title: Clostridium difficile glutamate dehydrogenase is a secreted enzyme that confers resistance to H2O2.
abstract: Clostridium difficile produces an NAD-specific glutamate dehydrogenase (GDH), which converts l-glutamate into α-ketoglutarate through an irreversible reaction. The enzyme GDH is detected in the stool samples of patients with C. difficile-associated disease and serves as one of the diagnostic tools to detect C. difficile infection (CDI). We demonstrate here that supernatant fluids of C. difficile cultures contain GDH. To understand the role of GDH in the physiology of C. difficile, an isogenic insertional mutant of gluD was created in strain JIR8094. The mutant failed to produce and secrete GDH as shown by Western blot analysis. Various phenotypic assays were performed to understand the importance of GDH in C. difficile physiology. In TY (tryptose yeast extract) medium, the gluD mutant grew slower than the parent strain. Complementation of the gluD mutant with the functional gluD gene reversed the growth defect in TY medium. The presence of extracellular GDH may have a functional role in the pathogenesis of CDI. In support of this assumption we found higher sensitivity to H2O2 in the gluD mutant as compared to the parent strain. Complementation of the gluD mutant with the functional gluD gene reversed the H2O2 sensitivity.
authors.affiliation: Division of Biology, Kansas State University, Manhattan, KS 66506, USA.
authors.forename: Brintha Prasummanna
authors.initials: BP
authors.lastname: Girinathan
authors.affiliation: Division of Biology, Kansas State University, Manhattan, KS 66506, USA.
authors.forename: Sterling E
authors.initials: SE
authors.lastname: Braun
authors.affiliation: Division of Biology, Kansas State University, Manhattan, KS 66506, USA.
authors.forename: Revathi
authors.initials: R
authors.lastname: Govind
mesh_terms: D016360:Clostridioides difficile / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D003470:Culture Media / Q000737:chemistry; D024881:Drug Resistance, Bacterial; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism* / Q000633:toxicity*; D016254:Mutagenesis, Insertional
chemical_list: D003470:Culture Media; D006861:Hydrogen Peroxide; D005969:Glutamate Dehydrogenase