The role of minor T4 pilins in activation of fibre assembly
In this study, we show that efficient assembly of E. coli type IV pilin PpdD can be promoted by the minor pseudopilins of the pullulanase T2SS. In the absence of minor pseudopilins, PpdD pilus assembly was promoted by the minor T4 pilins encoded by the ppdAB-ygdB-ppdC operon of E. coli (Fig. 1), suggesting that these proteins function as minor pilins in their native system. Their functional role remains to be confirmed in E. coli B or EHEC strains, in which PpdD is assembled into bona fide pili involved in adhesion, motility and signalling (Xicohtencatl-Cortes et al., 2007; 2009). Although silent in E. coli K-12 (Sauvonnet et al., 2000a), the four minor pilin genes are significantly induced (nine- to 100-fold) by Sxy (YccR), the global activator of competence genes in E. coli (Sinha et al., 2009), strongly suggesting their involvement in natural transformation. Interestingly, ppdC mutants have been isolated in a screen for defective swarming motility, although it is not known whether T4P are involved in this phenomenon (Inoue et al., 2007).
A set of four minor pilin genes has been implicated in T4P assembly in several well-studied systems, in which their deletion results in piliation defects (Alm and Mattick, 1995; Alm et al., 1996; Winther-Larsen et al., 2005; Carbonnelle et al., 2006; Giltner et al., 2010). These genes include pilHIJK in Neisseria meningitidis and N. gonorrhoeae and the fimU-pilVWX genes in P. aeruginosa. All these studies suggest that minor pilins favour efficient pilus assembly in the presence of retraction ATPase and it has been suggested that they counteract retraction (Carbonnelle et al., 2006; Pelicic, 2008). In Neisseria spp., deletion of the gene encoding the retraction ATPase PilT restores piliation in minor pilin mutants, although such double mutants show defective adherence to epithelial cells and abiotic surfaces (Winther-Larsen et al., 2005; Carbonnelle et al., 2006). In P. aeruginosa, pilus assembly is only partially restored in pilT/minor pilin gene double mutants, suggesting a defect in pilus assembly (Giltner et al., 2010). Using the T2SS as a heterologous assembly system we show here that both the E. coli T4 minor pilins and the T2S minor pseudopilins are functional in promoting assembly of T4P and T2S major pilins, suggesting a similar role and mechanism of action for both sets of proteins. Our results favour a model where T4 minor pilins act by promoting efficient initiation of pilus assembly allowing the assembly reaction to move in the forward direction.
As mentioned above, assembly of T4P in Neisseria spp. takes place in the absence of minor pilins in pilT mutants, which appears to contradict this model (Winther-Larsen et al., 2005; Carbonnelle et al., 2006). Although in T2SS minor pseudopilins are required for efficient pseudopilus assembly initiation, we showed that a very low basal level of PulG pilus assembly is observed even in the absence of minor pseudopilins (Cisneros et al., 2012). These ‘spontaneous initiation’ events may coincide with sporadic spontaneous activation of the assembly ATPase. The few pili assembled in these cases are typically very long, presumably due to the accumulation of pilins in the membrane, which drives the elongation of rare active assembly reactions forward (Cisneros et al., 2012). In T4P, the expression of major pilins is constitutive and their production levels and stability are much higher than in T2SS. In this context, inefficient piliation in the absence of minor pilins and PilT could still lead to accumulation of few long pili, masking the assembly defect. A similar accumulation of PulG pili can be observed over time in minor pseudopilin mutants (Fig. S2). In the presence of retraction forces that allow to reset the system, this assembly defect would become apparent, as it occurs for example in P. aeruginosa (Giltner et al., 2010). Therefore, it would be interesting to test this model by placing major pilins under control of an inducible promoter and performing the kinetic analysis of piliation, comparing the number and length of assembled pili in WT and minor pilin/pilT double mutants.
In addition to the cognate minor pilins, we show that minor pseudopilins PulHIJK are also capable of initiating PpdD pilus assembly. Furthermore, E. coli minor T4 pilins can initiate PulG pilus assembly, suggesting that they function by a similar mechanism, despite the lack of sequence similarity in their globular domains. So far, structural information is not available for minor T4 pilins of this class, but such data are available for the T2SS minor pseudopilins, whose biochemical and structural analysis indicates high affinity interactions between their globular domains. For example, globular domains of minor pseudopilins EpsI and EpsJ from Vibrio vulnificus could only be purified and crystallized after co-production, suggesting that they stabilize each other and possibly assist each other's folding (Yanez et al., 2008; Lam et al., 2009). Globular domains of the GspJ–GspI–GspK formed a quasi-helical hetero-trimer that may be localized at the tip of the pseudopilus (Korotkov and Hol, 2008). In vitro analysis of the P. aeruginosa Xcp T2SS showed that periplasmic domains of all four minor pseudopilins form a complex (Douzi et al., 2009). However, the major pseudopilin XcpT did not bind to this quaternary complex, suggesting that structural compatibility between major and minor pilins does not account for the assembly mechanism, consistent with the absolute requirement of the assembly platform components for the pseudopilus elongation (Sauvonnet et al., 2000b and Fig. S3).
How do these two different sets of pilins promote fibre assembly by T4 pilin PpdD and major pseudopilin PulG? We recently showed that the minor pseudopilins in T2SS initiate PulG polymerization by a mechanism involving the self-assembly of PulI, PulJ and PulK (Cisneros et al., 2012). In their native, membrane environment, formation of the minor pseudopilin complex, driven by the affinity of their globular domains, promotes the upward movement of their TM segments (Cisneros et al., 2012). This series of binding events between PulJ, PulI and PulK results in the formation of a pseudopilus-like structure on the periplasmic side of the IM, where each subunit is shifted with respect to its nearest neighbours by 1 nm (Fig. 7). The extent of this shift, demonstrated by position-specific cysteine cross-linking, corresponds to the axial rise between the major pilins in the assembled pseudopilus. We have proposed that the conformational changes during initiation complex formation activate the IM assembly machinery. Membrane extraction of PulK induced by its binding to the PulJ–PulI complex could transduce a signal to the assembly ATPase and pull it close to the membrane to activate ATP hydrolysis (Camberg et al., 2007; Cisneros et al., 2012). The capacity of minor T4 pilins to promote heterologous assembly of PulG, and the reciprocal induction of PpdD pilus assembly by the minor pseudopilins, lend further support to a model whereby minor (pseudo)pilin complex initiates assembly by triggering the elongation competent state of the IM assembly platform.
Figure 7. Model of (pseudo)pilus assembly.
Upper panel: 1. Minor pseudopilins self-assemble at the inner membrane into a pseudopilus-like structure without external energy (Cisneros et al., 2012). 2. Minor (pseudo)pilins transduce the signal to the assembly ATPase PulE (in red), priming it for major pilin elongation. 3. The active assembly ATPase catalyses the processive (pseudo)pilus elongation adding minor pseudopilin(s) PulH and multiple major subunits PulG coupled to ATP hydrolysis.
Lower panel: The analogous role is proposed for the E. coli minor T4 pilins YgdB-PpdA-PpdC in self-assembly (1), activating the assembly machinery (2) to promote addition of PpdB and PpdD in the elongation step (3). The assembly platform (AP) complex comprises PulF, PulL, PulM and PulC proteins.
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An alternative model would be that the self-assembly of the minor pseudopilins simply leads to the scaffolding of the major pseudopilin, which would require structural recognition between the minor pseudopilin globular domains and the major pilin. However, there is little sequence similarity between globular domains of the minor pilins and pseudopilins (Fig. S4). Therefore, it is reasonable to assume that there is no high-affinity interaction between PulG and T4 minor pilins or PpdD and T2S minor pseudopilins. Yet, both sets of minor pilins are functional for initiation of pilus assembly suggesting that they could exert their effect not on major pilins directly but on the assembly machinery. According to this model, initiation and elongation, at least in T2SS, are distinct processes, which are coupled at the level of the assembly platform. Therefore, it is tempting to speculate that the E. coli T4 minor pilins also undergo self-assembly that would promote PulG pilus assembly by the same mechanism. In support of this model, homologues of PulH, which are thought to structurally link the minor pseudopilin tip (Korotkov and Hol, 2008) to the major pilin (Douzi et al., 2009), are dispensable for pilus assembly (Sauvonnet et al., 2000b; Cisneros et al., 2012).
Unlike the globular domains of minor pseudopilins and minor T4 pilins, which are very different (Fig. S4), the TM segments of these proteins share certain conserved features (Fig. 1). All minor pseudopilins except one (PulK and its homologues) contain the conserved residue E5, which has been implicated in the interactions between neighbouring subunits, neutralizing the charge of the free N-terminal amine of the pseudopilin assembled upstream. The E5 residue would be dispensable in the tip subunit, and its absence is indeed conserved in all PulK homologues of T2SS, as well as in minor T4 pilins PilK of Neisseria spp. (Winther-Larsen et al., 2005), PilX in P. aeruginosa (Giltner et al., 2010) and YgdB of E. coli. In addition, in both PulK and YgdB, the more hydrophobic residues Ile or Val respectively, replace the N-terminal Phe residue. This feature could facilitate the extraction of these pilins from the membrane, which has been observed for PulK in silico by molecular dynamics simulations (Cisneros et al., 2012). The same feature is shared by PpdA and the central pseudopilin PulI, where the N-terminal Phe has been substituted by Tyr and Met residues respectively, again consistent with a partial membrane extraction of the central pilins in the tripartite ‘tip complex’. The minor pilin PpdB shares a conserved residue Pro25 with the pseudopilin PulH, which appears to be involved in a post-initiation step of assembly (Douzi et al., 2009). Based on these primary sequence features, it is tempting to speculate that the T4PS minor pilins PpdC, PpdA and YgdB form a tip complex that may nucleate pilus assembly (Fig. 7). Their globular domains are therefore predicted to interact strongly and sequentially to promote the conformational changes analogous to those that take place in the T2SS (Korotkov and Hol, 2008; Cisneros et al., 2012). In support of this model, N. gonorrhoeae minor pilins PilI and PilJ depend on each other for stability (Winther-Larsen et al., 2005), similar to their pseudopilin orthologues PulI and PulJ, and the P. aeruginosa minor pilins PilV and PilW follow the same pattern (Giltner et al., 2010). Interestingly, the order of minor pilin genes in the E. coli cluster is slightly different from that in Neisseria spp. or P. aeruginosa. In particular, in E. coli, it is the third gene in the cluster, ygdB and not the fourth, that encodes the minor pilin lacking the E5 residue. However, the same gene organization and linkage to recC is conserved in many species of γ-proteobacteria (Fig. S5).
Although minor T4 pilins have been proposed to promote pilus assembly initiation, the notion that they are localized at the pilus tip has been recently challenged (Giltner et al., 2010). On the other hand, distinct structures have been observed at the tip of gonococcal pili, and their size suggests that they may be composed of more than three protein subunits (Winther-Larsen et al., 2007). Clearly, further biochemical and structural analysis of minor T4 pilins will be necessary to test their assembly and function.
Assembly of T4 pili by the T2SS
It is interesting that the major subunits of the T2SS and T4PS, PulG and PpdD, are efficiently recognized and assembled by the same machinery. PpdD is an exception among T4 pilins in this regard, since PilE from Neisseria or PilA from P. aeruginosa could not be assembled in this context (Kohler et al., 2004). Homology between PulG and PpdD, which have a very similar size, is limited to their TM segments, which are 50% identical, and 85% similar or identical, while sequence identity of their periplasmic domains is only 13%. The highly conserved TM segments of major pilins are therefore likely to play a major role in assembly by interacting with the prepilin peptidase and with the components of the assembly platform. This is supported by the fact that PulL, PulE and PulF are essential for both PulG and PpdD assembly (Sauvonnet et al., 2000a). However, we provide evidence that pilus assembly also requires structural compatibility between pilins. When both PpdD and PulG pili were assembled in the same bacteria, we observed defined filaments composed of one or the other major pilin by electron microscopy. Similar results were previously observed in P. aeruginosa, where PAO strains expressing PAK pilA assemble only homopolymers of PAO or PAK pilins (Pasloske et al., 1989). Occasionally, some PulG subunits could be found in PpdD filaments, although in most cases this coincided with points of contact between distinct fibres. Nevertheless, most of the filaments are mainly constituted of one major pilin type. Consistent with this, PpdD could not be co-purified with PulG-His pili by affinity chromatography (Kohler et al., 2004). Interestingly, the same study has reported that, in the presence of the PulG : His6 variant, the Pul secretion system did not assemble PpdD as pili but rather as small aggregates (Kohler et al., 2004). Constitutive coexpression of pilin genes used in that study could have led PulG : His6 to outcompete PpdD for the assembly machinery. Alternatively, the two types of pili may have behaved differently in EM experiments. In the present study we used treatment of carbon grids with CaCl2 to improve pilus adsorption during EM sample preparation (see Experimental procedures). The formation of pilin homopolymers suggests that after the initiation step, filament elongation involves structural recognition between the incoming major pilin and the growing filament. This is consistent with the results of the structure–function analysis of PulG pili, which identified two specific and highly conserved salt bridge interactions between neighbouring protomers, both essential for pseudopilus assembly and for protein secretion (Campos et al., 2010).
Functional implications for protein secretion mechanism
One possible model for T2SS pseudopilus assembly is that the minor pseudopilins occupy the assembly platform to initiate the fibre elongation. However, the co-production of PpdD and the cognate minor pilins with the Pul system did not interfere with pullulanase secretion. This observation suggests that the self-assembly of minor (pseudo)pilins and their interactions with the assembly platform are transient and dynamic.
Interestingly, conditions favouring PpdD pilus assembly did not support pullulanase secretion, even in the presence of minor T2SS pseudopilins. Recently, it was shown that minor pseudopilin complex interacts in vitro with the specific substrate of the P. aeruginosa T2SS (Douzi et al., 2011). This result supports the piston model where the pseudopilus tip complex promotes secretion by direct contact with the substrate. Consistent with this model, E. coli minor pilins cannot substitute for the Pul T2SS minor pseudopilins in pullulanase secretion. One could hypothesize that, in the absence of the major pseudopilin (PulG), assembly of another pilin, (such as PpdD) should provide the driving force for the piston-like motion of the pseudopilin tip complex. However, this was not the case (Fig. 5), showing that, although necessary, minor pseudopilins are not sufficient for function. The functional defect could also be due to a lack of structural compatibility between minor pilins and any other component of the T2SS, since the sequence similarity between globular domains of T4P pilins and T2SS pseudopilins is low (see above). This could also explain why the E. coli T4PS minor pilins could not promote PulA secretion in the presence of PulG, and were clearly less efficient in initiating PulG pilus assembly compared with PulHIJK (Figs 6 and S1).
Alternatively, PulHIJK-promoted PpdD assembly by the Pul T2SS might not respect all aspects of the dynamics of pseudopilus assembly and disassembly that are required for pseudopilus function in secretion. A third explanation might invoke a direct and specific role for pseudopilin PulG, although PulG can be replaced by at least some major pseudopilins from other T2SS (Vignon et al., 2003). Further studies will be necessary to identify the qualitative difference between partial and fully efficient fibre assembly efficiency and the mechanistic link between assembly and protein secretion.
Structural and functional relatedness of T4PS and T2SS
Recent analyses revealed a higher degree of structural similarity between T2SS and T4PS than was suspected based on their composition and sequence (Golovanov et al., 2006; Ayers et al., 2010; Gu et al., 2012; Korotkov et al., 2012; McLaughlin et al., 2012). The present study further reinforces these similarities, showing that they extend to the functional level. We show that minor T4 pilins and T2SS pseudopilins initiate or activate the assembly of both major pilins and major pseudopilins in our heterologous system.
On the other hand, PpdD and PulG form fibres of different length and thickness apparent by immunofluorescence microscopy, implicating globular domains of major pilins as sole determinants of these properties. The major remaining difference between the T4PS and T2SS could lie in the intrinsic stability of pilins and the instability of pseudopilins. PpdD and its cognate minor pilins have two predicted disulfide bridges, which provide substantial stability. A recently characterized major pilin FimA from Dichelobacter nodosus also contains a disulfide bond in the αβ-loop as predicted for PpdD; however, its C-terminal d-loop, although well structured, is not stabilized by a disulfide bond but rather by a hydrogen bond network (Hartung et al., 2011). In T2SS, major pseudopilins do not contain cysteine residues and their globular domains are stabilized by a single Ca2+ ion in the C-terminal region that corresponds to the d-loop of T4 pilins (Korotkov et al., 2009). Therefore, pseudopilins might be more prone to degradation and to conformational changes leading to fibre dissociation. Indeed, in T2SS, pseudopili are short and remain periplasmic, although the disassembly ATPase is lacking. In contrast, the energy provided by the retraction ATPase of the PilT family present in T4PS is necessary to disassemble T4P, which are very stable. For example, gonococcal pili are heat resistant and remain intact in 8 M urea (Li et al., 2012). The analysis of the T4 pilin assembly in the reconstituted system could provide a valuable model for a comparative study of structure, dynamics and function of these two distinct types of filaments. Detailed structural analysis of PpdD pilins and the assembled pili currently under way will provide insight into the molecular bases underlying these distinct features.