Dual RNA-seq identifies genes and pathways modulated during Clostridioides difficile colonization

The gastrointestinal pathogen Clostridioides difficile is the most common cause of hospital-acquired diarrhea. Bacterial interactions with the gut mucosa are crucial for the establishment of C. difficile infection; however, key infection events like bacterial attachment and gut penetration are still poorly defined. To better understand the initial events that occur when this anaerobe interacts with the human gut epithelium, we employed a dual RNA-sequencing approach to study the bacterial and host transcriptomic profiles during C. difficile infection in a dual environment in vitro human gut model. Temporal changes in gene expression during infection were studied in bacterial and epithelial cells over 3-24 hours. While there were several common differentially expressed bacterial genes across different timepoints after infection, mammalian transcriptional profiles were quite distinct, with little overlap. Interestingly, an induction of colonic receptors for C. difficile toxins was observed, along with the downregulation of genes encoding immune response markers. Several cell wall-associated proteins were downregulated in C. difficile when associated with host cells, including slpA, which encodes the main S-layer protein. Gene function and pathway enrichment analyses revealed a potential modulation of the purine/pyrimidine synthesis pathways both in the mammalian and bacterial cells. We observed that proline-proline endopeptidase, a secreted metalloprotease responsible for cell surface protein cleavage, is downregulated during infection, and a mutant lacking this enzyme demonstrated enhanced adhesion to epithelial cells during infection. This study provides new insight into the host and bacterial pathways based on gene expression modulation during the initial contact of C. difficile with gut cells. IMPORTANCE The initial interactions between the colonic epithelium and the bacterium are likely critical in the establishment of Clostridioides difficile infection, one of the major causes of hospital-acquired diarrhea worldwide. Molecular interactions between C. difficile and human gut cells have not been well defined mainly due to the technical challenges of studying cellular host-pathogen interactions with this anaerobe. Here we have examined transcriptional changes occurring in the pathogen and host cells during the initial 24 hours of infection. Our data indicate several changes in metabolic pathways and virulence-associated factors during the initial bacterium-host cell contact and early stages of infection. We describe canonical pathways enriched based on the expression profiles of a dual RNA sequencing in the host and bacterium, and functions of bacterial factors that are modulated during infection. This study thus provides fresh insight into the early C. difficile infection process.