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The number of Neisseria meningitidis type IV pili determines host cell interaction

2014; Springer Nature; Volume: 33; Issue: 16 Linguagem: Inglês

10.15252/embj.201488031

1460-2075

Anne‐Flore Imhaus, Guillaume Duménil,

Pneumonia and Respiratory Infections

Article26 May 2014free access The number of Neisseria meningitidis type IV pili determines host cell interaction Anne-Flore Imhaus Anne-Flore Imhaus INSERM, U970, Paris Cardiovascular Research Center, Paris, France Faculté de Médecine Paris Descartes, Université Paris Descartes, Paris, France Search for more papers by this author Guillaume Duménil Corresponding Author Guillaume Duménil INSERM, U970, Paris Cardiovascular Research Center, Paris, France Faculté de Médecine Paris Descartes, Université Paris Descartes, Paris, France Search for more papers by this author Anne-Flore Imhaus Anne-Flore Imhaus INSERM, U970, Paris Cardiovascular Research Center, Paris, France Faculté de Médecine Paris Descartes, Université Paris Descartes, Paris, France Search for more papers by this author Guillaume Duménil Corresponding Author Guillaume Duménil INSERM, U970, Paris Cardiovascular Research Center, Paris, France Faculté de Médecine Paris Descartes, Université Paris Descartes, Paris, France Search for more papers by this author Author Information Anne-Flore Imhaus1,2 and Guillaume Duménil 1,2 1INSERM, U970, Paris Cardiovascular Research Center, Paris, France 2Faculté de Médecine Paris Descartes, Université Paris Descartes, Paris, France *Corresponding author. Tel: +33 1 53 98 80 49; Fax: +33 1 53 98 79 53; E-mail: [email protected] The EMBO Journal (2014)33:1767-1783https://doi.org/10.15252/embj.201488031 See also: V Karuppiah & JP Derrick (August 2014) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract As mediators of adhesion, autoaggregation and bacteria-induced plasma membrane reorganization, type IV pili are at the heart of Neisseria meningitidis infection. Previous studies have proposed that two minor pilins, PilV and PilX, are displayed along the pilus structure and play a direct role in mediating these effects. In contrast with this hypothesis, combining imaging and biochemical approaches we found that PilV and PilX are located in the bacterial periplasm rather than along pilus fibers. Furthermore, preventing exit of these proteins from the periplasm by fusing them to the mCherry protein did not alter their function. Deletion of the pilV and pilX genes led to a decrease in the number, but not length, of pili displayed on the bacterial surface indicating a role in the initiation of pilus biogenesis. By finely regulating the expression of a central component of the piliation machinery, we show that the modest reductions in the number of pili are sufficient to recapitulate the phenotypes of the pilV and pilX mutants. We further show that specific type IV pili-dependent functions require different ranges of pili numbers. Synopsis PilV and PilX pilins of Neisseria meningitidis type IV pili regulate pili number rather than their composition, suggesting that meningitidis infection-associated adhesion and plasma membrane re-organization require a minimum number of pili rather than specific subtypes. PilX and PilV minor pilins exert their effects for the periplasm rather than inserted in the pilus fiber. Inactivation of the pilV and pilX genes leads to altered pilus biogenesis resulting in a mild decrease in the number of pili. Decreasing the number of type IV pili is sufficient to recapitulate the defects observed in the pilV and pilX mutants. Introduction Type IV pili are filamentous organelles found on the surface of a large number of bacterial species including several pathogens (Pelicic, 2008). They can be found in different groups of proteobacteria in species such as Vibrio cholerae, enteropathogenic Escherichia coli, Pseudomonas aeruginosa, and Neisseria spp. More recently, they were also found in firmicutes such as Clostridium perfringens, illustrating the wide distribution of these structures (Varga et al, 2006). Type IV pili are part of a larger group of bacterial machineries. It is becoming increasingly clear that type II secretion systems share several features with type IV pili (Hobbs & Mattick, 1993; Giltner et al, 2012). Competence systems and flagella in archaea also appear to function through common mechanisms (Peabody et al, 2003). Type IV pili and related structures are thus one of the building blocks of the archeal and bacterial world. The diversity and importance of functions carried out by type IV pili are illustrated by the case of Neisseria meningitidis. In 5–30% of the total human population, N. meningitidis thrives in the nasopharynx without triggering any damage (Caugant et al, 1994). Bacteria proliferate in bacterial aggregates, referred to as microcolonies, in tight association with the epithelial surface (Stephens et al, 1983). In vitro studies indicate that both adhesion to epithelial cells and auto aggregation are mostly mediated by type IV pili (Carbonnelle et al, 2006). In addition, DNA exchange between different N. meningitidis strains occurs via the natural transformation properties mediated by type IV pili (Weyand et al, 2013). From the throat, bacteria can occasionally access the bloodstream and trigger septic shocks and meningitis. In the circulation, bacteria bind to the endothelium and proliferate, often filling the vessel lumen (Guarner et al, 2004; Mairey et al, 2006). A recent study involving a humanized model of infection points to the importance of type IV pili in the ability of the bacteria to adhere to human vessels and to trigger vascular damage (Melican et al, 2013). Following adhesion to endothelial cells an intense crosstalk is initiated by type IV pili that leads to the reshaping of the host cell plasma membrane with the formation of filopodia-like protrusions (Eugene et al, 2002; Soyer et al, 2014). Type IV pili are therefore involved in numerous steps of the N. meningitidis infection process, in particular those involving interaction with the host cells. Type IV pili are composed of one main component, the major pilin, PilE in Neisseria spp. This protein is composed of two domains, a hydrophobic alpha helix and a beta sheet head that contains a carboxy-terminal disulfide bond. The major pilin assembles into a helical fiber with the alpha helix buried inside the center of the structure (Parge et al, 1995; Craig et al, 2006). Determining how this fibrous organelle carries out such diverse functions remains a central challenge and requires a better understanding of pilus biogenesis. Genetic screens have revealed that building a fully functional type IV pilus requires over 20 proteins (Carbonnelle et al, 2006; Brown et al, 2010) although the function of most of these proteins remains unknown. After translocation to the periplasm via the general secretion pathway, pilins are cleaved by the PilD peptidase (Lory & Strom, 1997). Once cleaved, the major pilin, assembled in fibers in the periplasm, exits the outer membrane through a pore composed of the PilQ secretin (Wolfgang et al, 2000). The system is powered by several ATPases, which provide the energy for the extension or retraction of the pili fibers. A subclass of proteins referred to as 'minor pilins' or 'pilin-like proteins' share structural features with the major pilin. These proteins contain a hydrophobic amino-terminal alpha helix and a carboxy-terminal disulfide bond (Winther-Larsen et al, 2005). Although a clear homology can be detected in the first 20–30 amino acids of the minor pilins, the rest of the primary sequence bears no homology, either among them or with the major pilin. Among this subclass, a triad of proteins, PilV, PilX, and ComP, displays the unique feature of conferring specific functions to pili rather than promoting pilus biogenesis. The corresponding mutants lose specific functions but still display type IV pili on their surface and maintain other functions. These three proteins and the major pilin also have in common a similar molecular weight range (15–20 kDa). The pilV, pilX, and comP genes are located in unrelated regions of the bacterial chromosome, however. Because type IV pili are still expressed by the corresponding mutants, these genes were qualified as 'accessory' to pilus biogenesis (Brown et al, 2010). In the absence of PilX, bacteria fail to aggregate, adhere to host cells, and trigger plasma membrane reorganization although they maintain competence (Helaine et al, 2005; Brissac et al, 2012). In the absence of the ComP minor pilin, competence is lost but pili are otherwise functional (Wolfgang et al, 1999). Finally, the pilV mutant adheres to host cells and is competent but fails to trigger plasma membrane reshaping upon bacterial adhesion (Mikaty et al, 2009). An attractive hypothesis is that these minor pilins are localized in the pilus fiber to carry out their effect (Helaine et al, 2007). In this view, the minor pilins are of central importance in pilus biology as they are the 'effectors' of this complex organelle. A number of reports have provided arguments in favor of this hypothesis. In particular, PilX was detected by immuno-electron microscopy associated with type IV pili (Helaine et al, 2007). The crystallographic structure of PilX showed the presence of a hook that could explain its role in aggregation by a homodimerization process (Helaine et al, 2007). In addition, the purified globular domain of PilV coated on the surface of beads was sufficient to lead to the accumulation of the β2-adrenergic receptor under the beads suggesting a direct role in triggering intracellular signals (Coureuil et al, 2010). In the case of ComP, the purified globular domain was recently shown to directly interact with DNA in a sequence-specific manner (Cehovin et al, 2013). Although the genetic evidence is compelling, questions remain concerning the mode of action of these proteins. First, in terms of localization, pilus association was addressed by electron microscopy in the case of PilX but not for PilV or ComP. In addition, localization of a proportion of the protein at a given site does not necessarily reflect the actual site of action. Furthermore, recent evidence suggests that PilX has a global effect on the conformation of pili implying a possible indirect impact on pilus function (Brissac et al, 2012). Because PilV and PilX are key players in type IV pili biology, the objective of this study was to determine how these minor pilins exert their functions. We provide evidence that these two minor pilins are required for efficient initiation of pilus biogenesis and that this effect is sufficient to explain the phenotype of the corresponding mutants. Results The majority of PilV and PilX proteins is localized in the periplasm rather than associated with pilus fibers The low amounts of protein expression, together with structural similarities among the minor pilins and the major pilin, make determination of the localization of the minor pilins difficult. To address this technical difficulty, minor pilins were tagged with the Flag epitope (Munro & Pelham, 1984). This strategy offers high sensitivity and specificity due to the affinity of available antibodies directed against this peptide. To determine the localization of PilV and PilX, strains expressing the minor pilins with a Flag tag placed in the loop formed by the disulfide bond (D-region) were generated. Flagged constructs were placed under the control of the lac promoter in the mutant background (pilVpilVFlagind and pilXpilXFlagind strains). Bacteria were cultured in the presence of 25 μM IPTG to mimic endogenous expression (Supplementary Fig S1). As a positive control, a Flag tag was inserted in the same site in the major pilin, PilE, and expressed under the control of the lac promoter (pilEpilEFlagind strain). Staining of the pilEpilEFlagind strain immobilized on coverslips with the anti-Flag monoclonal antibody showed fibers typical of type IV pili (Fig 1A, left panel). In contrast, in the pilVpilVFlagind and the pilXpilXFlagind strain, Flag staining could not be detected despite repeated attempts using a highly sensitive back-illuminated camera (Evolve, Photometrics). Occasionally, a single bacterium on these samples showed a strong staining around the bacterial body, and we speculated that these bacteria may have ruptured outer membranes. Accordingly, bacteria with experimentally lyzed outer membranes showed intense staining around the bacterial body, indicative of a periplasmic localization of both of the tagged proteins (Fig 1A, spheroplasts). To demonstrate the sensitivity of the technique, we co-expressed the pilEFlag construct under the control of the lac promoter with the endogenous pilE gene (pilEFlagind strain). In the presence of 10 μM of IPTG, reflecting a lower expression level than pilX or pilV genes, the tagged major pilin could be readily detected along type IV pili in the form of aligned dots (Fig 1B). Importantly, in both cases, for pilV and pilX, complementation indicated that the Flag-tagged proteins were fully functional including at expression levels mimicking the endogenous expression. The Flag-tagged PilV was able to restore bacteria-induced plasma membrane reorganization upon adhesion to human umbilical endothelial cells (HUVECs, Fig 1C), and the Flag-tagged PilX construct provided normal adhesion levels on the same cells (Fig 1D). These results indicate that a large amount of these proteins are localized in the periplasm rather than inside the pilus fiber. Figure 1. Periplasmic localization of the minor pilins PilV and PilX Immunofluorescence detection of Flag-tagged PilE (left), PilV (middle), and PilX (right). Bacteria were visualized by staining their DNA with DAPI (blue), and anti-Flag detection appears in green. In the case of PilV and PilX, the outer membrane had to be removed thus generating spheroplasts to obtain a signal. Demonstration of the sensitivity of the immunofluorescence detection. Flag-tagged PilE was co-expressed with the endogenous PilE although at a low level. Detection was done in the presence of 10 μM IPTG generating a lower level of expression for the Flag-tagged protein than endogenous PilX or PilV levels. Bacteria are visualized in blue with DAPI, pili in red using the 20D9 monoclonal antibody, and the PilEFlag in green with the anti-Flag polyclonal antibody. Arrowheads indicate examples of co-localization of the PilEFlag signal and type IV pili. Ability of the Flag-tagged PilV (pilVpilVFlagind strain) to promote plasma membrane reshaping. Bacteria were incubated with endothelial cells for a period of 2 h, and the fraction of microcolonies with efficient plasma membrane reorganization was determined using ezrin as a marker. The amount of added inducer (IPTG, μM) is indicated. Arrows indicate conditions mimicking wild-type levels of expression. As in all the figures, each dot represents an independent experiment. Ability of the Flag-tagged PilX (pilXpilXFlagind strain) to promote adhesion. Bacteria were incubated with endothelial cells for a period of 4 h, and the number of cell-associated bacteria was determined. Pili from the wild-type strain were purified by mechanical shearing and ammonium sulfate precipitation, and the amounts of the proteins of interest were analyzed by Western blot. A total lysate diluted 100-fold from the starting bacterial suspension assesses the total amount of proteins, inside and outside of the bacteria. A total lysate from the corresponding mutant indicates the specificity of the detection. Different dilutions of the result of the pilus preparation were loaded (tenfold, 1/10; threefold 1/3, and undiluted, 1). Quantitative analysis of pilus association. Intensity of the bands on the immunoblots generated by pilus purification was analyzed, and the proportion of pilus-associated protein was plotted relative to the total. Each dot represents one experiment. Data information: Data represent mean ± SEM; ****P ≤ 0.0001; ns, not significant. Download figure Download PowerPoint In order to determine the proportion of periplasmic versus pili-associated minor pilins, we took advantage of a well-established pilus purification approach based on mechanical shearing and ammonium sulfate precipitation (Wolfgang et al, 1998). A number of reports describe the co-purification of a certain amount of PilV and PilX with type IV pili but the proportion of co-purified minor pilins relative to the total amount of these proteins is unknown (Winther-Larsen et al, 2001; Helaine et al, 2005). Pili were purified from wild-type bacteria, and the amount of PilV, PilX and the major pilin PilE were determined in the pilus fraction as well as in the initial bacterial suspension (Fig 1E). Quantitative analysis of Western blot signals determined that 5.6 ± 0.7% of the total major pilin could be purified in these conditions reflecting the purification efficiency and the balance between periplasmic and pili-associated PilE (Fig 1F). In contrast, in the case of PilV and PilX, the proportion of pilus-associated protein was much lower with 0.03 ± 0.01 and 0.9 ± 0.3%, respectively. These results were not significantly different from those obtained with PilU, a cytoplasmic protein not expected to associate with pili. These results confirm that, at best, only a minute proportion of minor pilins are associated with pili. PilV and PilX are necessary for optimal initiation of pilus biogenesis The presence of the minor pilins in the periplasm suggests a role in the pilus machinery and pilus biogenesis. In addition, given their common pilin-like structural features, we reasoned that PilV and PilX could exert redundant functions in pilus biogenesis. To evaluate this possibility, we reassessed the piliation levels of the pilV and pilX mutants and the double mutant by ELISA on whole bacteria, and the morphology of pili was observed using immunofluorescence. Inactivation of pilV led to a minor but reproducible defect in piliation with 61 ± 7% of piliation relative to the wild-type strain (Fig 2A). PilV overexpression not only rescued the amount of pili but increased pili levels above wild-type levels. Similar results were obtained for pilX with the mutant displaying a more severe phenotype with only 27 ± 4% of piliation and the complemented strain showing higher piliation than the wild type (Fig 2B). In addition, the pilV and pilX mutations had a synergistic effect. The double mutant could not be distinguished from the pilE deficient strain by ELISA (Fig 2C). These results were confirmed by quantitative biochemical pilus preparations (Supplementary Fig S2). Figure 2. Role of PilV and PilX in pilus biogenesis ELISA analysis of surface-exposed pilin in the pilV mutant and complemented strains. Results are presented relative to the wild-type strain, given the value of 1. ELISA analysis of surface-exposed pilin in the pilX mutant. ELISA analysis of surface-exposed pilin in the pilVpilX double mutant. Immunofluorescence analysis of pili on the surface of the indicated bacterial strains. DAPI is in blue and pilus staining in green (20D9). Quantitative determination of the number of pili detected per individual diplococcus. The results of 3 independent experiments each with over 50 bacteria are included. Frequency distribution of the number of pili per diplococcus expressed by the wild-type strain (black line), the pilV strain (blue line), and the pilX strain (red line). Pili length measurement (μm). The results of 3 independent experiments each with over 50 bacteria are included. Frequency distribution of the length of pili expressed by the wild-type, pilV and the pilX strains (same color code as in F). A typical track representing bacterial movement resulting from twitching motility over a 2-min period. Frequency representation of the different speeds of bacteria for the wild-type, pilV and pilX strains (same color code as in F). Data information: Data represent mean ± SEM; *P ≤ 0.05; ****P ≤ 0.001. Download figure Download PowerPoint Decreased steady state amount of pili can be explained by a decreased initiation of biogenesis, slower extension, or faster retraction. In the case of slower extension or faster retraction, pili should be shorter but with the same number per bacterium. In contrast, lower initiation rate would be predicted to produce a lower number of pili with the same length. To further characterize these piliation defects, pili were visualized on individual bacteria by immunofluorescence and their number and length determined (Fig 2D). While wild-type bacteria displayed an average of 5.5 ± 0.3 pili on their surface, pilV and pilX strains displayed 2.7 ± 0.08 and 1.6 ± 0.003, respectively (Fig 2E and F). Pili lengths, however, were similar with a trend toward longer pili in the pilV and pilX strains (Fig 2G and H). These results indicate that PilV and PilX exert their effect at the initiation of pilus biogenesis rather than extension or retraction. Since a role for PilV and PilX in opposing PilT-dependent pilus retraction was previously proposed (Carbonnelle et al, 2006), we sought an additional experimental approach to confirm the above result. If the effect of PilV and PilX was to slow down retraction, a prediction would be that twitching motility, a process powered by retraction, would be higher in the pilV and pilX mutants. Motility assays indicate that the twitching motility of the pilV mutant is indistinguishable from that of the wild-type strain (Fig 2I and J). The pilX mutant has a slower motility, probably due to the decreased number of pili expressed by this strain. These results confirm that PilV and PilX do not participate in the control of pilus retraction but rather play a role in the initiation phase of pilus biogenesis. PilV and PilX exert their functions from the periplasm As shown above, the PilV and PilX minor pilins are mainly localized in the periplasm. This does not however rule out the possibility that a small proportion of the proteins located in the pilus could carry out their functional roles, particularly in terms of interaction with host cells. To address this point, we designed a strategy to block these proteins in the periplasm and assessed their function under these conditions. Based on available structures of the secretin family of proteins (Korotkov et al, 2012), we reasoned that fusing the bulky mCherry protein to the carboxy-terminus of the minor pilins would prevent their exit from the periplasm through the PilQ outer-membrane pore. The mCherry fusion constructs with PilE, PilV, and PilX all localized in the bacterial periplasm and, as expected, no evidence of organization as pilus fibers could be seen (Fig 3A). To confirm that the PilE-mCherry fusion was retained in the periplasm, the amount of pili expressed at the surface of this strain was determined by ELISA on whole bacteria (Fig 3B). As previously described, the lac promoter was insufficiently strong to provide full complementation of pilE (Long et al, 2003). Nevertheless, expression of the unmodified pilE gene reached a piliation value of 0.21 ± 0.04 relative to wild-type level (value of 1). Validating our experimental strategy, expression of the pilEmCherry fusion did not lead to any detectable pili. In striking contrast, fusion of the mCherry protein to PilV and PilX did not have any obvious impact on the capacity of these proteins to initiate pilus biogenesis (Fig 3C). The functionality of the PilV-mCherry fusion protein was also tested for its ability to provide efficient crosstalk with host cells. At the concentration of inducer mimicking the wild-type level of PilV (25 μM, Supplementary Fig S1), the pilVmCherry construct fully complemented the deletion strain (Fig 3D). Similar results were obtained with the pilXmCherry construct, and full complementation was observed for adhesion and aggregation with 17 μM of IPTG (Fig 3E and Supplementary Figs S1 and S3). These results show that preventing the exit from the outer membrane of the minor pilins PilV and PilX does not affect their function. This demonstrates that PilV and PilX exert their functions, both in terms of pilus biogenesis and interaction with host cells, inside the periplasm and not from the bacterial surface. Figure 3. Fusion of PilV or PilX to mCherry does not prevent PilV or PilX function Fluorescence analysis of the localization of the mCherry fusions (mCherry is in red, DAPI is in blue), pilEmCherry (left), pilVmCherry (middle) and pilXmCherry (right). Impact of the mCherry fusion on the ability of PilE to form pilus fibers. The amount of surface-exposed pili was determined by ELISA on whole bacteria for the indicated strains. The amount of added IPTG to induce the Lac promoter is indicated (μM). Each dot represents one experiment. Impact of the mCherry fusion on the ability of PilV and PilX to promote pilus biogenesis. Ability of the PilV-mCherry fusion to promote plasma membrane reorganization following adhesion was determined using ezrin as a marker. Bacteria were incubated with endothelial cells for 2 h, and the proportion of microcolonies efficiently triggering plasma membrane reorganization was determined. Arrows point to the conditions mimicking wild-type levels of PilV. Ability of the PilX-mCherry fusion to promote adhesion. Bacteria were incubated with endothelial cells for a period of 4 h, and the number of cell-associated bacteria was determined. The amount of added inducer is indicated. Arrows point to the conditions mimicking wild-type levels of PilX. Data information: Data represent mean ± SEM; **P ≤ 0.01; ****P ≤ 0.0001. In (B–E) each dot represents one experiment. Download figure Download PowerPoint PilV and PilX do not require assembly into a fiber to exert their functions To further characterize the mechanism of action of PilV and PilX, we investigated whether assembly into a fiber was necessary to exert their functions. Mutation of glutamate at position 5 into an alanine (E5A) is a well-established way to block pilin-like monomer assembly into fibers in different piliation systems (Pasloske & Paranchych, 1988; Aas et al, 2007; Campos et al, 2010). We therefore generated E5A point mutations in pilV and pilX and evaluated the functionality of these mutants. While the pilEE5A mutant did not show any pili on its surface, the pilVE5A and pilXE5A constructs fully restored piliation (Fig 4A). Furthermore, PilVE5A was functional in mediating plasma membrane reshaping under microcolonies in conditions mimicking the endogenous expression level (Fig 4B). Similarly, PilXE5A restored bacterial adhesion to host cells and aggregation (Fig 4C and Supplementary Fig S3). These results show that PilV and PilX do not require the glutamate residue in position 5 to exert their functions. Since this residue is typically necessary for assembly into fibers, it is likely that PilV and PilX do not need to assemble into fibers to modulate piliation and to mediate interaction with host cells. Figure 4. The E5A mutation does not affect PilV or PilX function Effect of the E5A mutation in pilE, pilV, and pilX on pilus biogenesis determined by ELISA. Effect of the E5A mutation in PilV on its ability to promote plasma membrane reshaping. The amount of inducer is indicated (IPTG, μM). The arrow indicates the amount of inducer mimicking the wild-type level of expression. Effect of the E5A mutation on the ability of PilX to promote bacterial adhesion. The arrow indicates the amount of inducer mimicking the wild-type level of expression. Data information: Data represent mean ± SEM; ****P ≤ 0.0001. Download figure Download PowerPoint PilV and PilX require cleavage by the PilD peptidase to be functional Considering the evidence described above pointing to a possible site of action of the minor pilins PilV and PilX in the periplasm, we considered whether cleavage by the PilD peptidase was necessary. Point mutations in the PilD cleavage site, glycine in position −1 to asparagine (G-1N), were thus generated. First, the impact of the mutation on protein cleavage was checked (Fig 5A). Importantly, the G-1N mutations only affected the cleavage of the mutated protein but not the other minor or major pilins. As expected, the major pilin carrying the G-1N mutation was unable to form pilus fibers (Fig 5B). When pilVG-1N and pilXG-1N were expressed, not only were those constructs unable to complement the mutation but they appeared to block pilus formation. To confirm this observation, the pilVG-1N and pilXG-1N constructs were expressed in the wild-type background. Expression of the PilD-resistant PilV and PilX proteins restricted pilus formation thus exhibiting a dominant-negative effect (Fig 5C). It can be concluded that cleavage by PilD is necessary for proper function of PilV and PilX and that, surprisingly, the uncleaved forms are able to interact functionally with the piliation machinery and block biogenesis. These results highlight the tight functional association of PilV and PilX with the piliation machinery. Figure 5. Absence of PilD cleavage in G-1N mutants blocks the function of PilV and PilX Mobility shift due to the absence of cleavage by PilD of mutated PilE, PilV, and PilX. Western blots with polyclonal antibodies directed against PilE (top), PilV (middle), and PilX (bottom) are shown. Uncleaved proteins (UC) migrate slower than cleave proteins (C). Piliation levels determined by ELISA in the G-1N mutants. Dominant-negative effect of the G-1N mutation. Mutant proteins were expressed with intact endogenous protein and piliation levels were determined by ELISA. Data information: Data represent mean ± SEM; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.