SEARCH

SEARCH BY CITATION

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Although type IV pili (Tfp) are among the commonest virulence factors in bacteria, their biogenesis by complex machineries of 12–15 proteins, and thereby their function remains poorly understood. Interestingly, some of these proteins were reported to merely antagonize the retraction of the fibres powered by PilT, because piliation could be restored in their absence by a mutation in the pilT gene. The recent identification of the 15 Pil proteins dedicated to Tfp biogenesis in Neisseria meningitidis offered us the unprecedented possibility to define their exact contribution in this process. We therefore systematically introduced a pilT mutation into the corresponding non-piliated mutants and characterized them for the rescue of Tfp and Tfp-mediated virulence phenotypes. We found that in addition to the pilin, the main constituent of Tfp, only six Pil proteins were required for the actual assembly of the fibres, because apparently normal fibres were restored in the remaining mutants. Restored fibres were surface-exposed, except in the pilQ/T mutant in which they were trapped in the periplasm, suggesting that the PilQ secretin was the sole Pil component necessary for their emergence on the surface. Importantly, although in most mutants the restored Tfp were not functional, the pilG/T and pilH/T mutants could form bacterial aggregates and adhere to human cells efficiently, suggesting that Tfp stabilization and functional maturation are two discrete steps. These findings have numerous implications for understanding Tfp biogenesis/function and provide a useful groundwork for the characterization of the precise function of each Pil protein in this process.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Pili, or fimbriae, are hair-like appendages extending out from the surface of most bacterial types, which frequently mediate attachment to various surfaces (Soto and Hultgren, 1999). Among these organelles, type IV pili (Tfp) are likely to be the most widespread based on their detection in numerous and diverse Gram negative species (Mattick, 2002) and unexpectedly some Gram positive ones such as Ruminococcus albus in which their presence has been experimentally confirmed (Rakotoarivonina et al., 2002). In addition, genes specifically involved in Tfp biogenesis were discovered in tens of genome sequencing projects. Significantly, Tfp are found in human pathogens as diverse as enteropathogenic Escherichia coli (EPEC), pathogenic Neisseria species, Pseudomonas aeruginosa or Vibrio cholerae, where they play a key role in virulence (Soto and Hultgren, 1999; Mattick, 2002). Unlike other types of pili, however, Tfp not only participate in adhesion to host cells, but are also involved in a surprising number of functions (Merz and So, 2000), including competence for DNA transformation, the formation of bacterial aggregates and a form of surface translocation known as twitching motility, which is powered by fibre retraction (Merz et al., 2000).

Type IV pili are thin (50–80 Å), long (up to several μm) and flexible but strong fibres, which often interact laterally to form typical bundles (Craig et al., 2004). These highly dynamic organelles are mainly composed of thousands of copies of one protein, the pilin. Pilin is synthesized as a precursor and presents distinctive N-terminal sequence pattern and three-dimensional structure with an extended N-terminal spine followed by a globular head domain (Parge et al., 1995). This has led to helical assembly models for Tfp where the conserved N-terminal spines formed the hydrophobic core of the pilus and the variable globular heads were exposed on the surface. These shared sequence and structural characteristics, together with the ability of P. aeruginosa to assemble the Neisseria gonorrhoeae pilin into fibres(Hoyne et al., 1992), suggested that the machineries used by different bacteria to assemble Tfp were closely related, which was later confirmed by the identification of Tfp biogenesis genes. This process requires a strikingly large set of dedicated proteins, between 12 and 15, including the pilin (Yoshida et al., 1999; Anantha et al., 2000; Carbonnelle et al., 2005). Some of these have homologues in the type II secretion system used by a variety of Gram negative bacteria to transport exoproteins to the outside milieu (Nunn, 1999; Peabody et al., 2003). This suggested that these two systems have a common evolutionary origin and possibly similar modes of function, which fuelled important transfers of knowledge between the two fields of research (Pugsley, 1993; Nunn, 1999; Sauvonnet et al., 2000; Durand et al., 2005). However, a function could be determined for only a few of the above proteins and Tfp biogenesis remains a poorly understood process at a molecular level. Nevertheless, it is clear that upon synthesis, the prepilin is inserted in the inner membrane as a bitopic protein owing to its hydrophobic N-terminus, leaving the leader peptide on the cytoplasmic side (Strom and Lory, 1987). This leader peptide is then cleaved by a polytopic inner membrane protein (Lory and Strom, 1997), the prepilin peptidase (GspO in the unifying type II secretion nomenclature). This step is required for subsequent fibre assembly (Strom and Lory, 1991), which is powered by an oligomeric GspE-family cytoplasmic ATPase (Turner et al., 1993; Sakai et al., 2001). Finally, Tfp emerge on the cell surface through rings formed in the outer membrane by multimers of a protein belonging to the GspD-family of secretins (Collins et al., 2004; Chami et al., 2005), which sometimes depend on small outer membrane lipoproteins, known as pilotins, for their proper localization and stability (Hardie et al., 1996; Crago and Koronakis, 1998).

It seems obvious that a better understanding of Tfp biogenesis relies on our ability to determine the role in this process of each of the remaining proteins, i.e. an overwhelming majority of them. In this regard, several recent studies aimed at the identification of the interactions that occur among these proteins using different approaches (Ramer et al., 2002; Hwang et al., 2003; Crowther et al., 2004). This has led to a better knowledge of the corresponding machineries and some relevant functional information. For example, it was found in the EPEC that interactions of the GspE-family ATPase with two membrane proteins dramatically stimulated its activity (Crowther et al., 2005). This led the authors to propose a model in which the produced energy was converted into mechanical force allowing one of the interacting membrane proteins to act as a piston pushing the pilin out from the inner membrane, preparing it for pilus assembly. A powerful genetic approach also generated crucial information by providing evidence that the piliation defect in several Neisseria meningitidis and N. gonorrhoeae mutants could be suppressed by a mutation in pilT. This demonstrated that Tfp biogenesis can be resolved in discrete steps and that at least some proteins were actually dispensable for Tfp assembly and served to counteract PilT-mediated fibre retraction (Wolfgang et al., 1998a; Carbonnelle et al., 2005; Winther-Larsen et al., 2005). In other words, the corresponding mutants were non-piliated because pilus homeostasis was shifted towards retraction (Morand et al., 2004). Interestingly, the restored fibres were not functional, hence the definition of this step as a Tfp stabilization/functional maturation step. Similarly, fibres could be restored in a N. gonorrhoeae pilQ/T mutant but remained within the cell, providing the first evidence that Tfp were assembled in the periplasm before emerging on the surface through the PilQ secretin (Wolfgang et al., 2000).

In order to improve our understanding of Tfp biogenesis, we defined at which step of this process each Pil protein acts by implementing systematically, for the first time, the previous genetic approach. This recently became possible in N. meningitidis with the identification of all the piliation genes in this species (Carbonnelle et al., 2005). We therefore characterized in details a set of mutants in which each piliation gene was mutated together with pilT. This led to several important observations, which might have an impact on our understanding of the biology of one of the most widespread virulence factors in bacteria.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Only PilD, PilF, PilM, PilN, PilO and PilP are required to assemble the pilin into Tfp, while PilQ is the single protein required for the emergence of the fibres on the surface

We recently identified, by screening an almost exhaustive collection of N. meningitidis mutants, the genes that were necessary, and very likely sufficient, for Tfp biogenesis in this species (Carbonnelle et al., 2005). The complete set, which also comprises the double pilC1/C2 mutant in which the two alleles encoding PilC were sequentially mutated, consists of 15 non-piliated mutants (Table 1). Piliation status was unambiguously determined by immunofluorescence (IF) microscopy using a monoclonal antibody specific for the fibres (Carbonnelle et al., 2005). In order to genetically define the specific contribution in Tfp biogenesis for each of the above Pil proteins, we systematically introduced a pilT mutation into each of the corresponding non-piliated mutants and characterized them for the possible restoration of Tfp and Tfp-mediated virulence phenotypes. As non-piliated mutants are not competent for DNA transformation, the introduction of a pilT mutation in the above 15 mutants was performed by transforming the wild-type (WT) strain simultaneously with the chromosomal DNAs extracted from a pilT mutant and one of these non-piliated mutants. As above, the triple pilC1/C2/T mutant was constructed sequentially by transforming the pilC1 mutant simultaneously with chromosomal DNAs extracted from the pilC2 and pilT mutants. We then determined whether the double (triple) mutants were piliated or not, by scoring the presence or absence of Tfp by IF microscopy (Fig. 1). This showed that while variable amounts of Tfp could be easily seen in seven mutants (pilC1/C2/T, pilG/T, pilH/T, pilI/T, pilJ/T, pilK/T and pilW/T), not even trace amounts of fibres could be detected in the remaining eight mutants (pilD/T, pilE/T, pilF/T, pilM/T, pilN/T, pilO/T, pilP/T and pilQ/T).

Table 1.  Relevant characteristics of the corresponding gene products in the 15 non-piliated N. meningitidis pil mutants.
MutantProtein length (aa)Predicted localization/topologyaHomologuesbMain characteristics and features
  • a.

    Predictions of localization and topology were performed using computer methods available online such as PSORTb, TMHMM, Top Pred, etc.

  • b.

    Homologous proteins in the type II secretion pathway (unifying Gsp nomenclature) and/or the model Tfp biogenesis pathway of P. aeruginosa (Pil nomenclature).

pilC1/C21025 (PilC1)Outer membrane and pilusPilY1Contains a signal peptide, counteracts PilT-mediated Tfp retraction
1044 (PilC2)Outer membrane and pilusPilY1Contains a signal peptide, counteracts PilT-mediated Tfp retraction
pilD286Inner membrane/polytopicGspO, PilDPrepilin peptidase, processes and methylates the N-terminus of prepilins
pilE168Pilus Prepilin, is cleaved by PilD, is the main component of Tfp
pilF558CytoplasmGspE, PilBATPase, is likely to be involved in powering the assembly of Tfp
pilG410Inner membrane/polytopicGspF, PilCHighly conserved multispanning transmembrane protein
pilH221  Harbours a N-terminus conserved in prepilins, is cleaved by PilD
pilI206  Harbours a N-terminus conserved in prepilins, is cleaved by PilD
pilJ321  Harbours a N-terminus conserved in prepilins, is cleaved by PilD
pilK199  Harbours a N-terminus conserved in prepilins, is cleaved by PilD
pilM371CytoplasmPilM 
pilN199 PilNContains either a single transmembrane domain or a signal peptide (unclear)
pilO215 PilOContains either a single transmembrane domain or a signal peptide (unclear)
pilP181Membrane/anchoredPilPLipoprotein
pilQ761Outer membrane/integralGspD, PilQSecretin, forms multimeric channels necessary for Tfp translocation across the outer membrane
pilW253Outer membrane/anchoredPilFContains a lipoprotein signal peptide, counteracts PilT-mediated Tfp retraction and is important for the stability of PilQ multimers
image

Figure 1. Systematic IF microscopy detection of Tfp in the N. meningitidis pil mutants, which harbour mutations in genes specifically required for piliation, after the introduction of an additional mutation in the pilT gene in order to abolish Tfp retraction (bars represent 10 μm). The WT strain was included as a control. Bacteria (red) were stained with ethidium bromide. Tfp (green) were detected using first the 20D9 anti-Tfp mouse monoclonal antibody and then a goat anti-mouse antibody labelled with Alexa Fluor 488.

Download figure to PowerPoint

The presence of pilQ/T among these eight apparently non-piliated double mutants, which was previously reported to harbour intracellular fibres in N. gonorrhoeae (Wolfgang et al., 2000), suggested that the method we used to detect Tfp might not discriminate between mutants in which the fibres were actually absent from those in which they were restored but trapped within the cells. Consequently, no conclusions could be drawn at this point on whether the corresponding Pil proteins were involved in the assembly of the fibres or their emergence on the surface. Therefore, we designed a method allowing the distinction between these two classes of mutants. As the fibres in a N. gonorrhoeae pilQ/T mutant were very likely periplasmic (Wolfgang et al., 2000), we reasoned that similar fibres should be detectable after submitting the N. meningitidis mutants to a slightly modified cold osmotic shock treatment usually used to release periplasmic proteins (Neu and Heppel, 1965). As could be expected, although there was a clear effect of this treatment on cellular integrity as evidenced by the diffuse ethidium bromide staining of the bacteria (Fig. 2), no labelling was observed in non-piliated mutants such as pilD, which produces the main pilus subunit but is unable to assemble it in Tfp (Strom and Lory, 1991). In contrast, numerous short Tfp were visible by IF microscopy after submitting the pilQ/T mutant to osmotic shock treatment (Fig. 2), which confirmed the efficacy of this procedure for releasing intraperiplasmic fibres and demonstrated that N. meningitidis and N. gonorrhoeae pilQ/T mutants presented the same phenotype. We then re-analysed the piliation of the eight mutants in which no fibres could be detected in classical IF microscopy conditions (pilD/T, pilE/T, pilF/T, pilM/T, pilN/T, pilO/T, pilP/T and pilQ/T) after submitting them to this osmotic shock procedure. This demonstrated that pilQ/T was the only mutant in which Tfp could be released in this way and subsequently detected (Fig. 2).

image

Figure 2. Immunofluorescence microscopy detection of Tfp in representative N. meningitidis strains before and after osmotic shock release of their periplasmic content (bars represent 10 μm). As for the pilP/T mutant, no pili could be detected after osmotic shock for the pilD/T, pilE/T, pilF/T, pilM/T, pilN/T and pilO/T mutants (data not shown).

Download figure to PowerPoint

Next, in order to determine whether the seven mutants in which no Tfp could be restored (pilD/T, pilE/T, pilF/T, pilM/T, pilN/T, pilO/T and pilP/T) still expressed and matured the prepilin PilE, the main pilus component, whole-cell protein extracts were prepared, separated by SDS-PAGE and subjected to Western blotting using a monoclonal antibody directed against this protein. Besides the pilE mutant that obviously produced no pilin and the prepilin peptidase pilD mutant in which only the prepilin could be detected, no differences in the expression and/or maturation of PilE were observable in the pilF/T, pilM/T, pilN/T, pilO/T and pilP/T mutants (Fig. 3). This finding indicated that the absence of Tfp in these mutants was not due to an impaired expression or maturation of the main pilus component PilE and suggested that the corresponding proteins could actually be involved in the first step of Tfp biogenesis, i.e. the assembly of the fibres. However, as the pilM, pilN, pilO and pilP genes are likely to be cotranscribed, the possibility that at least some of the phenotypes we observed were due to polar effects of the mutations on distal gene expression could not be excluded at this point. Therefore, we complemented the pilM, pilN, pilO and pilP mutants by inserting in trans in their genomes an intact copy of the corresponding genes under the transcriptional control of an IPTG-inducible promoter. IF microscopy observation of the complemented mutants showed that they were all piliated (data not shown). This demonstrated that piliation in the pilM, pilN, pilO and pilP mutants could be restored by intact copies of the mutated genes, ruling out the possibility that the non-piliated phenotypes we observed resulted from polar effects on downstream gene expression and strengthening the hypothesis that the corresponding proteins were indeed involved in Tfp assembly.

image

Figure 3. Western blot detection of the pilin (PilE) in whole-cell extracts of the non-piliated mutants in which fibres could not be restored in the absence of PilT. The WT strain was included as a control. The protein concentrations in the extracts were quantified and adjusted, which was verified by detecting the RmpM protein as a control (data not shown). Equal amounts of proteins were present in each lane except for the pilD lane in which fivefold more proteins were loaded in order to detect the prepilin readily.

Download figure to PowerPoint

Taken together, these results suggested that a minority of the 15 N. meningitidis Pil proteins (PilD, PilF, PilM, PilN, PilO and PilP) were involved in the assembly stricto sensu of PilE into Tfp and that PilQ was likely to be the sole Pil protein involved in the emergence of the fibres on the surface.

PilW is the only protein that affects the stability of PilQ multimers

One possibility to learn more about the possible interactions between Pil proteins is to study the stability of one or more of these proteins in the absence of the others (Ramer et al., 2002). We previously reported that the PilQ multimers were strongly destabilized in the absence of PilW (Carbonnelle et al., 2005), which was recently confirmed in Myxococcus xanthus with the orthologous protein Tgl (Nudleman et al., 2006). This prompted us to test systematically by Western blotting, using an antibody directed against the N. meningitidis secretin, whether the absence of the other Pil components might have a negative effect on the stability of the PilQ multimers. These high molecular weight species of PilQ can be readily detected because they are SDS-resistant and retained in the stacking gel after SDS-PAGE separation of whole-cell protein extracts (Fig. 4). This systematic analysis indicated that an effect on secretin multimers could be seen only in the absence of PilW. Indeed, while no high molecular weight species of PilQ can be detected in a pilW mutant, as previously reported (Carbonnelle et al., 2005), all the other mutants presented amounts of both multimeric and monomeric forms of PilQ similar to those that could be detected in the WT strain (Fig. 4). This showed that, apart from PilW, no other N. meningitidis Pil protein was important for the stability of the PilQ multimers, suggesting that a specific interaction occurred between these two proteins.

image

Figure 4. Western blot detection of the PilQ monomers and oligomers in whole-cell extracts of each of the non-piliated mutants. The WT strain was included as a control. The protein concentrations in the extracts were quantified and equal amounts of proteins were loaded in each lane, which was verified by detecting the RmpM protein as a control (data not shown).

Download figure to PowerPoint

PilG and PilH are the only proteins involved in Tfp stabilization that are, at least partly, dispensable for fibre functionality

As seen above (Fig. 1), Tfp could be detected by IF microscopy in classical conditions in seven mutants (pilC1/C2/T, pilG/T, pilH/T, pilI/T, pilJ/T, pilK/T and pilW/T), including the previously characterized pilW/T (Carbonnelle et al., 2005). This indicated that the fibres in these mutants were surface exposed unlike the fibres in the pilQ/T mutant. As far as can be appreciated by transmission electron microscopy at high magnification, the fibres in these mutants were morphologically indistinguishable from those seen in the WT strain (Fig. 5). In each case, few long Tfp could be seen extending out from the cells as large bundles of laterally interacting fibres, although the bundles observed in the pilC1/C2/T and pilW/T mutants seemed somewhat thinner. These findings clearly indicated that the corresponding proteins (PilC, PilG, PilH, PilI, PilJ, PilK and PilW) were not canonical assembly components because biogenesis of apparently normal Tfp was possible in their absence. The availability of N. meningitidis strains expressing Tfp in the absence of the above proteins offered the unique opportunity to address their possible importance for Tfp-mediated phenotypes, as we previously did with the pilW/T mutant (Carbonnelle et al., 2005). We therefore characterized the ability of the above piliated mutants to promote adhesion to human umbilical vein endothelial cells (HUVEC) and to mediate the formation of aggregates, the two Tfp-linked properties that are not abolished in the absence of PilT unlike competence for DNA transformation and twitching motility (Wolfgang et al., 1998b).

image

Figure 5. Transmission electron microscopy analysis of Tfp in the double (triple) mutants in which the fibres were restored. The WT strain was included as a control. Fibres were visualized directly after negative staining with phosphotungstic acid (50 000× magnification).

Download figure to PowerPoint

First, we tested the adhesive abilities to HUVEC of each of the pilC1/C2/T, pilG/T, pilH/T, pilI/T, pilJ/T, pilK/T and pilW/T mutants using a classical 4 h adhesion assay (Fig. 6A). Despite their piliated phenotype, most of these mutants (pilC1/C2/T, pilI/T, pilJ/T, pilK/T and pilW/T) were unable to adhere to HUVEC, which was further confirmed by counting the adherent colony-forming units (cfu) (Fig. 6A). The adhesive abilities of these mutants were found to be as low as that of a non-piliated mutant with mean 1000-fold reduction in adherence when compared with the WT strain or a pilT mutant (approximately 105 versus 108 adherent cfu). Strikingly, the pilG/T and pilH/T mutants presented a phenotype previously never reported, i.e. Tfp that were restored were capable of mediating efficient adhesion to human cells. Although the adhesive abilities of the pilG/T and pilH/T mutants were slightly lower than that of the WT strain or a pilT mutant, they were able to adhere to HUVEC at least two orders of magnitude better than a non-piliated mutant, with 100-fold and 400-fold increases respectively (Fig. 6A). This was further evidenced by quantifying pilG/T and pilH/T adherence in more details over the course of the infection, which was found to present kinetics similar to that of the WT strain, from the very beginning of the adherence assay (Fig. 6B). Interestingly, however, the three-dimensional bacterial microcolonies that these two mutants formed on the cells, which did not disappear during the adhesion due to the absence of PilT (Pujol et al., 1999), were morphologically different from those seen with a pilT mutant. They seemed less compact and firm, which suggested that although adhesion to HUVEC occurred in the absence of PilG and PilH, these proteins might be required for full Tfp functionality.

image

Figure 6. Quantification of the adhesive abilities to human cells in the double (triple) mutants in which the fibres were restored. The WT strain, a non-piliated pilD mutant and a hyper-piliated pilT mutant were included as controls. After a 30 min contact during which bacteria adhered to the cells, the cells were incubated with regular washes every hour and the numbers of adherent bacteria were recovered by scrapping the wells at defined time points and counted. A. Numbers of bacteria adhering to a monolayer of HUVEC after 4 h of infection. Values are the means ± standard deviations of three to nine independent adhesion assays. B. Kinetics of the adherence to HUVEC of the pilG/T and pilH/T mutants in which adhesion was restored. The 0 h points in these graphs represent the sizes of the inocula that were adjusted and counted prior to the assay. Adherent bacteria were counted after the initial 30 min contact and after 2 and 4 h of incubation. Values are the means ± standard deviations of three independent assays.

Download figure to PowerPoint

Next, by observing liquid cultures of the above seven mutants by phase-contrast microscopy, we found that most of them were unable to form multicellular aggregates, with the notable exception of the pilG/T and pilH/T mutants that however, formed aggregates morphologically different from those highly irregular ones produced by a pilT mutant (Helaine et al., 2005). Using a method we developed recently, based on the measuring of the decrease in optical density (OD) that occurs in non-agitated liquid cultures upon sedimentation of the bacterial aggregates (Helaine et al., 2005), we precisely quantified the aggregative abilities of all these mutants (Fig. 7). The kinetics of aggregation that were measured for the various strains were consistent with the phenotypes that were observed by phase-contrast microscopy. Indeed, while pilC1/C2/T, pilI/T, pilJ/T, pilK/T and pilW/T presented a nil aggregation, just as a control non-piliated mutant, the pilG/T and pilH/T mutants presented aggregative abilities comparable to that of the WT strain (Fig. 7). This was coherent with the results of the adhesion assays in which pilG/T and pilH/T were the only mutants able to adhere to HUVEC and once again underscored the invariable direct link between Tfp-mediated aggregation and adhesion to human cells (Helaine et al., 2005). However, despite their PilT-negative background, the aggregative abilities of pilG/T and pilH/T were lower than that of the pilT mutant, which almost completely and rapidly sedimented (Fig. 7). Again, as with the adhesion assays, this indicated that PilG and PilH contributed to full Tfp functionality.

image

Figure 7. Quantification of the aggregative abilities in the double (triple) mutants in which the fibres were restored. The WT strain, a non-piliated pilD mutant and a hyper-piliated pilT mutant were included as controls. In brief, aggregation was quantified by measuring the decrease in OD600 that occurs upon sedimentation of bacterial aggregates in static liquid cultures. The aggregation of the non-adhesive pilC1/C2/T, pilI/T, pilJ/T, pilK/T and pilW/T mutants, were nil and indistinguishable from that of a non-piliated pilD mutant and we therefore display only one curve for the sake of legibility. Values are the means of three to nine independent experiments.

Download figure to PowerPoint

Together, these results indicated that as much as seven Pil proteins (PilC, PilG, PilH, PilI, PilJ, PilK and PilW) were involved in regulating pilus homeostasis by counteracting the PilT-mediated retraction of the fibres (Morand et al., 2004), which emphasized the extreme importance of this step in Tfp biogenesis. Importantly, although most of these proteins were also essential for Tfp functionality as previously reported for PilW (Carbonnelle et al., 2005), the absence of PilG and PilH was less deleterious, suggesting that the tightly linked events of stabilization and functional maturation could be genetically resolved.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

When compared with the well-characterized chaperone-usher pathway that relies on two proteins to assemble structural subunits into a pilus on the bacterial surface (Soto and Hultgren, 1999), the biogenesis of Tfp, which requires 11–14 dedicated proteins in addition to the pilin, seems utterly complex and remains poorly understood. Interestingly, however, it has been shown that in several Neisseria mutants the piliation defect could be suppressed by introducing a mutation in pilT (Wolfgang et al., 1998a; 2000; Carbonnelle et al., 2005; Winther-Larsen et al., 2005). This suggested that the corresponding Pil proteins acted after Tfp assembly either to stabilize the fibres by counteracting PilT-mediated retraction or to allow their emergence on the bacterial surface. It should be noted that all the proteins found to counteract PilT action were also necessary for the functionality of Tfp because the restored fibres were incapable of mediating well-known Tfp-linked phenotypes, adding to the complexity of Tfp biogenesis/function. Together, these findings led to a three-step model for Tfp biogenesis (Wolfgang et al., 2000; Carbonnelle et al., 2005), where fibres were first assembled in the periplasm, then altogether stabilized and functionally matured and finally emerged on the surface through a channel formed by the secretin. The recent identification of the complete set of N. meningitidis genes specifically involved in Tfp biogenesis (Carbonnelle et al., 2005), whose number and genomic organization were found to be strikingly similar to those in P. aeruginosa (Alm and Mattick, 1997), prompted us to implement, for the first time, the above genetic approach on a systematic basis in order to define at which step of Tfp biogenesis each of the corresponding proteins was involved.

One important finding in this study was that the first step of Tfp biogenesis, the actual assembly of the fibres, is simpler than could be anticipated because it required, in addition to the pilin, a core set of only six proteins (PilD, PilF, PilM, PilN, PilO and PilP). As the pilM to pilP genes are very likely to be cotranscribed, it is important to note that complementation experiments excluded the possibility that the piliation defects observed in the corresponding mutants were due to polar effects on the expression of downstream genes. Furthermore, the non-piliated phenotypes of these mutants were neither due to reduced amounts of pilin nor to an impaired maturation of this protein. Therefore, if the prepilin peptidase and PilF are only necessary, respectively, for the maturation of the prepilin and to provide the energy necessary to push the pilin outside the inner membrane, the four PilM, PilN, PilO and PilP proteins might constitute the essence of the machinery necessary for the mechanical assembly of Tfp. In accord with this hypothesis, these proteins were reported to be part of the core set of conserved Pil proteins in the cyanobacteria and β-, γ- and ∂-proteobacteria harbouring Tfp (Nudleman and Kaiser, 2004). These findings also point to the possibility of creating a minimal Tfp biogenesis system in a heterologous non-piliated organism by transferring the above genes on suitable expression vectors, which could be instrumental to the characterization of the underlying molecular mechanisms of pilus assembly.

A corollary of the above findings is that a significant proportion of the Pil proteins (PilC, PilG, PilH, PilI, PilJ, PilK and PilW) is involved in counteracting PilT action on the fibres, because morphologically normal Tfp can be expressed in their absence. This underscores the unexpectedly central role in Tfp biogenesis of pilus homeostasis, which results from the balance between retraction and counter-retraction (Morand et al., 2004). In accord with previous reports (Wolfgang et al., 1998a; Carbonnelle et al., 2005; Winther-Larsen et al., 2005), we found that this stabilization step is most often linked to what we previously defined as functional maturation of the fibres (Carbonnelle et al., 2005). Indeed, a precise quantification of Tfp-linked properties clearly demonstrated that the fibres in most of these mutants (pilC1/C2/T, pilI/T, pilJ/T, pilK/T and pilW/T) were not functional because they were incapable of mediating adhesion to human cells and the formation of bacterial aggregates. However, one of the most striking findings in this study was the discovery of a novel class of double mutants (pilG/T and pilH/T) where the fibres that were restored were able to mediate adhesion to human cells and the formation of bacterial aggregates. However, these mutants did not behave as pilT mutants, which indicated that although PilG and PilH role in Tfp functionality was minor when compared with the role of PilC, PilI, PilJ, PilK and PilW, these proteins nevertheless participated in the modulation of fibres functionality. This suggested that Tfp biogenesis is not a three-step pathway as previously thought because the counter-retraction and functional maturation activities could be genetically separated. Interestingly, one of the two proteins found to be associated with such a phenotype was the polytopic inner membrane protein PilG (GspF), which is one of the four extremely conserved proteins in both Tfp biogenesis and type II secretion (Peabody et al., 2003) and as such thought to be a key player or even the corner stone in both systems (Py et al., 2001; Crowther et al., 2004). Our data dot not support this possibility in Tfp biogenesis because piliation could be restored in N. meningitidis in the absence of PilG. This clearly indicates that PilG is not a canonical pilus assembly factor in N. meningitidis, which could be tested in other bacterial species using the same approach. However, it is unclear whether and how this finding might apply to type II secretion and the biogenesis of pilus-like structures by this machinery (Sauvonnet et al., 2000; Durand et al., 2005) because no PilT-like protein is present in this system. Concerning PilH, the second protein found to be associated with such a phenotype, our findings might be surprising in the first place because this protein belongs to a group of four related proteins, PilH to PilK, that were all shown to be cleaved by PilD in N. gonorrhoeae and because a pilH/T N. gonorrhoeae mutant apparently failed to adhere to human cells (Winther-Larsen et al., 2005). However, a possible clue as to PilH particular properties in N. meningitidis when compared with PilI, PilJ and PilK comes from clustalw multiple sequence alignments of these proteins, which show that while PilI, PilJ and PilK cluster together, PilH is clearly more distant and more closely related to PilE and the minor pilins (data not shown).

The design of a suitable method allowing the detection of intracellular fibres by IF helped us confirm that fibres were indeed restored intracellularly in a pilQ/T mutant, which was previously demonstrated in N. gonorrhoeae in which fibres embedded in membranous protrusions were seen by transmission electron microscopy (Wolfgang et al., 2000). Furthermore, the use of this method on a systematic basis demonstrated that intraperiplasmic fibres were seen exclusively in the absence of the secretin. Together, these findings suggested that PilQ's specific role was to provide a route to the surface for Tfp and that it was the only Pil protein implicated in this step of Tfp biogenesis, which has several important implications. First, our data argue against the possibility that the fibres in the pilW/T mutant, which are perfectly exposed on the surface despite an apparent absence of PilQ multimers, used a completely different route to the bacterial surface. Otherwise that alternative route would have also been available in the pilQ/T mutant and its fibres would have been exposed on the surface instead of being trapped in the periplasm. PilQ multimers were therefore likely to be still present in the absence of PilW, allowing the emergence of the fibres in the pilW/T mutant, but they might be too unstable to be detected after SDS-PAGE, which remains to be actually proven. This also suggested that the unstability of the PilQ multimers in the absence of PilW should merely be viewed as an indication that these proteins physically interact, rather than evidence that PilW takes an active part in the assembly of a functional secretin complex as recently proposed in M. xanthus for the orthologous protein Tgl (Nudleman et al., 2006). A second implication of our results was that PilQ in N. meningitidis probably does not rely on a dedicated pilot protein for its insertion in the outer membrane, similarly to the situation in EPEC (Schmidt et al., 2001). Indeed, it is likely that the introduction of a pilT mutation in a mutant missing such a pilot protein would lead to the restoration of intraperiplasmic fibres, because no secretin channel would be present in the outer membrane. However, this was never observed except for the pilQ/T mutant. Interestingly, it seems that in N. meningitidis PilQ multimerization and insertion in the outer membrane is rather under the dependence of Omp85, an evolutionarily conserved protein ubiquitous among Gram-negative bacteria thought to be involved in the membrane insertion and/or multimerization of most, if not all, bacterial outer membrane proteins (Voulhoux et al., 2003). Therefore, although this has been a matter of speculation for some time based on the report that PilP affects the expression of PilQ multimers in N. gonorrhoeae (Drake et al., 1997), which was not observed in N. meningitidis nor in M. xanthus (Nudleman et al., 2006), a role as a pilotin can a priori be excluded for this protein. Instead, PilP was found to be a member of the core set of proteins necessary for the mechanical assembly of Tfp.

In conclusion, the findings here offer some clues as to the mechanisms of Tfp biogenesis/function because they provide a four-step comprehensive scheme for the biogenesis of functional Tfp on which each of the 15 Pil proteins essential for piliation in N. meningitidis could be placed (Fig. 8). According to this model, which could be tested in other bacteria expressing Tfp, non-functional fibres are assembled in the periplasm by the PilD, PilF, PilM, PilN, PilO and PilP subset of proteins. Although the chronology of the steps after Tfp assembly remains to be precisely determined, one could speculate that these fibres are then taken in charge by the PilC, PilI, PilJ, PilK and PilW subset of proteins that make Tfp functional and then antagonize their retraction with the participation of PilG and PilH (although these two proteins also play a minor role in functional maturation). Finally, the fibres emerge on the surface through a channel formed by the secretin PilQ. This scenario could be instrumental in designing approaches aimed at understanding the above steps at a molecular level, for example, testing whether the functional maturation of the fibres might be the result of subtle alterations in Tfp structure and/or composition, which could have an impact on our understanding of what makes Tfp extremely efficient virulence factors.

image

Figure 8. Implication of the N. meningitidis Pil components, as inferred from this study, in the four steps of Tfp biogenesis: fibre assembly, functional maturation (PilG and PilH are not represented here because they only play a minor role in this step), counteraction of fibre retraction powered by the PilT protein and emergence of the fibres on the cell surface. Note that the order of the steps after Tfp assembly remains speculative.

Download figure to PowerPoint

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Bacterial strains: culture conditions and construction

Escherichia coli TOP10 (Invitrogen) was used for cloning experiments. It was grown at 37°C in liquid or solid Luria–Bertani medium (Difco), which contained 200 μg ml−1 erythromycin and 50 μg ml−1 kanamycin, when appropriate.

Neisseria meningitidis was grown at 37°C in a moist atmosphere containing 5% CO2 on GCB agar plates (Difco), which contained Kellogg's supplements and, when appropriate, 3 μg ml−1 erythromycin, 100 μg ml−1 kanamycin and 60 μg ml−1 spectinomycin (Pelicic et al., 1997). Liquid cultures were performed in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (both from PAA laboratories GmBH) as previously described (Helaine et al., 2005). The WT strain, a derivative of the serogroup C 8013 isolate, and most of its non-piliated mutants were described previously (Nassif et al., 1993; Carbonnelle et al., 2005). These mutants contain a mini-transposon, consisting of mariner Himar1-based inverted repeats flanking a kanamycin-resistance cassette, inserted in the N-terminal halves of the respective pil genes (Pelicic et al., 1997; Geoffroy et al., 2003). In addition, we constructed for this study the pilC1/C2 double mutant by transforming a pilC2 transposition mutant with the chromosomal DNA of a pilC1 mutant, in which the corresponding gene is partly deleted and replaced by a spectinomycin-resistance cassette (Morand et al., 2001). Chromosomal DNA preparation and transformation of N. meningitidis were done as previously described (Geoffroy et al., 2003). As all the above non-piliated mutants are not competent for transformation, the introduction of a pilT mutation in the various mutant backgrounds, where the pilT gene is interrupted by an erythromycin-resistance cassette (Pujol et al., 1999), was performed by transforming the WT strain simultaneously with the chromosomal DNAs extracted from the pilT mutant and the respective non-piliated mutants. Similarly to the above conditions, the pilC1/C2/T triple mutant was constructed by transforming the pilC2 mutant simultaneously with the chromosomal DNAs extracted from the pilT and pilC1 mutants. Primers specific for each pil gene, including pilT, were designed when they were not available and used to check by PCR that the mutants contained the expected mutant alleles. The pilC series mutants were also checked by Southern blotting, as previously described (Pelicic et al., 1997), using as a probe a PCR fragment in the region common to the pilC1 and pilC2 genes.

To complement the pilM, pilN, pilO and pilP mutants, the corresponding WT genes were amplified using primers pilM-IndF 5′-CCTTAATTAAGGAGTAATTTTATGCGCTTGTTTAAAAGCTTGA-3′ and pilM-IndR 5′-CCTTAATTAATTATAATCCCCGTACCGCCAA-3′, pilN-IndF 5′-CCTTAATTAAGGAGTAATTTTATGAACAATTTAATCAAAATCAAC-3′ and pilN-IndR 5′-CCTTAATTAATCAGTTTGCCTCCTGTGCGTT-3′, pilO-IndF 5′-CCTTAATTAAGGAGTAATTTTATGGCTTCTAAATCATCTAAAAC-3′ and pilO-IndR 5′-CCTTAATTAATTATTTTTGCTCGGCATTTTGTG-3′ and pilP-IndF 5′-CCTTAATTAAGGAGTAATTTTATGAAACACTATGCCTTACTCA-3′ and pilP-IndR 5′-CCTTAATTAATTAATTTTGTTCTGCGGCAGG-3′, which contained overhangs with underlined restriction sites for PacI. These PCR fragments were first cloned into pCRII-TOPO (Invitrogen), generating plasmids pYU21, pYU22, pYU23 and pYU24 respectively, and verified by sequencing to contain no errors. The fragments were excised from the above plasmids by PacI digestion and cloned into PacI-cut pGCC4 vector, adjacent to the lacIOP regulatory sequences, generating plasmids pYU25, pYU26, pYU27 and pYU28 respectively. This placed the pil genes under the transcriptional control of an IPTG-inducible promoter within a DNA fragment corresponding to an intragenic region of the gonococcal chromosome conserved in N. meningitidis (Mehr et al., 2000). The inducible alleles were then introduced into the chromosome of the corresponding mutants by homologous recombination. However, as these mutants are non-piliated and therefore not competent for transformation, this was performed in a reverse fashion by first transforming the WT strain with the NotI-cut pYU25, pYU26, pYU27 or pYU28 plasmids. Then the WT pil allele was interrupted in these transformants by a second transformation with the chromosomal DNAs extracted from the pilM, pilN, pilO or pilP non-piliated mutants respectively.

Tfp detection and morphological analysis

Neisseria meningitidis pili were detected by IF microscopy on bacteria fixed on coverslips as previously described (Carbonnelle et al., 2005). Fibres were specifically labelled with the anti-Tfp 20D9 mouse monoclonal antibody (Pujol et al., 1997) used at a 1/100 dilution, while the bacteria were stained with ethidium bromide at a 1/6000 dilution. The secondary antibody, also used at a 1/100 dilution, was a goat anti-mouse antibody conjugated with Alexa Fluor 488 (Molecular Probes). When indicated, bacteria were first submitted to a modified cold osmotic shock treatment (Neu and Heppel, 1965). They were resuspended in 30 mM Tris (pH 7.5), 20% sucrose, 2 mM EDTA, 1 mg ml−1 lysozyme and incubated 10 min at room temperature under constant agitation. Cells were then pelleted by centrifugation at 9000 g for 10 min and resuspended in an equal volume of cold 5 mM MgSO4. After a 10 min incubation at 4°C, bacteria were fixed on coverslips and their fibres detected by IF microscopy as above.

Fine structure of the fibres was determined by transmission electron microscopy, after negative staining with 1% phosphotungstic acid, using a JEOL JEM-100CX microscope operated at 80 kV as previously described (Carbonnelle et al., 2005).

Phenotypic analyses: aggregation and adhesion to HUVEC

Neisseria meningitidis ability to form aggregates was monitored by phase-contrast microscopy after resuspending the bacteria in RPMI-serum, as previously described (Helaine et al., 2005). Aggregation was quantified by measuring the changes in OD600 that occur in static cultures upon sedimentation of the aggregates, as previously described (Helaine et al., 2005).

Adhesion of N. meningitidis to HUVEC was quantified as described previously (Helaine et al., 2005). In brief, monolayers of 105 cells, in 24 wells plates, were infected with approximately 3 × 107 cfu ml−1. After 30 min of contact between the bacteria and the cells, unbound bacteria were removed by performing several washes and the infection was continued for 4 h with washes every hour. Adherent bacteria, recovered by scraping the wells, were then counted by plating appropriate dilutions on GCB agar plates.

Preparation of N. meningitidis protein extracts

Whole-cell extracts were prepared as described elsewhere (Winther-Larsen et al., 2005), using bacteria grown on GCB agar plates, which were first resuspended in cold PBS at an OD600 of 1. Ten millilitres of this suspension was then pelleted by centrifugation at 9000 g for 10 min and resuspended in 1 ml of cold lysis buffer containing 200 mM Tris (pH 7.5), 20% acetone, 40 mM EDTA, 0.1% Triton X100. This suspension was incubated 15 min at 4°C and the bacteria were briefly centrifuged for 20 s using a bench mini-centrifuge. The supernatant was recovered and solubilized proteins concentrations were determined using the Bio-Rad Protein Assay (Bio-Rad) following the manufacturer's instructions.

SDS-PAGE and Western blotting

Separation of the proteins by SDS-PAGE and subsequent transfer to nitrocellulose membranes were done as previously described (Carbonnelle et al., 2005). Blocking, incubation with primary/secondary antibodies and detection using ECL Plus (Amersham Biosciences) were done as outlined in the ECL Plus Western Blotting Detection Manual. When studying the oligomers formed by PilQ, which are resistant to SDS and are retained in the stacking gel due to their high molecular weight, the stacking gel was kept and transferred as well. All the antibodies were used at a 1/10 000 dilution. The class 4 outer membrane RmpM protein, used as a control to check the protein preparations, was detected using a mouse monoclonal serum (Morand et al., 2001). PilE and PilQ were detected, respectively, using the 5C5 mouse monoclonal serum (Marceau et al., 1998) and a rabbit polyclonal serum donated by T. Tønjum (University of Oslo, Norway). The secondary antibodies were anti-mouse or anti-rabbit horseradish peroxidase-linked IgG (Amersham Biosciences).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Tone Tønjum (University of Oslo, Norway) for the kind gift of anti-PilQ antibody. We are grateful to Guillaume Duménil, Patricia Martin and Chiara Recchi for critical reading of the manuscript. This work was supported by INSERM, Université Paris Descartes and Agence Nationale de la Recherche (JC05-44953 grant to V. Pelicic).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  • Alm, R.A., and Mattick, J.S. (1997) Genes involved in the biogenesis and function of type-4 fimbriae in Pseudomonas aeruginosa. Gene 192: 8998.
  • Anantha, R.P., Stone, K.D., and Donnenberg, M.S. (2000) Effects of bfp mutations on biogenesis of functional enteropathogenic Escherichia coli type IV pili. J Bacteriol 182: 24982506.
  • Carbonnelle, E., Helaine, S., Prouvensier, L., Nassif, X., and Pelicic, V. (2005) Type IV pilus biogenesis in Neisseria meningitidis: PilW is involved in a step occuring after pilus assembly, essential for fiber stability and function. Mol Microbiol 55: 5464.
  • Chami, M., Guilvout, I., Gregorini, M., Remigy, H.W., Muller, S.A., Valerio, M., et al. (2005) Structural insights into the secretin PulD and its trypsin-resistant core. J Biol Chem 280: 3773237741.
  • Collins, R.F., Frye, S.A., Kitmitto, A., Ford, R.C., Tonjum, T., and Derrick, J.P. (2004) Structure of the Neisseria meningitidis outer membrane PilQ secretin complex at 12 Å resolution. J Biol Chem 279: 3975039756.
  • Crago, A.M., and Koronakis, V. (1998) Salmonella InvG forms a ring-like multimer that requires the InvH lipoprotein for outer membrane localization. Mol Microbiol 30: 4756.
  • Craig, L., Pique, M.E., and Tainer, J.A. (2004) Type IV pilus structure and bacterial pathogenicity. Nat Rev Microbiol 2: 363378.
  • Crowther, L.J., Anantha, R.P., and Donnenberg, M.S. (2004) The inner membrane subassembly of the enteropathogenic Escherichia coli bundle-forming pilus machine. Mol Microbiol 52: 6779.
  • Crowther, L.J., Yamagata, A., Craig, L., Tainer, J.A., and Donnenberg, M.S. (2005) The ATPase activity of BfpD is greatly enhanced by zinc and allosteric interactions with other Bfp proteins. J Biol Chem 280: 2483924848.
  • Drake, S.L., Sandstedt, S.A., and Koomey, M. (1997) PilP, a pilus biogenesis lipoprotein in Neisseria gonorrhoeae, affects expression of PilQ as a high-molecular-mass multimer. Mol Microbiol 23: 657668.
  • Durand, E., Michel, G., Voulhoux, R., Kurner, J., Bernadac, A., and Filloux, A. (2005) XcpX controls biogenesis of the Pseudomonas aeruginosa XcpT-containing pseudopilus. J Biol Chem 280: 3137831389.
  • Geoffroy, M.-C., Floquet, S., Métais, A., Nassif, X., and Pelicic, V. (2003) Large-scale analysis of the meningococcus genome by gene disruption: resistance to complement-mediated lysis. Genome Res 13: 391398.
  • Hardie, K.R., Lory, S., and Pugsley, A.P. (1996) Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein. EMBO J 15: 978988.
  • Helaine, S., Carbonnelle, E., Prouvensier, L., Beretti, J.-L., Nassif, X., and Pelicic, V. (2005) PilX, a pilus-associated protein essential for bacterial aggregation, is a key to pilus-facilitated attachment of Neisseria meningitidis to human cells. Mol Microbiol 55: 6577.
  • Hoyne, P.A., Haas, R., Meyer, T.F., Davies, J.K., and Elleman, T.C. (1992) Production of Neisseria gonorrhoeae pili (fimbriae) in Pseudomonas aeruginosa. J Bacteriol 174: 73217327.
  • Hwang, J., Bieber, D., Ramer, S.W., Wu, C.Y., and Schoolnik, G.K. (2003) Structural and topographical studies of the type IV bundle-forming pilus assembly complex of enteropathogenic Escherichia coli. J Bacteriol 185: 66956701.
  • Lory, S., and Strom, M.S. (1997) Structure-function relationship of type-IV prepilin peptidase of Pseudomonas aeruginosa – a review. Gene 192: 117121.
  • Marceau, M., Forest, K., Beretti, J.-L., Tainer, J., and Nassif, X. (1998) Consequences of the loss of O-linked glycosylation of meningococcal type IV pilin on piliation and pilus-mediated adhesion. Mol Microbiol 27: 705715.
  • Mattick, J.S. (2002) Type IV pili and twitching motility. Annu Rev Microbiol 56: 289314.
  • Mehr, I.J., Long, C.D., Serkin, C.D., and Seifert, H.S. (2000) A homologue of the recombination-dependent growth gene, rdgC, is involved in gonococcal pilin antigenic variation. Genetics 154: 523532.
  • Merz, A.J., and So, M. (2000) Interactions of pathogenic Neisseriae with epithelial cell membranes. Annu Rev Cell Dev Biol 16: 423457.
  • Merz, A.J., So, M., and Sheetz, M.P. (2000) Pilus retraction powers bacterial twitching motility. Nature 407: 98102.
  • Morand, P.C., Tattevin, P., Eugène, E., Beretti, J.-L., and Nassif, X. (2001) The adhesive property of the type IV pilus-associated component PilC1 of pathogenic Neisseria is supported by the conformational structure of the N-terminal part of the molecule. Mol Microbiol 40: 846856.
  • Morand, P.C., Bille, E., Morelle, S., Eugène, E., Beretti, J.L., Wolfgang, M., et al. (2004) Type IV pilus retraction in pathogenic Neisseria is regulated by the PilC proteins. EMBO J 23: 20092017.
  • Nassif, X., Lowy, J., Stenberg, P., O'Gaora, P., Ganji, A., and So, M. (1993) Antigenic variation of pilin regulates adhesion of Neisseria meningitidis to human epithelial cells. Mol Microbiol 8: 719725.
  • Neu, H.C., and Heppel, L.A. (1965) The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem 240: 36853692.
  • Nudleman, E., and Kaiser, D. (2004) Pulling together with type IV pili. J Mol Microbiol Biotechnol 7: 5262.
  • Nudleman, E., Wall, D., and Kaiser, D. (2006) Polar assembly of the type IV pilus secretin in Myxococcus xanthus. Mol Microbiol 60: 1629.
  • Nunn, D. (1999) Bacterial type II protein export and pilus biogenesis: more than just homologies? Trends Cell Biol 9: 402408.
  • Parge, H.E., Forest, K.T., Hickey, M.J., Christensen, D.A., Getzoff, E.D., and Tainer, J.A. (1995) Structure of the fibre-forming protein pilin at 2.6 Å resolution. Nature 378: 3238.
  • Peabody, C.R., Chung, Y.J., Yen, M.R., Vidal-Ingigliardi, D., Pugsley, A.P., and Saier, M.H., Jr. (2003) Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella. Microbiology 149: 30513072.
  • Pelicic, V., Jackson, M., Reyrat, J.-M., Jacobs, W.R. Jr, Gicquel, B., and Guilhot, C. (1997) Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc Natl Acad Sci USA 94: 1095510960.
  • Pugsley, A.P. (1993) The complete general secretory pathway in gram-negative bacteria. Microbiol Rev 57: 50108.
  • Pujol, C., Eugène, E., De Saint Martin, L., and Nassif, X. (1997) Interaction of Neisseria meningitidis with a polarized monolayer of epithelial cells. Infect Immun 65: 48364842.
  • Pujol, C., Eugène, E., Marceau, M., and Nassif, X. (1999) The meningococcal PilT protein is required for induction of intimate attachment to epithelial cells following pilus-mediated adhesion. Proc Natl Acad Sci USA 96: 40174022.
  • Py, B., Loiseau, L., and Barras, F. (2001) An inner membrane platform in the type II secretion machinery of Gram-negative bacteria. EMBO Rep 2: 244248.
  • Rakotoarivonina, H., Jubelin, G., Hebraud, M., Gaillard-Martinie, B., Forano, E., and Mosoni, P. (2002) Adhesion to cellulose of the Gram-positive bacterium Ruminococcus albus involves type IV pili. Microbiology 148: 18711880.
  • Ramer, S., Schoolnik, G., Wu, C.-Y., Hwang, J., Schmidt, S., and Bieber, D. (2002) The type IV pilus assembly complex: biogenic interaction among the bundle-forming pilus proteins of enteropathogenic Escherichia coli. J Bacteriol 184: 34573465.
  • Sakai, D., Horiuchi, T., and Komano, T. (2001) ATPase activity and multimer formation of PilQ protein are required for thin pilus biogenesis in plasmid R64. J Biol Chem 276: 1796817975.
  • Sauvonnet, N., Vignon, G., Pugsley, A.P., and Gounon, P. (2000) Pilus formation and protein secretion by the same machinery in Escherichia coli. EMBO J 19: 22212228.
  • Schmidt, S.A., Bieber, D., Ramer, S.W., Hwang, J., Wu, C.Y., and Schoolnik, G. (2001) Structure-function analysis of BfpB, a secretin-like protein encoded by the bundle-forming-pilus operon of enteropathogenic Escherichia coli. J Bacteriol 183: 48484859.
  • Soto, G.E., and Hultgren, S.J. (1999) Bacterial adhesins: common themes and variations in architecture and assembly. J Bacteriol 181: 10591071.
  • Strom, M.S., and Lory, S. (1987) Mapping of export signals of Pseudomonas aeruginosa pilin with alkaline phosphatase fusions. J Bacteriol 169: 31813188.
  • Strom, M.S., and Lory, S. (1991) Amino acid substitutions in pilin of Pseudomonas aeruginosa. Effect on leader peptide cleavage, amino-terminal methylation, and pilus assembly. J Biol Chem 266: 16561664.
  • Turner, L.R., Lara, J.C., Nunn, D.N., and Lory, S. (1993) Mutations in the consensus ATP-binding sites of XcpR and PilB eliminate extracellular protein secretion and pilus biogenesis in Pseudomonas aeruginosa. J Bacteriol 175: 49624969.
  • Voulhoux, R., Bos, M.P., Geurtsen, J., Mols, M., and Tommassen, J. (2003) Role of a highly conserved bacterial protein in outer membrane protein assembly. Science 299: 262265.
  • Winther-Larsen, H.C., Wolfgang, M., Dunham, S., Van Putten, J.P., Dorward, D., Lovold, C., et al. (2005) A conserved set of pilin-like molecules controls type IV pilus dynamics and organelle-associated functions in Neisseria gonorrhoeae. Mol Microbiol 56: 903917.
  • Wolfgang, M., Park, H.-S., Hayes, S.F., Van Putten, J.P.M., and Koomey, M. (1998a) Suppression of an absolute defect in type IV pilus biogenesis by loss-of-function mutations in pilT, a twitching motility gene in Neisseria gonorrhoeae. Proc Natl Acad Sci USA 95: 1497314978.
  • Wolfgang, M., Lauer, P., Park, H.-S., Brossay, L., Hébert, J., and Koomey, M. (1998b) PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae. Mol Microbiol 29: 321330.
  • Wolfgang, M., Van Putten, J.P.M., Hayes, S.F., Dorward, D., and Koomey, M. (2000) Components and dynamics of fiber formation define a ubiquitous biogenesis pathway for bacterial pili. EMBO J 19: 64086418.
  • Yoshida, T., Kim, S.R., and Komano, T. (1999) Twelve pil genes are required for biogenesis of the R64 thin pilus. J Bacteriol 181: 20382043.