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Paul A. Manning,2 and Michael W. Heuzenroeder3*
Discipline of Microbiology and Immunology, Department of Molecular BioSciences, University of Adelaide, Adelaide SA 5005,1 Infectious Diseases Laboratories, Institute of Medical and Veterinary Science, Adelaide, SA 5000, Australia ,3 AstraZeneca R&D Boston, Waltham, Massachusetts 024512
Received 6 August 2001/ Accepted 13 November 2001
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A specific deletion in the csfD gene showed that CsfD is required for initiation of pilus biogenesis, since bacteria with this deletion showed no translocation of detectable CsfA subunits across the outer membrane and no visible cell surface pilus structures (6). CsfD was not required for the stability of CsfA in the periplasm. Morphological studies on purified CS5 by deep-etch freeze fracture electron microscopy showed that the pilus was a 2-nm flexible fibrillar structure, which was devoid of any tip-associated structures (6). CS5 pili are therefore morphologically similar to the K88 and K99 fibrillae of animal ETEC. This verified the conflicting reports as to the true morphology of the CS5 pilus, which ranged from a semirigid pilus structure to a 5- to 6-nm structure consisting of two fine fibrils wrapped around each other (11, 13). In the latter case, this was likely a consequence of the hydrophobicity of the pilus itself, causing multiple interactions between individual pili, which are frequently observed under the electron microscope (6, 7). Identification of either CsfA or CsfD as the adhesive component of CS5 pili remains unconfirmed.
Previous studies have shown that the csfC gene shares protein sequence similarity with the CooC and CfaC ushers from CS1 and colonization factor antigen I (CFA/I) pili, respectively, but does not share structural similarity by hydropathy plot alignments (6). Cell fractionation experiments and the construction of an in-frame deletion in the csfC gene showed that CsfC is outer membrane located and is responsible for directing both the CsfA and CsfD pilin subunits across the outer membrane, since release of these pilins did not occur in a csfC deletion strain (6). The absence of CsfC did not result in any alteration in the periplasmic accumulation of either pilin. As with the ushers of the Pap, CS1, and 987P biogenesis systems (3, 5, 16), CsfC has been hypothesized to direct the ordered translocation of pilin subunits across the outer membrane, since CsfD is translocated in a csfA deletion strain, while CsfA can be translocated only when CsfD is present (a csfD deletion strain does not permit the release of CsfA) (6). This implies that a specific recognition event between a CsfD-chaperone complex and CsfC is first required before CsfC is able to translocate CsfA subunits across the outer membrane. The functions of csfB, csfE, and csfF remain largely unknown, since no sequence homology with other proteins has been identified from database searches (7). However, CsfB is homologous to CsfF, sharing 30% identity and 51% sequence similarity as well as significant structural similarity, which suggests that these two proteins are likely to exhibit similar functions (7).
The purpose of the present study was to investigate the roles of the three unknown genes of the csf cluster, namely, csfB, csfE, and csfF, by creating a set of in-frame deletions in each gene and determining their effects on CS5 pilus biogenesis. A double in-frame deletion in the csfB and csfF genes was also constructed to examine the combined effect of deleting two genes with similar proposed functions in biogenesis.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(Bethesda Research Laboratories, Gaithersburg, Md.) harboring the various plasmid constructs was routinely grown in Luria-Bertani (LB) broth or agar plates, with aeration, at 37°C. For hemagglutination, immunogold electron microscopy, or other pilus expression experiments, strains were grown on CFA agar (9) at 37°C overnight. The E. coli K-12 strain BL21(DE3) (Novagen, Madison, Wis.) harboring the lacIq plasmid pREP4 (Qiagen, Hilden, Germany) (Table 1) was used as a source for specific His6-tagged protein expression. Antibiotics were used at the following concentrations: ampicillin, 100 µg/ml; kanamycin, 50 µg/ml. Both 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal) and isopropyl-ß-D-thiogalactopyranoside (IPTG) were used at 40 µg/ml.
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Construction of an in-frame deletion mutation in the csfE gene utilized a single NruI site located within the csfE gene across nucleotides (nt) 5597 to 5602 of the characterized sequence (GenBank accession no. AJ224079). This was utilized for exonuclease III-mediated bidirectional deletions using the csf operon plasmid pPM5631 (Table 1) (7), since NruI digestion produces blunt-end fragments that are susceptible to exonuclease III. One such sequenced deletion, which juxtaposed nt 5428 and 5806, thereby deleting 378 nt of the csfE gene and retaining the correct reading frame, was selected for further use and was designated pPM5654 (Table 1). A csfE complementing plasmid was constructed by using the wild-type csfE gene from pPM5631 in a PCR with oligonucleotides 2973 (5"-GGCTGCAGGGAAATATGTAAGCATTACC-3") and 2974 (5"-GGTCTAGACGATTAAAATCGCTCTAAAAA-3"), which contain PstI and XbaI sites, respectively (underlined); the fragment obtained was digested with the appropriate restriction enzymes and cloned into digested pWKS130 (22) to produce pPM5657, with the csfE gene oriented from the inducible lac promoter (Table 1). Plasmid pPM5684 was constructed by excising the csfE gene from pPM5633 (7) with KpnI and XbaI and then cloning it directly into digested pBAD18-Kan (10), such that the csfE gene was under the control of the PBAD promoter from the araBAD (arabinose) operon (Table 1).
An in-frame deletion mutation in the csfF gene was constructed from pPM5631 by utilizing a unique StuI site located across nt 6229 to 6234 (accession no. AJ224079) for exonuclease III-mediated deletion as described above. One such deletion obtained resulted in the juxtaposition of nt 6070 and nt 6397, thereby deleting 327 nt of the csfF gene and retaining the correct reading frame. This plasmid was selected for further use and was designated pPM5655 (Table 1). To construct a complementing plasmid, the wild-type csfF gene was PCR amplified from pPM5631 using oligonucleotides 2971 (5"-GGAATTCACTATGTAGGGGGAGTAT-3") and 2972 (5"-GGTCTAGATGAACCATAAAG GAAAAAAAG-3"), which contain EcoRI and XbaI sites, respectively (underlined). The PCR fragment obtained was digested with the appropriate restriction enzymes and cloned into EcoRI- and XbaI-digested pWKS130 to produce pPM5659, with the csfF gene oriented from the inducible lac promoter (Table 1). The plasmid was used in trans to complement the introduced in-frame deletion of csfF in pPM5655. To construct pPM5672 with the csfF gene under the control of the PBAD promoter of pBAD18-Kan (10), pPM5659 was digested with EcoRI and XbaI, and the csfF gene fragment obtained was cloned directly into EcoRI- and XbaI-digested pBAD18-Kan to produce pPM5672 (Table 1).
An in-frame deletion mutation was constructed in the csfB gene by utilizing a double PstI site located within the csfB gene at nt 2318 to 2323 and nt 2327 to 2232 of the characterized sequence (accession no. AJ224079). PstI restriction digestion produces 3" overhangs which are resistant to exonuclease III; therefore, to overcome this, the 3" overhangs were blunt ended by using the 3"-5" exonuclease activity of the Klenow enzyme (1 U/µg of DNA) without deoxynucleoside triphosphates for 10 min at 37°C. Blunt-end DNA fragments are susceptible to exonuclease III-mediated deletions. One csfB deletion mutant obtained resulted in the juxtaposition of nt 2157 and 2602, thereby deleting 444 nt of the csfB gene and retaining the correct reading frame. This plasmid was selected for further use and was designated pPM5662 (Table 1). To construct the csfB complementing plasmid pPM5665, the wild-type csfB gene was PCR amplified from pPM5631 using oligonucleotides 2944 (5"-GGAATTCCCAAGGCAGCTGCTGC-3") and 2945 (5"-GGTCTAGAGCCAGCTCACTTTATCAGC-3"), which contain EcoRI and XbaI sites, respectively (underlined). The PCR fragment obtained was restricted and cloned into EcoRI- and XbaI-digested pWKS130 to produce the csfB gene oriented behind the inducible lac promoter.
To construct an expression plasmid for the CsfB protein in order to generate a polyclonal antiserum, the csfB gene was PCR amplified from pPM5631 using oligonucleotides 3110 (5"-GGCGGATCCGTTCAGTGTTGATTCAATGATA-3") and 3111 (5"-GGGGTACCGGTGTCTTTTAGAGTCATA-3"), which contain BamHI and KpnI sites, respectively (underlined). The csfB fragment was then digested with the same restriction enzymes and cloned into BamHI- and KpnI-digested pQE-31 (Qiagen) such that the csfB gene was cloned in-frame with an N-terminal His6 tag to produce plasmid pPM5679 (Table 1).
Finally, a double in-frame deletion mutation of the csfB and csfF genes was constructed by utilizing the csfF deletion plasmid pPM5655 to construct a further deletion in the csfB gene. The generation of a specific in-frame deletion in csfB was carried out as described above. One such deletion obtained resulted in the juxtaposition of nt 2197 and 2591, thereby deleting 393 nt of the csfB gene and retaining the correct reading frame. This plasmid was selected for further use and was designated pPM5690 (Table 1).
Western blot analysis. Preparative protein samples were boiled for 5 min and then separated by sodium dodecyl sulfate (SDS)-15% polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany). For detection of CS5 pili, a rabbit anti-native CS5 pilus antiserum (11) was used at a dilution of 1:5,000. Rabbit anti-CsfE and rabbit anti-CsfB were used at a 1:1,000 dilution. Antigen-antibody complexes were reacted with a horseradish peroxidase-conjugated goat anti-rabbit antibody at a dilution of 1:10,000 and visualized by using a chemiluminescence kit according to the manufacturer's instructions (Roche). Blots were exposed to Cronex 10T medical X-ray film (Sterling Diagnostic Imaging, Stevenage, Hertfordshire, United Kingdom).
Overexpression and cell fractionation. Specific overexpression of His6-CsfB from pPM5679 initially involved transforming the plasmid into BL21(DE3) harboring pREP4. Bacteria were grown to the mid-exponential phase of growth (optical density at 600 nm, 0.6) with IPTG added to a final concentration of 1 mM for 3 h at 37°C with vigorous aeration. A 50-ml sample of induced bacterial cells was then fractionated into whole cells, periplasm, cytoplasm, and inclusion bodies as previously described (15). A sample of the inclusion body fraction (1 ml) was subjected to metal exchange chromatography using Ni-nitrilotriacetic acid agarose according to the manufacturer's instructions (Qiagen) in order to purify the His6-tagged CsfB protein.
Overexpression of CsfE from the T7 promoter has been described previously (7). Cells overexpressing CsfE were fractionated according to the procedure described by Morona et al. (15).
Antiserum preparation. Antibodies to both CsfE and CsfB were raised in New Zealand White rabbits. Samples of both CsfE and CsfB were run on SDS-15% PAGE gels, and bands corresponding to these proteins were excised from the gel and were each emulsified with 0.5 ml of Freund's complete adjuvant and an equal volume of 1x phosphate-buffered saline (PBS) in an Ultra-Turrax blender (Janke and Kunkel, Staufen, Germany). At day 0 each rabbit was immunized subcutaneously with 10 injections of the protein homogenate at different sites (100 µl per site). Periodic boosters were given in Freund's incomplete adjuvant. Each rabbit was exsanguinated by cardiac puncture, and the serum obtained was stored at -20°C. The antiserum was absorbed against live E. coli K-12 strains harboring the specific plasmid vectors used in the cloning process in the presence of 0.02% sodium azide. Absorbed antiserum was also incubated across nitrocellulose membranes impregnated with whole-cell extracts in order to further remove nonspecific antibodies. The specificities of the antiserum against CsfB and the antiserum against CsfE were examined by Western blot analysis.
Rapid pilus isolations. Bacterial strains were grown to confluence at 37°C on CFA plates (9), harvested in 1 ml of PBS, and incubated with vigorous shaking at 56°C for 20 min in an Eppendorf reaction tube. The cell suspension was clarified by centrifugation for 5 min at 8,000 x g. Samples of the heat extracts were then subjected to SDS-15% PAGE as previously described (6).
Hemagglutinations and slide agglutinations. Hemagglutination and slide agglutination experiments were performed as previously described (6).
Immunogold electron microscopy. Immunogold labeling and negative staining of bacterial strains were carried out essentially as previously described (6) by using an antiserum against CS5 pili added at a 1:10 dilution and protein A conjugated with 20-nm-diameter gold beads (ICN, Costa Mesa, Calif.) at a 1:40 dilution.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Western immunoblot analysis following SDS-15% PAGE using a rabbit anti-CS5 pilus antiserum was also performed on whole-cell, periplasmic, and heat extract samples prepared from E. coli K-12 harboring either pGEM-7Zf+, pPM5631, pPM5654, or pPM5654 complemented with pPM5657. E. coli K-12 harboring pPM5654 showed a small reduction in the accumulation of CsfA and CsfD pilin subunits in heat extract samples compared to E. coli K-12 harboring pPM5631 (Fig. 2). This reduction was examined further by preparing whole-cell and periplasmic extracts to ascertain whether the accumulation of CsfA and CsfD pilin subunits is reduced in a csfE deletion strain. E. coli K-12 harboring pPM5654 did not result in any significant change in the accumulation of either CsfA or CsfD in the periplasm compared to E. coli K-12 harboring pPM5631 or E. coli K-12 harboring pPM5654 complemented with pPM5657 (Fig. 2). This suggested that CsfE is not required for the stability of CsfA or CsfD in the periplasm of E. coli K-12. Increased amounts of CsfA and CsfD were detected in whole cells of E. coli K-12 with pPM5654 compared to E. coli K-12 harboring pPM5631 or pPM5654 complemented with pPM5657 (Fig. 2). This is likely a reflection of the overall hydrophobicity of CS5 pili (6, 7), where detached longer CS5 pili reassociate with cell surface pili and are centrifuged with the whole cells. Examination of electron micrographs of the csfE mutant strain shows a mesh-like network of associated pili on these strains (Fig. 1D and E), which provides some evidence for long, cell-free pili reassociating with cell surface pili attached to the bacteria.
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The question of whether CsfE is assembled as a minor component of the CS5 pilus was investigated. CsfE could not be detected in denatured pure CS5 pili weighing up to 100 µg or in heat extracts representing 5 x 109 cells, a number that has been shown by Coomassie brilliant blue gel analysis to release more than 100 µg of CS5 pili (data not shown). Instead, CsfE remains in the outer membranes of these cells, which indicates either that the association of CsfE with the CS5 pilus is a relatively weak interaction or that CsfE directly associates with the CsfC usher (6) in the outer membrane rather than with the pilus itself.
csfF deletion mutants produce longer CS5 pili morphologically similar to those produced by csfE deletion mutants. The effect of a deletion mutation in the csfF gene on CS5 pilus biogenesis was initially assessed by slide agglutinations using a rabbit anti-CS5 pilus antiserum. E. coli K-12 harboring pGEM-7Zf+ or pPM5655 did not agglutinate with the antiserum. Conversely, E. coli K-12 with pPM5631 or E. coli K-12 with pPM5655 complemented with pPM5659 showed immediate agglutination at room temperature. This indicates that the csfF deletion mutation may alter the cell surface assembly of CS5 pili. Examination of the same strains by immunogold electron microscopy revealed that E. coli K-12 harboring pPM5655 showed cell surface assembly of CS5 pili, but in far lower numbers, while the pili appeared to be far longer overall than those of the wild-type or the complemented mutant (Fig. 4A and B). This phenotype was very similar to that observed in a csfE deletion strain (Fig. 1D and E), which suggested that both CsfE and CsfF may control pilus length. E. coli K-12 containing pPM5655 complemented with pPM5659 produced peritrichous CS5 pili (Fig. 4C). No hemagglutination of the erythrocytes was observed with E. coli K-12 harboring pPM5655; however, hemagglutination of the erythrocytes was restored when E. coli K-12 harboring pPM5655 was complemented with pPM5659. The reduction in the number of cell surface pili, along with the increased susceptibility to shearing forces on longer pili, may also explain the negative slide agglutination and hemagglutination results obtained for the csfF deletion mutant strain.
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Expression of CsfE and CsfF and their effects on pilus length and number. The results have indicated that CsfE and CsfF appear to play a role in regulating the length of the CS5 pilus. csfE and csfF were cloned into pBAD18-Kan under the control of the tightly regulated PBAD promoter from the araBAD (arabinose) operon to produce pPM5684 and pPM5679, respectively. The presence of arabinose (at 0.002, 0.01, or 0.05%) in CFA agar induced expression of the genes. Following overnight incubation, bacteria were examined under the electron microscope after immunogold labeling and negative staining with 1% uranyl acetate, and numbers and lengths of pili were recorded. The mean number of surface-located pili ± standard error of the mean (SEM) was calculated on 50 randomly selected bacteria, while the mean length of the pili ± SEM was calculated by recording the lengths of 5 pili on each of 10 randomly selected bacteria (n = 50). Since CS5 pili bundle, the mean number and length are representative of discrete bundles of pili.
It must be noted that the presence of the pBR322 ori of pBAD18-Kan leads to a reduction in copy number when coexpressed with the incompatible pGEM-7Zf+ ColE1 ori. When this incompatibility was examined in the wild-type E. coli K-12 strain harboring pPM5631 and pBAD18-Kan in the presence of 0.01% arabinose (Table 2), it led to a reduction in the mean number of cell surface pili (11.82 versus 41.34 in the wild type) but did not significantly affect mean pilus length (0.522 µm versus 0.55 µm in the wild type) (P = 0.42). Similarly, no reduction in pilus length was observed in the presence of increasing or decreasing concentrations of the inducer (data not shown). Therefore, this two-plasmid system is suitable for examining the effects of modulating CsfE and CsfF expression on pilus length.
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When the csfE mutant was complemented in trans with pPM5657 in the presence of IPTG, the mean number of pili per cell increased (22.72 ± 1.085), while the mean pilus length decreased (0.771 ± 0.045 µm), compared to those for E. coli K-12 harboring pPM5654 (Table 2). However, neither the mean number of pili nor the mean pilus length was restored to that observed in the wild-type strain (P < 0.0001), which may be a consequence of expression of csfE from a low-copy-number plasmid versus the high copy number of pPM5631. Expression of CsfE from the PBAD promoter on pPM5684 was carried out by addition of either 0.002, 0.01, or 0.05% arabinose, which specifically induces CsfE expression. For E. coli K-12 harboring pPM5654 and pPM5684 in the presence of 0.002% arabinose, the mean pilus length was 1.036 µm; this decreased further when the concentration of arabinose was increased to 0.01% (0.721 ± 0.035 µm) or 0.05% (0.735 ± 0.038 µm). The pili produced were still significantly longer than those observed in the wild type (P < 0.0001). When pPM5684 was expressed with pPM5631 in the presence of 0.01% arabinose, the mean pilus length was 0.539 ± 0.018 µm, which was not significantly different from that for pPM5631 alone (P = 0.68) (Table 2). The data showed that CsfE is specifically involved in length regulation but is not rate-limiting for length control, since increased expression of CsfE does not reduce mean pilus length below that observed in the wild-type strain.
In contrast, CsfF is rate-limiting for determination of pilus length. E. coli K-12 harboring both pPM5655 and the compatible low-copy-number plasmid pPM5659 grown in the presence of IPTG reduced the mean pilus length to 0.771 ± 0.023 µm, but pili were still significantly longer than those observed in the wild type (P < 0.0001) (Table 2). However, E. coli K-12 harboring pPM5655 complemented with pPM5672 grown in the presence of 0.002% arabinose resulted in a significant decrease in mean pilus length, to 0.247 ± 0.015 µm (P < 0.0001). When the same strain was grown in the presence of 0.01% arabinose, no surface-associated pili were evident. Moreover, when E. coli K-12 harboring pPM5631 was complemented with pPM5672 in the presence of 0.002% arabinose, the bacteria were virtually devoid of pili (mean number per cell, 0.480 ± 0.104), and thus no mean pilus length was recorded (Table 2). Taken together, these results indicate a direct association between the level of expression of CsfF in the periplasm and the mean length of the pilus. Therefore, CsfF is rate-limiting for the determination of pilus length.
Effect of an in-frame csfB deletion mutation on CS5 pilus biogenesis. To examine the role of csfB in pilus assembly, an in-frame deletion mutation in the csfB gene was constructed in E. coli K-12 and designated pPM5662. Initial slide agglutination experiments using an anti-CS5 antiserum showed that strains harboring pPM5662 did not agglutinate. When the same strains were examined by immunogold electron microscopy, E. coli K-12 harboring pPM5662 was devoid of CS5 pili, while E. coli K-12 containing pPM5662 complemented with pPM5665 produced peritrichous CS5 pili (Fig. 6). The csfB deletion mutant strain was also tested for the ability to cause mannose-resistant hemagglutination of human group A+ erythrocytes. As expected from the immunogold electron microscopy data, no hemagglutination ability was conferred by E. coli K-12 with pPM5662, while hemagglutination of the human erythrocytes was restored when the csfB mutant was complemented with pPM5665. Therefore, CsfB is required for cell surface assembly of CS5 pili.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A constructed in-frame deletion mutation of the csfE gene resulted in pili that were more than three times longer than those of the wild-type strain, highlighting a specific role for CsfE in controlling the length of assembled CS5 pili. However, expression of CsfE did not reduce the length of the pilus from that of the wild type, which suggested that CsfE is not rate-limiting for pilus length regulation. Specific overexpression of other, analogous proteins such as PapH from Pap pili and MrpB from MR/P pili of Proteus mirabilis resulted in significant reductions in pilus length compared to that of the wild type (1, 14). A recent study has shown that the use of araBAD promoters to express genes of interest may not be modulated as first described (10); rather, at subsaturating levels of induction of the araBAD promoter, gene expression is not uniform with respect to individual cells (19). Therefore, a certain amount of caution must be applied to these results; however, a previous study using the minor pilin gene cooD under the control of the araBAD promoter found induction of CS1 pili from a mean of 28 pili/cell to >150 pili/cell in the majority of cells examined, suggesting that induction may have been uniform (16).
CsfE was localized to the outer membrane in E. coli K-12, as with other pilus length regulators, and as such is predicted to encode a minor pilin subunit, although no CsfE could be detected in pure CS5 pilus preparations. The difficulty in this study was determining a link between CsfE and the anchoring of CS5 pili to the cell surface. In other systems such as Pap, the PapH protein was shown to regulate the length of the pilus and was predicted to anchor the pilus to the cell surface, since an increased number of pili was detected in the supernatant of a papH mutant (1). Although this may be the case for such studies, the increased shearing forces on longer pili must be taken into account in harvesting the bacteria. The longer a pilus becomes, the greater, presumably, is the corresponding increase in shearing forces on the pilus. Based on the results obtained and on analogy with other systems, it is hypothesized that the incorporation of CsfE with the outer membrane usher CsfC results in a stable binding complex which cannot be displaced by further chaperone-subunit complexes, thereby leading to termination of assembly. This event may also provide a stable platform for association of the pilus with the cell envelope. The low level of expression of CsfE in the cell suggests that association of CsfE with other pilins would be a rare event.
A deletion mutation in the csfB gene was found to completely abolish CsfA subunit accumulation in the periplasm, and therefore bacteria with this deletion did not produce detectable cell surface CS5 pili. However, the deletion in CsfB did not affect the transport of the minor pilin subunit CsfD into the periplasm and across the outer membrane, since CsfD subunits were detected in heat extract samples from the csfB deletion strain. CsfB is therefore likely to function as the major subunit-specific chaperone. Localization studies using an anti-CsfB antiserum on fractions derived from the wild-type strain confirmed the periplasmic location of CsfB and showed that CsfB did not form part of the mature pilus structure.
A deletion mutation constructed in the csfF gene resulted in two distinct changes in the pilus structure. Firstly, the pili produced in this strain were approximately three times longer than those observed in the wild type, and secondly, no CsfD was detected in the longer pilus structures. Specific modulation of CsfF expression in a csfF deletion strain resulted in significantly shorter pili, and when CsfF was introduced into the wild-type strain, cell surface pilus expression was abolished. This showed that CsfF is rate-limiting for the control of pilus length, and unlike CsfE, increasing expression of CsfF results in pili that are significantly shorter than those of the wild type. Based on its amino acid similarity to the predicted CsfB chaperone, CsfF is predicted to function as a specific chaperone for delivering CsfE to the outer membrane assembly protein CsfC in order to terminate pilus elongation.
Although no CsfD minor subunits could be detected in the pili produced in the csfF deletion strain, CsfD was detected at levels comparable to those of the wild type in the periplasm. Therefore, the absence of csfF abrogates delivery of CsfD across the outer membrane. This implies that CsfF may also function as the specific chaperone for CsfD but may not be required for the stability of CsfD in a csfF deletion strain. Clearly, some CsfD must be assembled onto the cell surface, since CsfD has previously been shown to be absolutely required for initiation of CS5 pilus biogenesis (6). However, the level of CsfD is undetectable under the conditions used.
To explain this observation, a double in-frame deletion mutation in the csfB and csfF genes was constructed, thereby establishing directly whether some functional redundancy may exist between CsfB and CsfF. This double-deletion mutant resulted in the absence of CsfA and a large decrease in the level of detectable CsfD subunits in the periplasm. When a wild-type copy of csfF was provided in trans, CsfD levels in the periplasm were stabilized. Likewise, when csfB was reintroduced in trans, CsfA accumulation in the periplasm was restored. Therefore, CsfF appears to be directly responsible for the stability of CsfD in the periplasm, but probably also for that of CsfE, and thus functions as a chaperone.
So why is CsfD detected in the periplasm, but not in the final pilus structure, in the absence of CsfF but not in the absence of CsfB? The presence of CsfD in the periplasm in a csfF mutant can be explained in terms of the relative copy number of the plasmid system used and the altered binding specificity of CsfD in the absence of CsfF. Since the regulator of CS5 pili expression has never been directly demonstrated, significant expression of this pilus can be achieved only from high-copy-number plasmids (7). In the absence of CsfF, CsfD may interact weakly with CsfB alone or possibly with CsfB-CsfA complexes in the periplasm. It is unclear how pilus initiation occurs in this case, but it is possible that a CsfB-CsfA-CsfD complex, or indeed CsfD aggregates alone, may be able to associate with CsfC in the outer membrane, resulting in subunit translocation and the targeting of further complexes to the usher. Clearly, complexes of the CsfA-CsfB type are favored for assembly in this case, since CsfD is not detected. When the double deletion was complemented with csfB and csfF on low-copy-number plasmids in trans, the decreases in levels of these proteins relative to the subunits originating from the high-copy-number plasmids could lead to the chaperones binding only the pilins for which they have the greatest affinity. This may explain why only CsfA accumulates when CsfB is provided in trans and likewise why CsfD predominates when CsfF is provided. Since no CsfA is retained in a single csfB mutant, it is unlikely that CsfF shows any binding specificity for CsfA to help stabilize this protein in the periplasm.
The CsfB and CsfF chaperones do not show any discernible sequence or structural similarity with the prototypic member of the immunoglobulin-like chaperones, PapD (P = 0.89). CsfB and CsfF did, however, share 4 out of 10 and 3 out of 10, respectively, of the invariant residues associated with the 26 known members of the superfamily of the immunoglobulin-like chaperones (12). The present report on the CS5 pilus biogenesis operon is the first description of a dual-chaperone system for any human ETEC pilus system, in which one chaperone appears to be directly responsible for stabilizing and delivering the major subunit protein and the other is specifically responsible for stabilizing and delivering minor subunits to the outer membrane. Only one other pilus biogenesis system, 987P, which is an important colonization factor on porcine ETEC strains, contains two chaperones with similar functions (8). FasB is the periplasmic chaperone for the major pilin subunit FasA, and similarly, FasC chaperones the 987P adhesin FasG (8). The CsfB and CsfF chaperones bear no homology to the 987P chaperones. Binding experiments will need to be performed to specifically show that a protein interaction occurs between the CS5 chaperones and their cognate pilins.
Interestingly, the csf cluster also shows a genetic organization similar to that of 987P from porcine ETEC (18). Both systems utilize two chaperones, one for minor subunits and one for major subunits, and if the fasH regulatory gene is not considered, both contain the same number of genes encoding structural and assembly proteins. Furthermore, a large stem-loop structure (
G = -21.1) is located between the fasA major-subunit gene and the fasB chaperone gene (8). This is identical in organizational terms to the stem-loop structure located between the csfA major-subunit gene and the csfB major-subunit chaperone gene, which is predicted to act as an attenuator sequence to reduce expression of downstream genes (4).
A summary of the proposed model for CS5 assembly is shown in Fig. 10. All of the Csf proteins are translocated across the inner membrane via the Sec-dependent pathway. The major pilin subunit, CsfA, is then bound by the CsfB chaperone, and the minor pilin subunits, CsfD and CsfE, are bound by the CsfF chaperone. This serves to protect the pilin subunits from misfolding, premature aggregation, or degradation by periplasmic proteases. Pilus biogenesis is thought to be initiated by CsfD-CsfF complexes binding to the outer membrane assembly protein CsfC, which results in the translocation of CsfD across the outer membrane and leaves CsfC with an altered conformation, in an assembly-competent state. Pilus elongation occurs by means of multiple CsfA-CsfB interactions with CsfC in the outer membrane, driving the assembly of CsfA into the growing pilus, along with further CsfD-CsfF interactions. The rate of incorporation of CsfA versus CsfD is thought to depend on the stoichiometric ratio of the two pilins in the periplasm. CsfD is thought to add flexibility to the CS5 structure (Fig. 10, magnified region). Pilus termination occurs when CsfE-CsfF complexes are targeted to CsfC, which is predicted to result in the irreversible association of CsfE with CsfC, thereby preventing further polymerization of the pilus.
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ACKNOWLEDGMENTS |
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We thank Lothar Staendner for useful discussions.
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FOOTNOTES |
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Department of Microbiology and Infectious Diseases, Flinders Medical Centre, Bedford Park, South Australia, 5041, Australia.
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