Clostridium difficile MazF Toxin Exhibits Selective, Not Global, mRNA Cleavage

Abstract

e Clostridium difficile is an important, emerging nosocomial pathogen. The transition from harmless colonization to disease is typically preceded by antimicrobial therapy, which alters the balance of the intestinal flora, enabling C. difficile to proliferate in the colon. One of the most perplexing aspects of the C. difficile infectious cycle is its ability to survive antimicrobial therapy and transition from inert colonization to active infection. Toxin-antitoxin (TA) systems have been implicated in facilitating persistence after antibiotic treatment. We identified only one TA system in C. difficile strain 630 (epidemic type X), designated MazE-cd and MazF-cd, a counterpart of the well-characterized Escherichia coli MazEF TA system. This E. coli MazF toxin cleaves mRNA at ACA sequences, leading to global mRNA degradation, growth arrest, and death. Likewise, MazF-cd expression in E. coli or Clostridium perfringens resulted in growth arrest. Primer extension analysis revealed that MazF-cd cleaved RNA at the five-base consensus sequence UACAU, suggesting that the mRNAs susceptible to cleavage comprise a subset of total mRNAs. In agreement, we observed differential cleavage of several mRNAs by MazF-cd in vivo, revealing a direct correlation between the number of cleavage recognition sites within a given transcript and its susceptibility to degradation by MazF-cd. Interestingly, upon detailed statistical analyses of the C. difficile transcriptome, the major C. difficile virulence factor toxin B (TcdB) and CwpV, a cell wall protein involved in aggregation, were predicted to be significantly resistant to MazF-cd cleavage.

A regulatory role for Staphylococcus aureus toxin–antitoxin system PemIKSa

“…A simple mechanism such as that described here could potentially regulate all TA systems where the toxin is a sequencespecific RNAse. Indeed, the pentad sequences recognized by MazF-mt3, MazF-mt7, MazF Sa , MazF Bs and MazF-cd are absent in the transcripts of their respective antitoxins [15][16][17]35 . Moreover, toxins other than PemK Sa do not interfere with cell translation machinery 17,36 .…”

“…For example, the MazF-mt3 or MazF-mt7 recognition sequence is underrepresented in a subset of genes of the PE and PPE families, which are important in the immunopathogenicity of M. tuberculosis 16 . Further, the recognition sequence for MazF-cd is underrepresented in toxB and cwpV genes, encoding factors implicated in virulence 17 . We argue that PemIK Sa is involved in virulence modulation in S. aureus, based on the fact that the PemK Sa recognition sequence is underrepresented in a range of genes encoding virulence factors.…”

“…However, many class II toxins are sequence-specific ribonucleases. For example, the MazF toxin from Escherichia coli hydrolyses mRNA at the AkCA sequence 14 , where the arrow denotes the site of hydrolysis; the MazF homologues MazF-mt3, MazF-mt7, MazF Sa and MazF-cd from Mycobacterium tuberculosis, S. aureus and Clostridium difficile recognize pentad target sequences (UUkCCU or CUkCCU, UkCGCU, UkACAU and UkAkCkAU, respectively) [15][16][17] ; whereas MazF-hw from Haloquadra walsbyi recognizes seven consecutive nucleotides (UUkACUCA) 18 . The activity of highly sequence-specific toxins leads to selective degradation and, as a result, inhibition of translation from only a limited pool of transcripts; this process is termed RNA interference 19 , and probably constitutes an efficient means of global regulation of gene expression.…”

“…TA-driven RNA interference has been implicated in the regulation of virulence and antibiotic resistance in numerous pathogens including M. tuberculosis 16 , S. aureus 15,20 , Yersinia pestis 21 , Helicobacter pylori 22 , Streptococcus mutans 23 and C. difficile 17 , and also among enterococci 24 . However, the occurrence of this mechanism remains largely speculative, and the very attractive concept of global regulation of virulence factor expression by TA systems in pathogenic bacteria still awaits thorough experimental characterization.…”

Regulatory crosstalk between type I and type II toxin-antitoxin systems in the human pathogen Enterococcus faecalis

“…DNA palindromes that encompass the TSS region and the minimal promoter sequence (i.e., ¡10 and ¡35 boxes, and the spacer), have been shown to be recognized and bound by MazE and MazEF. 43,[52][53][54] We sought for palindromes within Figure 7. Regulatory model of the TA crosstalk at the TAI-II locus in E. faecalis V583.…”