Ramifications of kinetic partitioning on usher-mediated pilus biogenesis
1998; Springer Nature; Volume: 17; Issue: 8 Linguagem: Inglês
10.1093/emboj/17.8.2177
ISSN1460-2075
Autores Tópico(s)Biochemical and Structural Characterization
ResumoArticle15 April 1998free access Ramifications of kinetic partitioning on usher-mediated pilus biogenesis E. T. Saulino E. T. Saulino Department of Molecular Microbiology and Microbial Pathogenesis, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8230, St Louis, MO, 63110 USA Search for more papers by this author D. G. Thanassi D. G. Thanassi Department of Molecular Microbiology and Microbial Pathogenesis, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8230, St Louis, MO, 63110 USA Search for more papers by this author J. S. Pinkner J. S. Pinkner Department of Molecular Microbiology and Microbial Pathogenesis, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8230, St Louis, MO, 63110 USA Search for more papers by this author S. J. Hultgren Corresponding Author S. J. Hultgren Department of Molecular Microbiology and Microbial Pathogenesis, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8230, St Louis, MO, 63110 USA Search for more papers by this author E. T. Saulino E. T. Saulino Department of Molecular Microbiology and Microbial Pathogenesis, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8230, St Louis, MO, 63110 USA Search for more papers by this author D. G. Thanassi D. G. Thanassi Department of Molecular Microbiology and Microbial Pathogenesis, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8230, St Louis, MO, 63110 USA Search for more papers by this author J. S. Pinkner J. S. Pinkner Department of Molecular Microbiology and Microbial Pathogenesis, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8230, St Louis, MO, 63110 USA Search for more papers by this author S. J. Hultgren Corresponding Author S. J. Hultgren Department of Molecular Microbiology and Microbial Pathogenesis, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8230, St Louis, MO, 63110 USA Search for more papers by this author Author Information E. T. Saulino1, D. G. Thanassi1, J. S. Pinkner1 and S. J. Hultgren 1 1Department of Molecular Microbiology and Microbial Pathogenesis, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8230, St Louis, MO, 63110 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:2177-2185https://doi.org/10.1093/emboj/17.8.2177 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The biogenesis of diverse adhesive structures in a variety of Gram-negative bacterial species is dependent on the chaperone/usher pathway. Very little is known about how the usher protein translocates protein subunits across the outer membrane or how assembly of these adhesive structures occurs. We have discovered several mechanisms by which the usher protein acts to regulate the ordered assembly of type 1 pili, specifically through critical interactions of the chaperone–adhesin complex with the usher. A study of association and dissociation events of chaperone–subunit complexes with the usher in real time using surface plasmon resonance revealed that the chaperone–adhesin complex has the tightest and fastest association with the usher. This suggests that kinetic partitioning of chaperone–adhesin complexes to the usher is a defining factor in tip localization of the adhesin in the pilus. Furthermore, we identified and purified a chaperone–adhesin–usher assembly intermediate that was formed in vivo. Trypsin digestion assays showed that the usher in this complex was in an altered conformation, which was maintained during pilus assembly. The data support a model in which binding of the chaperone–adhesin complex to the usher stabilizes the usher in an assembly-competent conformation and allows initiation of pilus assembly. Introduction A critical virulence mechanism common to most pathogenic bacteria is adherence to host tissues. Initial colonization events have been shown to be crucial in many different infectious processes, e.g. the binding of Escherichia coli to Galα(1–4)Gal sugar moieties in the kidney prior to development of pyelonephritis (Roberts et al., 1994). Bacterial adhesins mediate attachment by binding with stereochemical specificity to complementary receptors present on eukaryotic cells. Adhesins can be assembled onto the surfaces of bacteria as monomers, simple oligomers or as parts of supramolecular fibers (Hultgren et al., 1996). The most well-characterized adhesive fibers are known as pili or fimbriae, which are rod-like organelles 5–7 nm in diameter (Mitsui et al., 1973; Hultgren et al., 1996). Type 1 pili are produced by the entire Enterobacteriaceae family. Their expression requires at least eight genes organized in the type 1 gene cluster (Hull et al., 1981; Orndorff and Falkow, 1984) (Figure 1). Type 1 pili consist of two distinct subassemblies: a thin tip fibrillum which is joined to the distal end of a pilus rod (Jones et al., 1995). The 7 nm wide, 5–7 μm long rod is comprised of FimA subunits arranged in a right-handed helical conformation with 3.125 residues per turn (Mitsui et al., 1973). The tip fibrillum is ∼16 Å long and primarily contains the adhesive moiety of the pilus, FimH (Jones et al., 1995). In uropathogenic E.coli, FimH mediates binding to mannose-containing receptors (Minion et al., 1989; Krogfelt et al., 1990) exposed on the luminal surface of both the human and mouse bladder epithelium (Langermann et al., 1997). This binding event has been shown to be critical in the ability of uropathogenic E.coli strains to colonize the bladder and cause cystitis in mice (Langermann et al., 1997). A FimH-based vaccine has been shown to elicit IgG antibodies that block colonization in vivo and protect mice against a mucosal infection of the bladder and subsequent ascending urinary tract infections (Langermann et al., 1997). FimF and FimG have been suggested to play roles in initiation and termination of pilus assembly, respectively (Russell and Orndorff, 1992). Figure 1.Schematic representation of the type 1 (fim) and P (pap) gene clusters listing the functions of the various gene products. Download figure Download PowerPoint The assembly of >27 different adhesive organelles in diverse Gram-negative bacteria requires a periplasmic chaperone and an outer membrane usher (Hung et al., 1996). FimC is the periplasmic chaperone required for type 1 pilus assembly (Klemm et al., 1992; Jones et al., 1993). It is one of the 27 members of the PapD chaperone family which are 30–60% identical to one another in amino acid sequence (Hung et al., 1996). The crystal structure of PapD, the chaperone involved in P-pilus assembly, has been solved to 2.5 Å resolution. The structure revealed a two-domain boomerang-shaped molecule with each domain having an immunoglobulin-like topology (Holmgren and Brändén, 1989). Molecular modeling revealed that all of the PapD-like chaperones adopt similar immunoglobulin-like two-domain structures (Hung et al., 1996). PapD-like chaperones bind to the C-terminus of subunits via a beta zippering interaction (Kuehn et al., 1993; Bullitt et al., 1996). This interaction results in the partitioning of a subunit from a membrane-associated state to a chaperone-associated periplasmic complex (Jones et al., 1997). The maintenance of the C-terminus of the subunit in an extended conformation in the context of the chaperone may predispose subsequent folding reactions of the subunit on the chaperone template (Jones et al., 1997). Chaperone–subunit complexes are targeted to outer membrane ushers where the chaperone is dissociated. This exposes the conserved C-terminal region which is involved in mediating subunit–subunit interactions within the assembled organelle (Soto,G., Dodson,K., Liu,C., Jones,C.H., Ogg,D., Knight,S. and Hultgren,S.J., in preparation). FimD is the outer membrane usher required for type 1 pilus assembly. When fimD is disrupted with a Tn5 insertion, bacteria are unable to produce type 1 pili (Klemm and Christiansen, 1990). Recently it was shown that the usher forms a channel, possibly providing for the translocation of subunits across the outer membrane (Thanassi et al., 1998); however, little is known about the structure, function or mechanism of action of usher proteins. PapC, the usher in the P-pilus system (Figure 1), has been shown to differentially recognize chaperone–subunit complexes relative to the final position of the subunit in the pilus (Dodson et al., 1993). This information has led to the hypothesis that the final position of a subunit in the pilus is determined in part by the strength with which the subunits (complexed with the chaperone) bind to the usher assembly site. The usher is thus a multifunctional protein that presumably has a number of functional regions including membrane-spanning, pore-forming and periplasmic domains. The periplasmic domain presumably interacts with incoming chaperone–subunit complexes. Molecular modeling and topology studies suggest that the FaeD usher has a middle transmembrane portion, and that the N- and C-terminal regions of the protein form periplasmic domains (Valent et al., 1995). Despite the critical role of pili in colonization and virulence, the usher-mediated mechanism by which bacteria are able to incorporate an adhesin into the pilus is poorly understood. The experiments reported here have uncovered critical chaperone–subunit–usher interactions that have revealed a kinetic partitioning mechanism by which the usher mediates ordered pilus assembly. Due to the almost ubiquitous use of membrane-associated adhesive organelles and secreted proteins by pathogenic bacteria, understanding the events leading to the biogenesis of adhesive organelles and protein translocation across the outer membrane is of fundamental importance. Utilizing the tools we have developed in the type 1 pilus system, we have the unique opportunity to dissect the initial events in the process by which a virulence-associated adhesin is translocated across the outer membrane and incorporated into an adhesive organelle. Results Purification and characterization of the usher and chaperone–subunit complexes A histidine tag was genetically engineered onto the 3′ end of the fimD (usher) gene to facilitate purification. This gene product was fully functional in vivo as it was able to complement a fimD− type 1 operon (pETS6) using a standard hemagglutination assay (see Materials and methods). The plasmid containing His-tagged fimD, pETS4, was transformed into the type 1− strain, ORN103, and protein production was induced. The 92 kDa FimD protein was present in outer membrane preparations as determined by SDS–PAGE (Figure 2A) and N-terminal sequencing. FimD was purified to >90% homogeneity by subjecting the outer membrane preparations to Ni2+ chromatography (Figure 2A, lane 5). Figure 2.Purification of usher and chaperone–subunit complexes. (A) Coomassie Blue-stained 12.5% SDS–PAGE of ORN103/pETS4 membrane fractions. FimD expression was induced with 20 μM IPTG (lanes 2 and 4). Lanes 1 and 3 are the uninduced controls. Lanes 1 and 2 are inner membrane fractions; lanes 3 and 4 are outer membrane fractions. FimD was only observed in the induced outer membrane fractions (lane 4, arrow). Lane 5 shows FimD purified from the outer membrane extracts by Ni2+ chromatography (see Materials and methods). Molecular weight markers in all the figures are in kilodaltons. (B) Coomassie Blue-stained 12.5% SDS–PAGE showing the FimC chaperone alone (lane 5), and FimCH (lane 1), FimCG (lane 2), FimCF (lane 3) and FimCA (lane 4) complexes purified by Ni2+ chromatography and FPLC as described in Materials and methods. Download figure Download PowerPoint A histidine tag was also genetically engineered onto the 3′ end of the fimC (chaperone) gene to facilitate purification of chaperone–subunit complexes. The FimC-His tag protein was functional in assembling type 1 pili as it was able to complement a fimC− type 1 operon (pETS12) (see Materials and methods). We transformed the plasmid pETS1000 containing His-tagged fimC into ORN103 alone or in combination with pETS2A (fimG), pETS1 (fimF), pHJ20 (fimH) or pETS5 (fimA). Following protein induction, periplasmic extracts containing chaperone–subunit complexes were subjected to Ni2+ chromatography and FPLC. Subunits bound to FimC were co-purified as chaperone–subunit (CH, CG, CF and CA) complexes as determined by SDS–PAGE (Figure 2B). These results reveal that FimC binds to and forms chaperone–subunit complexes with minor subunits (FimG and FimF) and the major subunit (FimA). FimC and FimC–FimH complexes have been identified and purified previously (Jones et al., 1993). Chaperone–subunit–usher interactions We used the purified proteins shown in Figure 2 in an enzyme-linked immunosorbent assay (ELISA) to investigate potential interactions between the FimD usher and the chaperone or the various chaperone–subunit complexes. A similar ELISA procedure was performed previously in the study of the PapC usher (Dodson et al., 1993). FimC–FimH complexes had the highest affinity for FimD (Figure 3). Binding of all of the other complexes and of FimC alone to FimD also occurred, but at a very low level. The strong binding of FimC–FimH complexes to FimD may in part facilitate the localization of the FimH adhesin at the distal end of the tip fibrillum of type 1 pili. This is consistent with the model that Dodson et al. (1993) proposed for differential recognition of chaperone–subunit complexes in P-pilus assembly. It is not apparent from the ELISA data what roles might be played by the low level of FimC, FimC–FimG, FimC–FimF and FimC–FimA binding. Figure 3.Chaperone–subunit–usher interactions measured via ELISA. Microtiter plate wells were coated overnight with 5 μg/ml of pure FimD. Wells were blocked with 3% BSA/PBS and then various concentrations of pure FimCA (-□-), FimCF (-⋄-), FimCG (-×-), FimCH (-●-), or FimC alone (-○-) were incubated in the wells for 45 min at room temperature. Controls were done with no FimD and with no complexes added (see Materials and methods). Binding to FimD was detected with anti-FimC antibody followed by alkaline phosphatase-linked anti-rabbit IgG. Absorption at 405 nm was read after addition of p-nitrophenol phosphate. Points shown represent an average value from experiments done in triplicate. Download figure Download PowerPoint Investigation of real time binding events We reasoned that a study of association and dissociation events in real time would yield additional insight into the ordered mechanism of pilus assembly. Using surface plasmon resonance technology (Karlsson et al., 1991), we attempted to determine both kinetic and equilibrium association and dissociation constants. Using a Biacore 2000 (Biosensor) machine, we immobilized the FimD usher on the surface of a research grade CM5 Biosensor chip using a standard amide cross-linking procedure. Then, we injected various concentrations of purified FimC chaperone or the various chaperone–subunit complexes over the immobilized usher. Representative binding curves for FimCH are shown in Figure 4A. Equilibrium evaluation showed that the FimC chaperone alone was unable to bind to FimD under these experimental conditions. FimCA, FimCG and FimCF bound relatively weakly to the usher, with calculated equilibrium constants (KD) of 176 nM (FimA), 670 nM (FimCG) and 1.37 μM (FimCF) (Figure 4B). In contrast, chaperone–adhesin (FimCH) complexes bound to FimD 20- to 150-fold better than the other complexes, having a KD = 9.1 nM. The kinetic basis of the higher affinity of FimCH resided in the kon rate (4.3×105 M−1 s−1) which was ∼8- to 35-fold faster than the kon rates of FimCG, FimCF and FimCA complexes (Figure 4C). In each interaction of the usher with the chaperone–subunit complexes, the koff was very slow, as only ∼1.0% of the total number of chaperone–subunit–usher complexes dissociated per second (Figure 4D). Complete pilus assembly occurs in minutes (Jacob-Dubuisson et al., 1994); therefore, in physiological terms, individual chaperone–subunit complexes essentially remain attached to the usher once the initial interaction occurs. The differences in rates of dissociation would not necessarily affect the order of events in pilus assembly because before FimCH could dissociate from FimD, other subunits would presumably be added to FimH, resulting in the growth of a pilus. Accordingly, the ability of FimCH complexes to associate first or fastest with FimD appears to be a critical factor in insuring the tip localization of the FimH adhesin in the pilus. Figure 4.Real-time analysis of chaperone–subunit–usher interactions. (A) Representative binding curves from FimCH–D interactions. The curves (from top to bottom) represent decreasing concentrations of FimCH complex (from 1.0 to 0.075 μM) injected over immobilized FimD. Binding of FimCH to FimD is detected by the Biacore machine and converted to an arbitrary response unit (RU) over time (s) that can be analyzed to obtain kinetic and equilibrium constants. The regions representing real-time association and dissociation events are labeled. (B) Log scale graphs showing equilibrium dissociation constants (KD in molar concentration units) for various chaperone–subunit–usher interactions in the type 1 (left graph) and P-pilus (right graph) systems. For (B–D), the numbers listed above each bar in the graph represent the actual binding constant for that particular chaperone–subunit complex. The columns labeled PapDA* represent experiments done with PapAG150T (Bullitt et al., 1996) complexed with PapD. Standard errors shown graphically for koff and kon are an average of the standard errors from all measurements taken for a particular complex. The standard error for KD is a combination of the errors from the koff and kon (see Materials and methods). The chaperone–adhesin (FimCH or PapDG) complex in each system bound to the usher with a much greater affinity than any of the chaperone–subunit complexes. The chaperone alone (FimC or PapD) did not show any binding to the usher (FimD or PapC, respectively) when injected (up to 10.0 μM). The chaperone–adhesin complex (PapDG or FimCH) that was 'mismatched' (to FimD or PapC, respectively) also did not show any binding when injected (up to 2.0 μM). (C) Log scale graphs showing kinetic association constants (kon in M−1 s−1) for type 1 and P-pilus systems. The chaperone–adhesin complexes in both systems bound fastest. (D) Log scale graphs showing kinetic dissociation constants (koff in s−1) for type 1 and P-pilus systems. The 'off' rate is slow for all of the complexes (∼1% of the complexes dissociating per second). Download figure Download PowerPoint Biacore binding experiments were also done using the P-pilus system to determine if the principle of a rapid association and slow dissociation of chaperone–adhesin complexes to the usher is a general phenomenon. Analogous to the experiments with the type 1 system, the PapC usher was the immobilized ligand. The binding analytes were the PapD chaperone, a PapDG chaperone–adhesin complex and a chaperone–subunit complex (PapDAG150T). PapA subunits make up the pilus rod (Bullitt et al., 1996). The G150T PapA mutant was used because it does not self-aggregate like wild-type PapA (Bullitt et al., 1996). Self-association could possibly interfere with the Biacore analysis. As was found in the type 1 system, the PapDG chaperone–adhesin complex had a 16-fold greater affinity (KD = 90 nM) (Figure 4B) and a 29-fold faster kinetic rate of association (kon = 5.7×104 M−1 s−1) (Figure 4C) with the usher in comparison with the PapDAG150T complex. Additionally, the kinetic dissociation rates (koff) remained slow (Figure 4D) and the PapD chaperone alone was unable to interact with the usher (Figure 4B). Therefore, analogous to what was seen in the type 1 pilus system, the faster association rate of the PapDG complex with the PapC usher may play a critical role in enabling the PapG adhesin to take its place as the first subunit incorporated into the pilus. In previous experiments (Dodson et al., 1993), the chaperone–major subunit complex (PapDA) was not shown to bind to the usher (PapC). The fact that we were able to detect binding of PapDA and of FimCA to their respective ushers (Figure 4B) is probably due to the use of pure protein and to the increased sensitivity of the surface plasmon resonance technique. The inability to detect binding of the chaperone alone or the mismatched chaperone–adhesin complex to the usher (Figure 4B) argues that even the weak interactions observed are biologically relevant. Changes in trypsin susceptibility of FimD upon FimCH binding Next, we wanted to determine if usher–chaperone–subunit interactions could be detected in vivo. We hypothesized that these interactions might result in conformational changes in the usher which could be detected by differences in sensitivity to trypsin. This assay was used successfully for studying surface-exposed loops and mapping the topology of the K88 usher, FaeD (Valent et al., 1995). Trypsin digestion of whole cells expressing the FimD usher alone or co-expressed with either FimC, FimCF, FimCG, FimCH or FimCA was followed by Western blotting of outer membrane preparations using anti-FimD antiserum (Figure 5). When FimD was expressed alone or with FimC, FimCF, FimCG or FimCA individually, it was susceptible to complete proteolytic degradation by trypsin under the conditions used. It was somewhat surprising to find the outer membrane usher protein to be completely susceptible to externally added trypsin. This indicates that FimD must have a high number of surface-exposed trypsin-cleavable sites or that cleavage at one or a few sites leads to destabilization and further degradation of the protein. However, as seen in Figure 5, lane 13, a large portion of the FimD usher was protected from proteolytic digestion when the chaperone–adhesin complex, FimCH, was co-expressed. The same results were obtained when whole cell suspensions were probed by Western blot analysis (data not shown), arguing that additional digestion did not occur during preparation of the outer membranes. The FimD fragment protected by co-expression of FimCH was purified by Ni2+ chromatography (Figure 7) and N-terminally sequenced, demonstrating that it represents a 40 kDa C-terminal domain of FimD beginning with amino acid K477 of the mature protein. Cleavage occurred in an extracellular region of FimD, because the periplasm was not accessible to trypsin in these whole cell experiments. This was confirmed by showing that the periplasmic FimC chaperone, which is trypsin-sensitive (unpublished data), was not cleaved by trypsin in this experiment (data not shown). Finally, if we lowered the concentration of trypsin or decreased the digestion time, a small amount of this protected 40 kDa fragment could be seen in an anti-FimD Western blot even when FimD was expressed by itself or with any of the other Fim proteins (data not shown). This suggests that the FimD usher may exist in an equilibrium between protected and unprotected conformations, and that interaction of FimCH with FimD drives the usher into the protected form. Figure 5.Susceptibility of FimD to cleavage by extracellular trypsin. The presence of FimD in outer membrane extracts was analyzed by Western blotting with anti-FimD antiserum before (lanes 1–7) or after (lanes 9–15) the cells were treated with trypsin. Lane 8 shows molecular weight markers. FimD was expressed by itself (lanes 1 and 15) or co-expressed with FimC (lanes 2 and 14), FimCH (lanes 3 and 13), FimCG (lanes 4 and 12), FimCF (lanes 5 and 11) or FimCA (lanes 6 and 10). As a negative control, FimCH alone was expressed (lanes 7 and 9). The N-terminal sequence of the trypsin-protected portion of FimD (lane 13) was FTDYYN. This placed the trypsin cleavage site at K477 of the mature protein. Download figure Download PowerPoint We next investigated whether the trypsin-protected form of FimD induced by interaction with the FimCH complex is maintained throughout pilus biogenesis. The state of type 1 pilus-assembling FimD was investigated using ORN103 cells containing the plasmid pSH2, which carries the entire type 1 operon (Orndorff and Falkow, 1984), or pETS6, a FimD knock-out of pSH2 that does not produce pili (unpublished data). We also used the clinical isolate NU14 (Hultgren et al., 1986) and a fimH− mutant of NU14, NU14-1 (Langermann et al., 1997). The cells were grown under conditions that resulted in the expression of type 1 pili, as confirmed by their ability to hemagglutinate guinea pig erythrocytes (see Materials and methods), and then subjected to the trypsin treatment described above. In all cases, the 40 kDa portion of the FimD usher that was protected by the co-expression of FimCH was also protected during expression of type 1 pili (Figure 6). This indicates that the conformation of the usher that is stabilized by its interaction with the chaperone–adhesin complex is maintained throughout pilus assembly. It should be noted that in these experiments a substantial portion of full-length FimD remained undigested by trypsin. This could be due to steric inhibition of the protease by the 7 nm wide pilus rod. Figure 6.Trypsin susceptibility of FimD in cells producing pili. Outer membrane preparations of piliated NU14 or NU14-1 (fimH−) cells (left gel) or ORN103 cells containing the type 1 pilus operon [pSH2 or pETS6 (fimD−)] (right gel) were analyzed for the presence of FimD by Western blotting with anti-FimD antiserum before (lanes 1, 3, 5 and 7) or after (lanes 2, 4, 6 and 8) the cells were treated with trypsin. The abnormally wavy pattern of FimD migration (lanes 1–4) is probably due to the presence of wild-type lipopolysaccharide in the clinical strain NU14. The different mobility of FimD in NU14 (lane 1) versus FimD in NU14-1 (lane 3) may be due to the assembly state of the usher as pili are being assembled in NU14 but not in NU14-1 (Langermann et al., 1997). Download figure Download PowerPoint Figure 7.Purification of the ternary FimC–FimH–FimD assembly intermediate. (A) Coomassie Blue-stained 12.5% SDS–PAGE of outer membrane preparations after Ni2+ chromatography. The preparations were from cells in which His-tagged FimD (from pETS4) was co-expressed with non-His tagged FimCG (lane 1), FimCF (lane 2), FimCA (lane 3) or FimCH (lane 5). Lane 4 shows that non-His-tagged FimCH alone does not stick to Ni2+ beads. Lane 6 shows that the 40 kDa C-terminal fragment protected from extracellular trypsin digestion can be purified by Ni2+ chromatography as a ternary complex with FimCH. (B) Western blots of the FimD–FimC–FimH complex eluted as a single peak from a Superdex 200 column after Ni2+ chromatography and developed with anti-FimD (lanes 1) or anti-FimCH (lanes 2) antibodies. Western blot of an extract from trypsin-treated cells identical to that shown in (A), lane 6, developed with anti-FimD (lane 3) or anti-FimCH (lane 4). Download figure Download PowerPoint Isolation of a FimD–FimC–FimH assembly intermediate formed in vivo The values derived from the Biacore experiments suggest a strong and long-lasting interaction between FimCH and FimD, and the trypsin digestion results show that this interaction occurs in vivo. Therefore, we attempted to isolate a pilus assembly intermediate targeting the step at which the FimD usher interacts with FimC–FimH complexes. We co-expressed His-tagged FimD (pETS4) with non-His-tagged FimC and FimH (pETS1004) in ORN103 cells, and purified FimD using Ni2+ chromatography as discussed above. We found that FimC and FimH co-purified with FimD, suggesting a stable, ternary, usher–chaperone–adhesin (FimD–FimC–FimH) complex (Figure 7A, lane 5). Non-His-tagged FimCH complexes expressed in the absence of FimD could not be purified by Ni2+ chromatography (Figure 7A, lane 4). This is the first demonstration of the in vivo formation of an usher intermediate in pilus assembly. When FimCA, FimCG or FimCF were co-expressed with FimD, none of these chaperone–subunit complexes co-purified with the usher (Figure 7A, lanes 1–3), consistent with their weak interactions as seen in the Biacore experiments and their inability to protect FimD from trypsin. The putative FimD–FimC–FimH complex was analyzed by FPLC gel filtration chromatography (Superdex 200) and was shown to elute as a single peak (Figure 7B, lanes 1 and 2). The sensitivity of the ternary complex to trypsin was also analyzed. We co-expressed the FimD usher with FimCH and subjected the cells to the tryptic digestion reaction as discussed above. The outer membrane fraction was then subjected to Ni2+ chromatography which resulted in the co-purification of the 40 kDa C-terminal fragment of FimD with FimCH (Figure 7A, lane 6, and B, lanes 3 and 4). These data demonstrates that the probable binding site for the FimCH chaperone–adhesin complex is in the C-terminal portion of FimD. Discussion Presentation of a pilus-bound adhesin protein for optimal binding to eukaryotic cells is a critical event in bacterial pathogenesis (Roberts et al., 1994; Langermann et al., 1997). The assembly of >27 diverse adhesive fibers in many different Gram-negative bacteria occurs via a multistep chaperone–usher pathway (reviewed in Hultgren et al., 1996). In the terminal step, protein subunits are translocated across the outer membrane and assembled into composite heteropolymeric organelles in a defi
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