Gram-positive bacteria utilize a highly conserved family of sortase enzymes to anchor on the cell wall of many surface proteins that are important for bacterial virulence and fitness. The so-called housekeeping sortase (such as SrtA in S. aureus
) anchors a number of LPXTG-containing proteins to the cell wall, while a handful of other sortases (such as SrtABCDE in C. diphtheriae
) are involved in the assembly of various pili (Ton-That and Schneewind, 2004
). In this study, we demonstrated that although the housekeeping sortase of C. diphtheriae
does not participate in pilus polymerization, the enzyme clearly plays a physiological role in facilitating the efficient cell wall anchoring of pilus polymers. The cellular requirement for the housekeeping sortase for pilus assembly is most pronounced at higher levels of the major pilin subunit, which drives the extent of pilus polymerization (Swierczynski and Ton-That, 2006
). Our studies showed conclusively that a pilus-specific sortase and the housekeeping sortase together produce wild-type levels of the different pili on bacterial surface, and they suffice for optimal binding of corynebacteria to pharyngeal epithelial cells, a specific adhesion that is mediated by the SpaA-type pili (Mandlik et al., 2007
). Together, these findings establish for the first time a function of the housekeeping sortase in the final step of pilus assembly. Our findings also provide important evidence for the hypothesis that pilus polymerization precedes the cell wall-anchoring step during pilus biogenesis.
That the housekeeping sortase SrtF of C. diphtheriae is not required for pilus polymerization is evident from genetic and biochemical analysis of mutant strains, which express individual sortases. The strain expressing SrtA only (SrtA+) is able to catalyse not only pilus polymerization, but also the cell wall anchoring of some of the resulting pilus fibres (). Thus, the pilus-specific sortase SrtA is both necessary and sufficient for pilus assembly (see also and ). Furthermore, we have shown that none of the other sortases, including the housekeeping SrtF, is able to catalyse SpaA polymerization.
The vital role of the housekeeping sortase in pilus biogenesis is clearly evident from the striking fact that, in its absence, only a small fraction of the SpaA polymers are anchored to the cell wall. Inspection of the SrtA-only strain revealed that although the efficiency of SpaA polymerization in this strain is comparable to the wild type, most of the polymers made in the mutant strain are secreted out in the culture medium, and little is anchored to the cell wall ( and ). Clearly, there must be a factor missing in this strain that is normally required for the efficient cell wall anchoring of the SpaA pili. That the housekeeping sortase is the missing component is established by the fact that an identical phenotype is observed with the strain that is devoid of SrtF alone (). Importantly, we have demonstrated that in the strain that expresses both SrtA and SrtF, but none of the other sortases, the assembly and cell wall anchoring of the SpaA pili is comparable to the wild type (–). This requirement of both the pilus-specific sortase and the housekeeping sortase for optimal cell wall anchoring of the SpaA pili is further underscored by our demonstration that the two sortases are indeed sufficient to allow corynebacterial adherence to human pharyngeal epithelial cells to a level that is comparable to the wild-type bacteria (). Finally, the role of the housekeeping sortase in pilus biogenesis is not restricted to the SpaA pili. The deletion of srtF
alone significantly reduces the cell wall anchoring of both SpaD- and SpaH-type pili ( and , and S1 and S3
). Therefore, the housekeeping sortase plays a prominent physiological role in the pilus biogenesis in corynebacteria.
The critical question that now arises is precisely how the housekeeping sortase facilitates pilus biogenesis. An obvious function of this sortase is that it directly participates in the catalysis of cell wall anchoring of the pilus, as was pointed out in a recent review article (Scott and Zahner, 2006
). An alternate possibility is that the housekeeping sortase acts indirectly, by catalysing the surface localization of an accessory component that is required for pilus anchoring to the cell wall. Fortunately, we obtained evidence that helps to distinguish these possibilities. Immunofluorescence experiments, which measured surface display of pilin antigens quantitatively, revealed clearly that a corynebacterial mutant that expresses SrtF only displays an appreciable amount of the SpaA pilin on the cell surface (see ). Immunofluorescence analysis showed that SpaB/SpaC is also linked to the cell wall in this strain (Fig. S2
). These results demonstrate the catalytic competence of the housekeeping sortase in anchoring of specific pilus proteins to the cell wall. We, therefore, favour the model that the housekeeping sortase directly catalyses the cell wall anchoring of both pilins and pilus fibres ().
Fig. 9 The two-stage mechanism of pilus biogenesis: function of the housekeeping sortase. This working model, built on the most significant findings reported here, depicts the two stages of pilus polymerization and cell wall anchoring catalysed by a pilus-specific (more ...)
Our finding that pilus polymers are secreted in the culture medium in the absence of the housekeeping sortase has another important implication. If pilus polymerization were carried out on a cell wall-anchored pilin, polymers would never be free to leak out into the medium. Thus, our data provide critical evidence for a biphasic pathway of pilus biogenesis in which cell wall anchoring is the terminal step (). We envision that the pathway of pilus assembly involves two discrete stages – one allowing the joining of pilin monomers to form an extended pilus fibre, and the other terminating this process by cell wall anchoring of the fibre (Mandlik et al., 2007
). Upon synthesis in the cytoplasm, pilin precursors are translocated across the membrane by the Sec machinery. The translocated pilin precursor is captured within exoplasm by a sortase embedded in the nearby membrane, which cleaves the LPXTG motif between the Thr and Gly, and forms an acyl-enzyme substrate intermediate involving the catalytic Cys residue and the Thr residue of the sorting signal. Importantly, the pilus polymerization reaction requires appropriate juxtaposition of two of these intermediates, one of which must be the pilus-specific sortase. The pilin protein moiety from this intermediate is then transferred to the other sortase intermediate through the formation of a Thr–Lys linkage, which is triggered by the nucleophilic attack by an invariant Lys residue that characterizes the conserved pilin motif (). Additional cycles of this reaction can continue pilus growth so long as pilins are available and the sessile Thr–Cys bond in the last subunit added in the polymer is presented by the pilus-specific sortase for nucleophilic attack by another pilin subunit. This is obviously ensured when each of the two sortases participating in the reaction are both pilus-specific, namely SrtA for the SpaA pilus ().
The pilus polymerization stage is terminated when the polymer is transferred to the lipid II precursor, which is in turn cross-linked to the growing cell wall. Based on the data presented here, we suggest that the housekeeping sortase performs this reaction predominantly. For that to happen, the housekeeping sortase must be located in the site of pilus assembly, and it must first form an acyl-enzyme intermediate and provide the Lys nucleophile to receive and secure the pilus polymer (). Once this has occurred, further growth of the pilus is irreversibly blocked because the housekeeping sortase is not a pilin polymerase. This ensures the subsequent transfer of the pilus to lipid II and in turn the cross-linking of the product to the cell wall. Our data suggest that the pilin-specific sortase also carries out this last step physiologically, albeit inefficiently (). Unless there is some form of an organized localization of the different machineries involved, for which there is no evidence, the pilus assembly centre could not always harbour the housekeeping sortase. This necessitates that the pilus-specific sortases perform dual function catalysing polymerization as well as surface anchoring, potentially a fail-safe mechanism evolved to optimize pilus assembly in Gram-positive bacteria.
The participation of the housekeeping sortase in the cell wall-anchoring step of pilus biogenesis helps to explain how pilins are anchored to the cell wall in a pilus-independent fashion. When a pilin precursor is captured by the housekeeping sortase, there is a definite probability that the pilin monomer is incorporated to the cell wall before another subunit or an oligomer is joined by a pilus-specific sortase (). This provides the cell an efficient mechanism to display both pilins and pili on the envelope, a scenario that has important implication in pathogenesis. While pili must aid in the tethering of the pathogen to the appropriate host cells, the surface-displayed pilins would enable the bacterial cell to create intimate zones of adhesion with the host cells. This tight adhesion may trigger not only appropriate host cell signalling but also an efficient toxin delivery (see Mandlik et al., 2007
During infection, many virulence factors are likely to be abundantly produced by corynebacteria for efficient colonization and invasion of the human nasopharynx, and the expression of SpaA pili required for specific adhesion of corynebacteria to the pharyngeal cells may also be increased. Because the requirement of housekeeping sortase in cell wall anchoring is most pronounced when SpaA level is sufficiently high (see ), it is tempting to suggest that the function of the housekeeping sortase might be most crucial in the infection setting. In this regard, it is noteworthy that a binding site for the diphtheria toxin repressor, DtxR, has been identified upstream of the srtF
gene (Yellaboina et al., 2004
). DtxR is an iron-activated transcription regulator that controls the expression of diphtheria toxin, and possibly many additional genes encoding virulence factors. Work is now underway to test whether DtxR regulates pilus gene expression as well as pilus assembly during corynebacterial infection. Considering the fact that three distinct classes of pili are assembled in corynebacteria, and the apparent specificity of the sortases involved in each of these systems, the findings of the present study underscore how little is known so far about the mechanisms by which pilus assembly is co-ordinated in the cell envelope and the factors that must regulate this process temporally and spatially.