Clostridium difficile surface proteins are anchored to the cell wall using CWB2 motifs that recognise the anionic polymer PSII

Gram-positive surface proteins can be covalently or non-covalently anchored to the cell wall and can impart important properties on the bacterium in respect of cell envelope organisation and interaction with the environment. We describe here a mechanism of protein anchoring involving tandem CWB2 motifs found in a large number of cell wall proteins in the Firmicutes. In the Clostridium difficile cell wall protein family, we show the three tandem repeats of the CWB2 motif are essential for correct anchoring to the cell wall. CWB2 repeats are non-identical and cannot substitute for each other, as shown by the secretion into the culture supernatant of proteins containing variations in the patterns of repeats. A conserved Ile Leu Leu sequence within the CWB2 repeats is essential for correct anchoring, although a preceding proline residue is dispensable. We propose a likely genetic locus encoding synthesis of the anionic polymer PSII and, using RNA knock-down of key genes, reveal subtle effects on cell wall composition. We show that the anionic polymer PSII binds two cell wall proteins, SlpA and Cwp2, and these interactions require the CWB2 repeats, defining a new mechanism of protein anchoring in Gram-positive bacteria.