Importance of Glutamate Dehydrogenase (GDH) in Clostridium difficile Colonization In Vivo

Citation: Girinathan, B. P., Braun, S., Sirigireddy, A. R., Lopez, J. E., & Govind, R. (2016). Importance of Glutamate Dehydrogenase (GDH) in Clostridium difficile Colonization In Vivo. Plos One, 11(7), 18. doi:10.1371/journal.pone.0160107Clostridium difficile is the principal cause of antibiotic-associated diarrhea. Major metabolic requirements for colonization and expansion of C. difficile after microbiota disturbance have not been fully determined. In this study, we show that glutamate utilization is important for C. difficile to establish itself in the animal gut. When the gluD gene, which codes for glutamate dehydrogenase (GDH), was disrupted, the mutant C. difficile was unable to colonize and cause disease in a hamster model. Further, from the complementation experiment it appears that extracellular GDH may be playing a role in promoting C. difficile colonization and disease progression. Quantification of free amino acids in the hamster gut during C. difficile infection showed that glutamate is among preferred amino acids utilized by C. difficile during its expansion. This study provides evidence of the importance of glutamate metabolism for C. difficile pathogenesis

Correction: Importance of Glutamate Dehydrogenase (GDH) in Clostridium difficile Colonization In Vivo.

[This corrects the article DOI: 10.1371/journal.pone.0160107.]

<i>C</i>. <i>difficile gluD</i> mutant complemented with secretable forms of GDH colonized better and induced more inflammation than the mutant expressing nonsecretable GDH.

<p><b>A</b>. Representative image of H&E stained colonic specimens. <b>B</b>. Histology scores evaluated as described in the Materials and Methods section. <b>C</b>. <i>C</i>. <i>difficile</i> colonization levels for each of the groups (CFU per gram of cecal content at the time of necropsy). Unpaired <i>t</i> test was performed for statistical analysis (n = 7 per group). Error bars represent SD.</p

<i>C</i>. <i>difficile</i> JIR8094::<i>gluD</i> mutant does not colonize or induce inflammation in hamsters.

<p><b>A.</b> Representative colonic histologic images (hematoxylin and eosin (H&E) staining). Cecal tissues from parental strain-infected hamsters were harvested at the time of sacrifice. Cecal tissues from surviving <i>gluD</i> mutant-infected (and uninfected) hamsters were harvested 15 days post-infection. <b>B.</b> Histology scores were evaluated as described in the Materials and Methods. <b>C.</b> <i>C</i>. <i>difficile</i> colonization levels for each of the two groups in CFU per gram of cecal content at the time of necropsy. In three of seven <i>gluD</i> mutant-infected animals, <i>C</i>. <i>difficile</i> was not detected and are not represented in the figure (n = 7 per group). Error bars represent SD.</p

Amino acid analyses of hamster cecal contents.

<p>Amino acid analyses of hamster cecal contents.</p

Complementation of <i>gluD</i> mutant with various <i>gluD</i> constructs.

<p>The <i>gluD</i> homologues from closely related bacterial species were expressed in the <i>C</i>. <i>difficile gluD</i> mutant strain, and their secretion from <i>C</i>. <i>difficile</i> was analyzed. Cytosolic (cyt) and concentrated supernatants (sup) from the bacterial cultures expressing various <i>gluD</i> constructs were separated by SDS-PAGE, and were analyzed by Coomassie staining <b>(A)</b> and by zymogram <b>(B)</b>. <i>C</i>. <i>difficile gluD</i> constructs with deletions of their N-terminus (panels <b>C</b>, <b>D,</b> and <b>E</b>) or of C-terminus (panels <b>F</b>, <b>G</b> and <b>H</b>) were expressed in <i>C</i>. <i>difficile gluD</i> mutant, and their secretion from <i>C</i>. <i>difficile</i> was analyzed by zymogram (<b>D</b>&<b>G</b>) and ELISA (<b>E</b>&<b>H</b>).</p

GDH is required for <i>C</i>. <i>difficile</i> virulence.

<p>Kaplan-Meier survival curve of clindamycin-treated Syrian hamsters inoculated with 2,000 vegetative cells of <i>C</i>. <i>difficile</i> JIR8094 (Parent) or <i>C</i>. <i>difficile</i> JIR8094::<i>gluD</i> (mutant). Animals (n = 7 per group) were monitored every four hours for the symptoms of wet tail, poor fur coat, lethargy, or hunched posture. Moribund animals were euthanized. Log rank statistical analysis was performed; <i>p</i> <0.0001.</p

Bacterial strains/ plasmids used in this study.

<p>Bacterial strains/ plasmids used in this study.</p