Milk Exosomes Select Mutations That Decrease the Toxicity of Clostridioides difficile

Abstract

ObjectivesBackground: Exosomes are natural nanoparticles that play an essential role in cell-to-cell communication. Exosomes are present in all body fluids and their concentration is particularly high in milk. We previously found that bovine milk exosomes (BMEs) select polymorphisms and mutations in gut bacteria, potentially affecting the synthesis of microbial metabolites. Clostridioides difficile (C. difficile) is the primary cause of infectious diarrhea in the United States and it is unknown whether BMEs impact its pathogenicity through the selection of mutations. Objective: Assess the effect of chronic BME exposure on C. difficile growth and pathogenicity. MethodsC. difficile was cultured in BME-supplemented and -free media under continuous culture conditions for 21 days. Strains were isolated from both conditions. Doubling times and maximal density were assessed by monitoring optical density (OD600). Toxicity was assessed by Vero cell rounding and qRT-PCR of toxin genes (tcdA and tcdB) mRNA. Whole-genome sequencing was used to identify mutations in C. difficile strains. Effects of gene mutations on growth and toxicity were determined by overexpression of the mutants in C. difficile. Mann-Whitney test was used for statistical analysis; P < 0.05 was considered significant. ResultsBMEs selected for mutations that reduced doubling time by 15% (P < 0.05), decreased tcdA and tcdB production by 92% and 93%, respectively, (P < 0.05) and relative toxin activity reduced by 74% (p = 0.055). BME-selected strains contained a 25-bp deletion in the gene encoding the glucose-specific PTS transporter subunit IIBCA (ptsG). As a result, the translated ptsG (ptsG-indel) protein is truncated by 49 amino acids with 5 mismatches compared with wild-type. Overexpression of ptsG-indel in a wild-type strain of C. difficile increased maximum OD600 by 34% (P < 0.05), lowered relative toxin activity by 88% (P < 0.001), and reduced tcdB expression by 94% (P < 0.05) compared to C. difficile that overexpress wild-type ptsG. ConclusionsBMEs decreased the pathogenicity of C. difficile. Decreased pathogenicity was likely due to mutations in ptsG selected by growth in the presence of BMEs. Funding SourcesNIH P20GM104320, NIFA 2020–67,017-30,834, USDA Hatch and W-4002. J.Z is a consultant for PureTech Health, Inc.