AsnB is responsible for peptidoglycan precursor amidation in Clostridium difficile in the presence of vancomycin

Abstract

<i>Clostridium difficile</i> 630 possesses a cryptic but functional gene cluster <i>vanG</i>_Cd homologous to the <i>vanG</i> operon of <i>Enterococcus faecalis</i>. Expression of <i>vanG</i>_Cd in the presence of subinhibitory concentrations of vancomycin is accompanied by peptidoglycan amidation on the <i>meso</i>-DAP residue. In this paper, we report the presence of two potential asparagine synthetase genes named <i>asnB</i> and <i>asnB2</i> in the <i>C. difficile</i> genome whose products were potentially involved in this peptidoglycan structure modification. We found that <i>asnB</i> expression was only induced when <i>C. difficile</i> was grown in the presence of vancomycin, yet independently from the <i>vanG</i>_Cd resistance and regulation operons. In addition, peptidoglycan precursors were not amidated when <i>asnB</i> was inactivated. No change in vancomycin MIC was observed in the <i>asnB</i> mutant strain. In contrast, overexpression of <i>asnB</i> resulted in the amidation of most of the <i>C. difficile</i> peptidoglycan precursors and in a weak increase of vancomycin susceptibility. AsnB activity was confirmed in <i>E. coli</i>. In contrast, the expression of the second asparagine synthetase, AsnB2, was not induced in the presence of vancomycin. In summary, our results demonstrate that AsnB is responsible for peptidoglycan amidation of <i>C. difficile</i> in the presence of vancomycin.