Clostridioides difficile para -Cresol Production Is Induced by the Precursor para -Hydroxyphenylacetate

Clostridioides difficile is an etiological agent for antibiotic-associated diarrheal disease. C. difficile produces a phenolic compound, para -cresol, which selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. C. difficile decarboxylates para -hydroxyphenylacetate ( p -HPA) to produce p -cresol by the action of the HpdBCA decarboxylase encoded by the hpdBCA operon. Here, we investigate regulation of the hpdBCA operon and directly compare three independent reporter systems; SNAP-tag, glucuronidase gusA , and alkaline phosphatase phoZ reporters to detect basal and inducible expression. We show that expression of hpdBCA is upregulated in response to elevated p -HPA. In silico analysis identified three putative promoters upstream of hpdBCA operon-P 1 , P 2 , and Pσ 54 ; only the P 1 promoter was responsible for both basal and p -HPA-inducible expression of hpdBCA We demonstrated that turnover of tyrosine, a precursor for p -HPA, is insufficient to induce expression of the hpdBCA operon above basal levels because it is inefficiently converted to p -HPA in minimal media. We show that induction of the hpdBCA operon in response to p -HPA occurs in a dose-dependent manner. We also identified an inverted palindromic repeat (G-N 13 -C) upstream of the hpdBCA start codon (ATG) that is essential for inducing transcription of the hpdBCA operon in response to p -HPA, which drives the production of p -cresol. This provides insights into the regulatory control of p -cresol production, which affords a competitive advantage for C. difficile over other intestinal bacteria, promoting dysbiosis. IMPORTANCE Clostridioides difficile infection results from antibiotic-associated dysbiosis. para -Cresol, a phenolic compound produced by C. difficile , selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. Here, we demonstrate that expression of the hpdBCA operon, encoding the HpdBCA decarboxylase which converts p -HPA to p -cresol, is upregulated in response to elevated exogenous p -HPA, with induction occurring between >0.1 and ≤0.25 mg/ml. We determined a single promoter and an inverted palindromic repeat responsible for basal and p -HPA-inducible hpdBCA expression. We identified turnover of tyrosine, a p -HPA precursor, does not induce hpdBCA expression above basal level, indicating that exogenous p -HPA was required for p -cresol production. Identifying regulatory controls of p -cresol production will provide novel therapeutic targets to prevent p -cresol production, reducing C. difficile 's competitive advantage.