Development of a high-throughput screening platform for <i>C. difficile</i> toxin synthesis inhibitors unveils meclizine as an antivirulence agent

<i>Clostridioides difficile,</i> a leading cause of hospital-acquired diarrhea, exerts its virulence through two co-regulated toxins, TcdA and TcdB. Despite their pivotal roles, the discovery of inhibitors targeting their biosynthesis is underexplored. Here, we present a high-throughput screening (HTS) platform designed to identify toxin synthesis inhibitors (TSIs) that minimally impact bacterial growth. The primary screen utilized a <i>C. difficile</i> reporter strain expressing secreted Nano-luciferase (secNluc) under the <i>tcdA</i> promoter, whereby inhibition of secNluc production indicates toxin biosynthesis inhibition. Screening the Prestwick Chemical Library at 10 and 100 µM identified several compounds that reduced secNluc activity. Through counter-screening, we eliminated compounds that caused spectral interference. Orthogonal dose-response assays assessing the effectiveness of inhibiting toxin production without affecting growth identified meclizine, an antihistamine, as the primary antivirulence candidate. Meclizine was confirmed as a TSI by showing that it reduced TcdA and TcdB protein levels, the cytopathic potential of cultures, and <i>tcdA</i> and <i>tcdB</i> transcription as determined by ELISA, cell-rounding assays, and RT-qPCR, respectively. Meclizine significantly altered central carbon metabolism in <i>C. difficile</i>, upregulating carbohydrate transport systems and the conversion of lactate to pyruvate, while downregulating glycolytic genes. These changes were associated with intracellular accumulation of glucose and pyruvate, metabolites known to negatively impact toxin production. Taken together, our findings underscore the utility of the above HTS platform to identify anti-<i>C</i>. <i>difficile</i> TSIs, which can serve as molecular and cellular probes, as well as chemical starting points for developing novel therapeutics for <i>C. difficile</i> infection.