Glycine fermentation by C. difficile promotes virulence and spore formation, and is induced by host cathelicidin

Abstract

Clostridioides difficile is a leading cause of antibiotic-associated diarrheal disease. C. difficile colonization, growth, and toxin production in the intestine is strongly associated with its ability to use amino acids to generate energy, but little is known about the impact of specific amino acids on C. difficile pathogenesis. The amino acid glycine is enriched in the dysbiotic gut and is suspected to contribute to C. difficile infection. We hypothesized that the use of glycine as an energy source contributes to colonization of the intestine and pathogenesis of C. difficile. To test this hypothesis, we deleted the glycine reductase (GR) genes grdAB, rendering C. difficile unable to ferment glycine, and investigated the impact on growth and pathogenesis. Our data show that the grd pathway promotes growth, toxin production, and sporulation. Glycine fermentation also had a significant impact on toxin production and pathogenesis of C. difficile in the hamster model of disease. Furthermore, we determined that the grd locus is regulated by host cathelicidin (LL-37) and the cathelicidin-responsive regulator, ClnR, indicating that the host peptide signals to control glycine catabolism. The induction of glycine fermentation by LL-37 demonstrates a direct link between the host immune response and the bacterial reactions of toxin production and spore formation.

Keywords: Clostridioides; Clostridium difficile; LL-37; Stickland metabolism; cathelicidin; cationic antimicrobial peptides; glycine.

Fig 1

The host peptide LL-37 and glycine catabolism enhance growth of C. difficile. (A) Active cultures of strain 630∆erm (WT) and the grdAB mutant (MC1576) were grown in CDMM, or CDMM supplemented with LL-37 (0.5 µg/mL), glycine (30 mM), or both LL-37 and glycine. Graphs are plotted as the means ± SD from four independent replicates. Data were analyzed by Student’s t-test comparing the mean values of grdAB to WT at each time point. (B) Maximum cell density of WT or grdAB mutant cultures in different conditions. The mean value and SD of four replicates for each condition and strain are shown. Data were analyzed by two-way analysis of variance followed by Tukey’s multiple comparisons test. *, P < 0.05; **P < 0.01; ***, P < 0.001; ****P < 0.0001.

Fig 2

Glycine utilization promotes sporulation and spore development. (A) Representative phase-contrast micrographs of 630∆erm and the grdAB mutant (MC1576) grown on sporulation agar for 24 h. White arrowheads indicate phase bright spores, and dark arrowheads indicate phase dark spores. (B) Ethanol-resistant spore formation for 630∆erm and the grdAB mutant. ****P < 0.0001 by a two-tailed Student’s t-test. (C) Quantitative reverse transcription PCR analysis of transcripts of the major sporulation regulators spo0A, sigF, sigE, sigG, and sigK. The means and individual values for four biological replicates are shown. *, P < 0.05 using the two-tailed Student’s t-test comparing each transcript in the grdAB mutant to the isogenic parent strain.

Fig 3

Glycine catabolism impedes spore germination. Purified spores of C. difficile 630∆erm (WT) and the grdAB mutant (MC1576) were assessed for germination in BHIS ±5 mM taurocholate. The optical densities of spore samples were measured and the ratio of the optical density at 600 nm (OD₆₀₀) at each timepoint was plotted against the density observed prior to the addition of germinant. The means for four biological replicates are shown. *, P < 0.05 using the two-tailed Student’s t-test comparing the grdAB mutant to the isogenic parent strain.

Fig 4

Glycine catabolism promotes virulence. (A) Kaplan-Meier survival curve depicting the time to morbidity of Syrian golden hamsters infected with 630∆erm (n = 12) or the grdAB mutant (MC1576; n = 13). The mean times to morbidity were: 630∆erm, 48.7 ± 10.7 h; grdAB, 70.9 ± 44.2 h. ***, P < 0.01, by log-rank test. (B) The total C. difficile CFU and (C) toxin recovered from cecal content post-mortem. Solid lines represent the median value for each strain. ns = not significant by Mann-Whitney test (B) or unpaired t-test (C).

Fig 5

LL-37 activates transcription of glycine catabolism from the grdX promoter. (A) Schematic of DNA fragments (F1–F10) of the grd region transcriptionally fused to phoZ. (B) AP activity detected from C. difficile strain 630∆erm carrying different grd region fragments fused to phoZ and grown in CDMM + 2 µg mL⁻¹ thiamphenicol with or without LL-37 and glycine, as indicated. Statistical significance was assessed by one-way analysis of variance within each growth condition compared to the promoterless ::phoZ control. (C) AP activity from the F1 promoter (PgrdX::phoZ) in WT (MC1649) or the clnR mutant (MC1650). Statistical significance was assessed by Student’s t-test, comparing activity in the WT and clnR mutant from each condition. The mean values with standard deviation of a minimum of three biological replicates are shown.

Fig 6

Transcriptional start site mapping and ClnR binding to PgrdX. (A) Schematic of the grdX promoter region. Transcriptional start sites identified by 5’ RACE are underlined in black, a predicted SigA promoter is marked with gray dashed lines, and an inverted repeat is shaded. (B) and (C) Electrophoretic mobility shift assays were performed using N-terminally His-tagged ClnR and fluorescein-labeled DNA. Binding of ClnR to the (B) PgrdX-1 or (C) PgrdX-2 DNA probe with increasing concentrations of ClnR protein. Competitive EMSAs performed with the addition of unlabeled specific or unlabeled nonspecific (Pspo0A) DNA at either 10x or 100x the concentration of labeled probe. n.d.: not determined due to instability.