Clinical disease from Clostridioides difficile infection can be mediated by two toxins and their neighboring regulatory genes encoded within the five-gene pathogenicity locus (PaLoc). We provide several lines of evidence that the toxin activity of C. difficile may be modulated by genomic variants outside of the PaLoc. We used a phylogenetic tree-based approach to demonstrate discordance between toxin activity and PaLoc evolutionary history, an elastic net method to show the insufficiency of PaLoc variants alone to model toxin activity, and a convergence-based bacterial genome-wide association study (GWAS) to identify correlations between non-PaLoc loci with changes in toxin activity. Combined, these data support a model of C. difficile disease wherein toxin activity may be strongly affected by many non-PaLoc loci. Additionally, we characterize multiple other in vitro phenotypes relevant to human infections including germination and sporulation. These phenotypes vary greatly in their clonality, variability, convergence, and concordance with genomic variation. Lastly, we highlight the intersection of loci identified by GWAS for different phenotypes and clinical severity. This strategy to identify the overlapping loci can facilitate the identification of genetic variation linking phenotypic variation to clinical outcomes.

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This paper describes an adjuvant-free immunization regimen that results in the priming of T cells but not B cells. B10.A mice were primed s.c. with syngeneic spleen cells that had been pulsed with the peptide 81-104 derived from pigeon cytochrome c. The T cell response was measured by using a sensitive limiting dilution assay that measures lymphokine production. The precursor frequency of Ag-specific cells found in these mice was indistinguishable from the frequency found in mice primed in the footpads with 81-104 in CFA. A striking difference in antibody induction was found, however, when these two immunization regimens were compared. Mice primed with 81-104 in CFA developed significant serum antibody responses against the peptide, whereas mice primed with Ag-pulsed spleen cells produced no detectable anti-peptide antibodies. This lack of antibody did not result from detectable differences in the T cells that were primed: no differences were seen in IL-2 and IL-4 production or in the ability to provide help to B cells in vitro. In vitro stimulation with LPS suggested that the B cells were not primed by the Ag-pulsed spleen cells. The B cells were not tolerized, however, because boosting the mice with Ag in CFA resulted in the induction of an antibody response. The failure to induce an antibody response by priming with Ag-pulsed spleen cells was not caused by the site of immunization or the total amount of Ag used for priming. The critical variable may be the introduction of the Ag on the surface of an APC; in this form, B cell Ag recognition was apparently inefficient, whereas T cell Ag recognition was optimal.