Increased toxin expression in a Clostridium difficile mfd mutant.

Stephanie E Willing,Lluis Sempere,Emma J Richards,Simon M Cutting,Neil F Fairweather,Aaron G Dale

doi:10.1186/s12866-015-0611-5

Abstract

BackgroundThe symptoms of Clostridium difficile infection are mediated primarily by two toxins, TcdA and TcdB, the expression of which is governed by a multitude of factors including nutrient availability, growth phase and cell stress. Several global regulators have been implicated in the regulation of toxin expression, such as CcpA and CodY.ResultsDuring attempts to insertionally inactivate a putative secondary cell wall polysaccharide synthesis gene, we obtained several mutants containing off-target insertions. One mutant displayed an unusual branched colony morphology and was investigated further. Marker recovery revealed an insertion in mfd, a gene encoding a transcription-coupled repair factor. The mfd mutant exhibited pleiotropic effects, in particular increased expression of both toxin A and B (TcdA and TcdB) compared to the parental strain. Western blotting and cellular cytotoxicity assays revealed increased expression across all time points over a 24 h period, with inactivation of mfd resulting in at least a 10 fold increase in cell cytotoxicity. qRT-PCR demonstrated the upregulation of both toxins occurred on a transcriptional level. All effects of the mfd mutation were complemented by a plasmid-encoded copy of mfd, showing the effects are not due to polar effects of the intron insertion or to second site mutations.ConclusionsThis study adds Mfd to the repertoire of factors involved in regulation of toxin expression in Clostridium difficile. Mfd is known to remove RNA polymerase molecules from transcriptional sites where it has stalled due to repressor action, preventing transcriptional read through. The consistently high levels of toxin in the C. difficile mfd mutant indicate this process is inefficient leading to transcriptional de-repression.

Highlights

The symptoms of Clostridium difficile infection are mediated primarily by two toxins, TcdA and TcdB, the expression of which is governed by a multitude of factors including nutrient availability, growth phase and cell stress
On TY agar, the mfd mutant colonies appeared slightly smaller than the parental strain, but on TYG agar the mutant colonies were considerably larger and displayed a highly branched structure (Fig. 1a)
The mfd mutant does not exhibit a pleiotropic phenotype As transcription-repair coupling factor (TCRF) can be involved in a diversity of cellular activities [24, 25] we investigated whether our C. difficile mfd mutant exhibited phenotypes other than the unusual colony morphology and toxin upregulation

Summary

Introduction

The symptoms of Clostridium difficile infection are mediated primarily by two toxins, TcdA and TcdB, the expression of which is governed by a multitude of factors including nutrient availability, growth phase and cell stress. All toxigenic strains produce toxin B and a large percentage of strains produce toxin A [7]. These toxins have a similar structure and mode of action; the toxins are large, multi-domain proteins encoding glucoslytransferase and cysteine protease activities together with a repetitive receptor binding domain [8]. The toxins are released from bacteria in the gut lumen and are taken up by receptor-mediated endocytosis into enteric cells. The Willing et al BMC Microbiology (2015) 15:280 cysteine protease activity is required to release the Nterminal glucosyltransferase domain from the adjacent protease domain, an activity dependent on cytosolic inositol-6-phosphate [10]

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