A novel genetic switch controls phase variable expression of CwpV, a Clostridium difficile cell wall protein.

Jenny E Emerson,Helen A Shaw,Catherine B Reynolds,Neil F Fairweather,David Goulding,Robert P Fagan

doi:10.1111/j.1365-2958.2009.06812.x

Jenny E Emerson, Helen A Shaw + Show 4 more

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https://doi.org/10.1111/j.1365-2958.2009.06812.x

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Journal: Molecular microbiology	Publication Date: Aug 6, 2009
Citations: 99	License type: unspecified-oa

Affiliation: University of Oxford, Imperial College London

Abstract

Clostridium difficile is a nosocomial pathogen that can cause severe gastrointestinal infections. C. difficile encodes a family of cell wall proteins, some of which are implicated in pathogenesis. Here we have characterized CwpV, the largest member of this family. CwpV is surface expressed and post-translationally processed in a manner analogous to the major S-layer protein SlpA. Expression of cwpV is phase variable, with approximately 5% of cells in a population expressing the protein under standard laboratory growth conditions. Upstream of cwpV, inverted repeats flank a 195 bp sequence which undergoes DNA inversion. Use of a gusA transcriptional reporter demonstrated that phase variation is mediated by DNA inversion; in one orientation cwpV is expressed while in the opposite orientation the gene is silent. The inversion region contains neither the promoter nor any of the open reading frame, therefore this system differs from previously described phase variation mechanisms. The cwpV promoter is located upstream of the inversion region and we propose a model of phase variation based on intrinsic terminator formation in the OFF transcript. A C. difficile site-specific recombinase able to catalyse the inversion has been identified.

Highlights

Clostridium difficile is a Gram-positive, spore-forming anaerobe that causes a range of gastrointestinal diseases, ranging from diarrhoea to pseudomembraneous colitis, collectively termed Clostridium difficile-associated disease (CDAD) (Poxton et al, 2001; Bartlett, 2006)
We propose a mechanism for this phase variation and have identified one C. difficile site-specific recombinase that mediates DNA inversion
To further characterize cwpV, the gene from C. difficile 630 was cloned into pMTL960, an Escherichia coli – C. difficile shuttle vector

Summary

Introduction

Clostridium difficile is a Gram-positive, spore-forming anaerobe that causes a range of gastrointestinal diseases, ranging from diarrhoea to pseudomembraneous colitis, collectively termed Clostridium difficile-associated disease (CDAD) (Poxton et al, 2001; Bartlett, 2006). After cleavage of the SlpA precursor, the resulting HMW and LMW SLPs interact via defined domains to form a stable heteromeric complex (Fagan et al, 2009). Antibodies against some of these proteins are found in serum of CDAD patients (Wright et al, 2008) implying at least some of this family of cell wall proteins (CWPs) are expressed in vivo during infection. All of these CWPs contain two or three cell wall binding motifs (Pfam PF04122) in addition to a unique domain that is proposed to specify function

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