 
58-kDa band is evident in both lanes. Though this appears to have comigrated with BfpB, the use of the BfpB-specific antibody in immunoblotting experiments did not detect the BfpB protein in lane 7, which contains proteins from the BfpB negative-control strain. Accordingly, this protein likely interacts nonspecifically with both matrices and thus is not a component of the BFP assembly complex. Moreover, none of the other proteins identified in lane 7 were detected by antisera to 10 proteins encoded by the bfp operon and therefore are not likely to be components of the BFP assembly complex.
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60 kDa) (Table 1) (21, 23). Because BFP assembly complex proteins might have been associated with proteins that were not encoded by the BFP operon, lane 8 was also inspected for bands that are not present in lane 7 and do not correspond in size with any of the BFP proteins. Two such proteins were identified (Fig. 2A), including one represented by a prominent and well-resolved band of 100 kDa. The identities and possible functional significance of these non-BFP operon-encoded proteins were not further investigated. Characterization of the affinity-purified BFP assembly complex by immunoblot analysis. To determine whether the cross-linked protein complex isolated by the combination of sequential Ni and immunoaffinity chromatography contains proteins encoded by the BFP operon, immunoblot assays of the eluted complex were performed with antibodies to each of the 14 BFP proteins, excepting BfpP (13). Antibodies to BfpI, BfpK, and BfpH failed to detect the corresponding protein in the isolated cross-linked complex. Results obtained with antibodies to the 10 remaining bfp operon-encoded proteins are shown in Fig. 2B, in which lanes 1 and 2 represent proteins isolated from the BfpB negative-control strain and lanes 3 and 4 represent proteins from the strain that expresses the BfpB-His-tagged fusion protein and produces functional BFP. The protein complexes represented in lanes 2 and 4 were treated with 5% (vol/vol) 2-mercaptoethanol to reduce the cross-linker, thus dissociating the complex and allowing the proteins to migrate as monomers. By contrast, the proteins represented in lanes 1 and 3 were not treated with 2-mercaptoethanol; thus, these lanes depict nondissociated complexes.
Treatment of the cross-linked complex that was isolated by sequential Ni and BfpB antibody affinity chromatography with 2-mercaptoethanol allowed each of the 10 detected proteins to migrate in accordance with its predicted molecular mass. By contrast, none of these proteins was detected in lanes 1 and 2, indicating that in this bfpB deletion strain the remaining 13 bfp operon-encoded proteins that are expressed by this mutant do not nonspecifically bind and elute from the affinity columns. These Western blot results and the small number of proteins detected in lane 8 of Fig. 2A provide compelling evidence that at least 10 of the 14 proteins encoded by the bfp operon form a macromolecular complex. Prior localization studies show that two of these proteins (BfpB and BfpG) reside exclusively in the outer membrane and that one (BfpE) resides exclusively in the inner membrane (3, 13). Thus, this complex appears to span the periplasmic space, a conclusion that is supported by the finding that BfpU, a soluble protein that is principally located in the periplasmic space, is also a component of the cross-linked complex (13, 18). Not surprisingly, BfpA, the principal repeating subunit of the pilus fiber, was also identified in the complex, as was the pilin-like protein BfpJ. Likewise, BfpD and BfpF were found in the complex, thus demonstrating a close physical association between proteins required for the kinetic properties (extrusion and retraction, respectively) of the pilus with structural features of the assembly complex (2).
To investigate the role of cross-linking per se in the results depicted in Fig. 2, non-cross-linked B171-8B/pApB-His was subjected to all steps in the affinity purification protocol and the proteins that were eluted from the Ni and BfpB antibody affinity columns were characterized by immunoblot analysis using the same antisera. Only BfpB and BfpG were copurified in the absence of chemical cross-linking under the conditions used in this study (data not shown). Because both of these proteins reside in the outer membrane, this control experiment shows that cross-linking is required to copurify components of the assembly complex that reside in the periplasmic space and inner membrane.
More-slowly migrating bands are evident in lane 4 of the BfpE, BfpF, and BfpJ immunoblots, indicating the presence of a molecular species that is larger than the expected molecular mass of the assayed protein. The identification of these higher-molecular-mass bands was not investigated, but they could be oligomeric forms of the protein or complexes that contain the protein together with other components of the assembly complex.
BfpH was not detected in the complex with these antibodies. This was an expected finding, since the BfpH anti-peptide antibody used here failed to detect this protein in the wild-type parent strain (13). We were able to use an antibody to the His6 epitope to localize a His-tagged variant of this protein to the periplasmic space only recently (data not shown). Failure to detect BfpI and BfpK in the complex requires the consideration of other explanations, since both were detected in the wild-type parent by anti-peptide antibodies (13). One possibility is that the stoichiometric ratios of these pilin-like proteins to other components of the complex are too low for detection by this method. A second possibility is that they are not sufficiently near other components of the complex to be cross-linked by the reagent (Pierce) used here, which has an estimated effective length of 12 angstroms. Finally, it is possible that the use of the His-tagged BfpF fusion protein (which also yielded functional BFP), rather than that of the His-tagged BfpB fusion protein, would have provided a different portrait of complex membership.
The BFP assembly complex localizes to the poles of the cell. The well-studied type IV pili of Caulobacter crescentus, Myxococcus xanthus, and Pseudomonas aeruginosa are located at the poles of these cells, a topographical feature that is likely critical for the functions they confer in these species (19, 20, 28). By contrast, the type IV pili of Neisseria gonorrhoeae are believed to be peritrichous (25, 27). The polar or peritrichous location of BFP pili is unresolved: electron micrographs of BFP-expressing EPEC have failed to disclose where they emanate from the cell surface, because BFP from adjacent cells tend to aggregate, forming a meshwork that obscures their site of origination. To address this question, the BFP assembly complex was localized on the cell surface by immunofluorescence and immuno-gold electron microscopy through the use of antibodies to the proteins shown in Fig. 2B to be part of the assembly complex.
Immunofluorescence microscopy (7) was conducted by incubating an anti-penta-His murine monoclonal antibody of the immunoglobulin G class with the BfpF deletion strain B171-8F/pApF-His that had been functionally complemented for the AA and LA phenotypes with a plasmid expressing the His6-tagged BfpF fusion protein. B171-8dbfp (Table 2), which lacks part of the principal promoter of the bfp operon and thus produces none of the BFP proteins, served as a negative control for these studies. Detection of the anti-penta-His antibody with a fluorescein-conjugated anti-mouse immunoglobulin G antibody showed distinctive polar staining of this recombinant strain, which produces functional BFP assembly complexes containing the His-tagged BfpF fusion protein (Fig. 3B). By contrast, B171-8dbfp showed only faint circumferential staining (Fig. 3A).
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The ability to detect the BfpF-His6-tagged fusion protein within BFP assembly complexes at the cell pole led us to initiate studies aimed at identifying determinants of this organelle's polar localization. To learn whether BfpA is required for the polar localization of BfpF, a derivative of the wild-type strain B171-8 was prepared that produced all of the bfp operon proteins except BfpA (2). Immunofluorescence microscopy (using the anti-penta-His antibody) of this strain (B171-8A/pApF-His) showed clear polar localization of the His-tagged BfpF fusion protein (Fig. 3E). Thus, the polar localization of BfpF does not require BfpA or the production of pilus filaments per se, since such cells are nonpiliated.
Conclusion. Chemical cross-linking experiments and immunofluorescence and immuno-gold transmission electron microscopy have shown that proteins encoded by the bfp operon form a hetero-oligomeric complex that spans the periplasmic space and is preferentially localized to one pole of the cell. These results provide the basis for ongoing studies that seek to determine how the individual proteins are organized in the complex, how this organizational plan contributes to their biogenic roles, and by what mechanism they are targeted to the pole. From a pathogenic perspective, future studies will examine whether the polar location of the organelle favors the formation of infectious units and epithelial cell colonization.
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ACKNOWLEDGMENTS |
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We thank Jon Mulholland for assistance with fluorescence microscopy.
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FOOTNOTES |
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Present address: San Jose State University, San Jose, CA 95192.
Present address: Genencor International, Inc., Palo Alto, CA 94304.
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REFERENCES |
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