C. difficile infection is invariably associated with the disruption of the normal intestinal microflora by the administration of broad spectrum antibiotics. Thus there is a pressing need to develop therapies that selectively target C. difficile while leaving the intestinal microflora intact. The C. difficile reference strain 630 encodes a single predicted sortase, CD630_27180, which has strong amino acid similarity with SrtB of S. aureus and B. anthracis . Sortase substrates frequently contribute toward pathogenesis via their involvement in attachment to specific tissues during infection [17,41–44], as well as the bacteria’s ability to evade the immune response of the host [32,36]. Sortases, although not essential for growth or viability of the organism, are often essential for virulence in Gram-positive organisms; inactivation of sortases reduces colonization in mice [8,13,44,45], and decreases adhesion and invasion in vitro [8,10,14,46,47]. Sortases and their substrates are considered promising targets for the development of new anti-infective compounds [10,14,48]. Unusually for Gram-positive bacteria, C. difficile appears to possess a single sortase enzyme that is likely to be important for the viability of the pathogen as we have been unable to construct a C. difficile strain 630 SrtB defined mutant (unpublished data). Inhibiting the C. difficile sortase could prove to be a strategy to specifically target C. difficile.
In this study, we cloned, expressed and characterized the sortase encoded by CD630_27180 of C. difficile 630, a predicted class B sortase (SrtB). Sortase nomenclature is based on sequence similarity to the known classes of sortase, A-F . Sortases of class B typically are involved in heme-iron uptake and tend to be expressed in operons with their substrates [17,18]. Genes encoding class A sortases are not found in proximity to their substrates, which consist of a variety of surface proteins with diverse biological functions. Several exceptions to these rules have already been described, notably a class B sortase that polymerizes pilin subunits in S. pyogenes , and a class E sortase from C. diphtheriae that serves a housekeeping function . The potential C. difficile sortase substrates identified in this paper comprise a diverse range of surface proteins, suggesting that SrtB may serve as a housekeeping sortase in C. difficile, a function usually reserved for class A sortases.
These potential sortase substrates in C. difficile strain 630 comprise of seven proteins, all containing an (S/P)PXTG motif, that are predicted to be surface localized and are conserved across C. difficile strains. Recently it was proposed that a C. difficile collagen binding protein, CbpA, may be sorted to the cell surface by sortase recognizing an NVQTG motif . In this study, we developed a FRET-based assay to demonstrate that SrtB of C. difficile recognizes and cleaves the (S/P)PXTG motif between the threonine and glycine residues, and that cleavage is dependent on a single cysteine residue at position 209. SrtBΔN26 does not appear to cleave the S. aureus SrtA and SrtB motifs, LPXTG and NPQTN, respectively, nor the NVQTG motif in vitro, suggesting that CbpA from C. difficile may be attached to the cell surface by another mechanism.
The FRET-based assay enabled us to determine kinetic parameters for the recombinant C. difficile SrtB. Although the catalytic activity appears low, low catalytic efficiency is observed for most sortases in vitro [40,51]. The kinetic and cleavage data we report for SrtBΔN26 is consistent with this trend. In vivo, the sorting motifs are part of a larger protein, and the transpeptidation substrates are part of a cell wall precursor or mature peptidoglycan [5,6,39]. The transpeptidation reaction has been observed in vitro for sortases from bacteria with a Lys-type peptidoglycan, where cross-linking occurs through a peptide bridge [52,53] such as S. aureus and Streptococcus species [4,40,54], but not for bacteria with Dap-type peptidoglycan such as Bacillus with direct cross-linkages through m-diaminopimelic acid . The likely cell wall anchor of the C. difficile SrtB substrates is the diaminopimelic acid cross-link , similar to Bacillus. When transpeptidation is observed in vitro, the cleavage efficiency of sortase increases.
This study revealed that recombinant SrtBΔN26 cleaves the (S/P)PXTG motifs with varying levels of efficiency, cleaving the sequences PPKTG and SPQTG with the greatest efficiency. Apparent preferential cleavage efficiency of certain substrate sequences in vitro has been observed in other sortases. For example, in B. anthracis, BaSrtA cleaves LPXTG peptides more readily than a peptide containing the sequence LPNTA . The biological significance of this peptide sequence preference is unknown.
Small-molecule inhibitors with activity against SrtA and SrtB have been reported that prevent cleavage of fluorescently-labelled peptide compounds by sortase in vitro . These compounds inhibit cell adhesion to fibronectin, yet, they have no effect on in vitro growth. Inhibitors tested against SrtA, SrtB and SrtC in B. anthracis irreversibly modified the active cysteine residue . Several compounds identified in this study had an inhibitory effect on C. difficile SrtB activity. However, these lead compounds had no direct effect on in vitro C. difficile growth (data not shown), which is consistent with observations in S. aureus . Inhibition of bacterial growth is not considered vital in the development of sortase-based drug therapies. In both Staphylococcus and Bacillus, sortase inhibitors show good suitability for further development as therapeutics despite their lack of bactericidal activity. When mice challenged with S. aureus were treated with sortase inhibitor compounds, infection rates and mortality were reduced , despite these compounds having no effect on staphylococcal growth . The use of in silico approaches such as the LeadBuilder method employed by this study to screen databases of putative small-molecule inhibitors for further analysis has been validated. Further analysis of the structural similarities between the hit compounds could lead to a refinement of SrtB inhibitor design and increased potency in vitro.