Increased toxin expression in a Clostridium difficile mfd mutant

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.

The Bacterial Mfd Protein Prevents DNA Damage Induced by the Host Nitrogen Immune Response in a NER-Independent but RecBC-Dependent Pathway

“…subtilis Δ mfd mutants a high UV mutability despite a minimal UV or mitomycin sensitivity [ 34 , 35 ]. In Clostridium difficile , toxin production is modified in the Δ mfd mutant [ 36 ]. We assessed whether in our mutant mfd deletion could have a role in the induction of cell toxicity as we have previously shown that toxicity is due to the production of extracellular toxins in B .…”

The Regulatory Networks That Control Clostridium difficile Toxin Synthesis

“…The removal of stalled RNAP is not confined to DNA lesions but also occurs for RNAP that is stalled for other reasons, such as nucleotide starvation or blockage by other DNA proteins, such as transcriptional repressors [ 176 ]. The inactivation of mfd in C. difficile resulted in an unusual colony morphology and increased expression of TcdA and TcdB, which occurs at the transcriptional level [ 177 ]. In B. subtilis , Mfd inactivation partially relieved the CodY- or CcpA-mediated transcriptional repression of genes with binding sites downstream of the promoters [ 178 , 179 , 180 ].…”

Identification of Genes Regulating Cell Death inStaphylococcus aureus

“…It is well known that Mfd is involved in prevent DNA damage from oxidative stress, immune response, and drugs [47, 50]. Decreased expression of Mfd in S. aureus led to decreased biofilm formation [51] and a mfd mutant of C. difficle has increased toxin production [52]. For B. subtilis , Mfd-deficient cells formed less endospores [53] and resulted in a 35-fold overexpression of OhrR, a transcription factor involved in peroxide stress response [54].…”