Abstract
Clostridium difficile is a gastrointestinal pathogen but how the bacterium colonises this niche is still little understood. Sortase enzymes covalently attach specific bacterial proteins to the peptidoglycan cell wall and are often involved in colonisation by pathogens. Here we show C. difficile proteins CD2537 and CD3392 are functional substrates of sortase SrtB. Through manipulation of the C-terminal regions of these proteins we show the SPKTG motif is essential for covalent attachment to the cell wall. Two additional putative substrates, CD0183 which contains an SPSTG motif, and CD2768 which contains an SPQTG motif, are not cleaved or anchored to the cell wall by sortase. Finally, using an in vivo asymmetric cleavage assay, we show that despite containing a conserved SPKTG motif, in the absence of SrtB these proteins are localised to disparate cellular compartments.
Highlights
Clostridium difficile is an intestinal pathogen that causes significant mortality and morbidity throughout the world[1]
CD3392 has the classic structure of sortase substrates with an N-terminal secretion signal, and a C-terminal cell wall sorting signal (CWSS) comprising an “LPXTG” like motif, a hydrophobic region and a charged tail (Fig. 1A)
Secretion implies that proteins need to cross the cell wall, and bands still observed in the cell wall fraction of the ΔsrtB mutant likely correspond to residual un-cleaved CD3392 retained in the cell wall in a non-covalent manner
Summary
By c-di-GMP which regulates both PPEP-1 and CD2831 expression[19], with a c-di-GMP controlled self-splicing ribozyme controlling CD3246 expression[23]. Small RNAs have been shown to control expression of the putative sortase substrate CD018324. A PPEP-1mutant exhibits increased collagen binding activity, implicating sortase substrates CD2831 and CD3246 in this phenotype[22]. CD3246 is predicted to form an internal thioester bond which may be involved in binding to host proteins[26]. In this study we confirm the in vivo cellular activity in C. difficile of sortase SrtB in strain 630 on substrates containing the motif SPKTG, while those containing SPSTG and SPQTG motifs are uncleaved and we characterise disparate cellular localisation phenotypes of sortase substrates
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