Disruption of dnaK results in transcriptional reprogramming of biofilm-associated pathways in Clostridioides difficile

Introduction. Clostridioides difficile is an opportunistic enteric pathogen posing significant challenges in human and animal healthcare due to the recurrent nature of infections and associated financial burden. The molecular chaperone protein, DnaK (Hsp70), encoded by dnaK , plays a central role in the heat stress response and protein homeostasis. Disruption of dnaK in C. difficile 630Δ erm has previously been associated with increased biofilm formation and multiple phenotypic alterations. Gap Statement. Although disruption of dnaK has been shown to alter biofilm formation and multiple phenotypes in C. difficile , the global transcriptional mechanisms linking DnaK to biofilm development and stress adaptation remain poorly understood. Aim. This study aimed to characterize the global transcriptional response associated with dnaK disruption during planktonic and biofilm growth to identify pathways linking the DnaK-mediated stress response with biofilm formation. Methods. This study investigated the global transcriptional changes associated with dnaK disruption during planktonic and colony biofilm culture. Colony biofilms of C. difficile strains 630, 630Δ erm and 630Δ erm:dnaK were cultured on mixed nitrocellulose ester semi-permeable membranes and examined structurally using low-vacuum scanning electron microscopy. Total RNA was extracted from both planktonic cultures and 24 h colony biofilms, followed by Illumina RNA sequencing. Differential gene expression analysis was performed using DESeq ( P adj <0.05). Gene set enrichment analysis (Kyoto Encyclopedia of Genes and Genomes [KEGG] and Gene Ontology) was conducted to identify significantly enriched metabolic pathways and biological processes. Results. Disruption of dnaK resulted in global transcriptomic remodelling, particularly during biofilm culture, where 72.4% of genes were significantly differentially expressed relative to planktonic culture. A core number of pathways were involved in biofilm formation in all strains, including increased sporulation with reduced translation, amino acid biosynthesis and flagellar assembly. The dnaK mutant exhibited a unique pattern of enrichment of pathways associated with cell division, peptidoglycan synthesis, carbon metabolism and amino acid biosynthesis, alongside a coordinated reduction of motility and chemotaxis pathways. Conclusion. Disruption of the dnaK gene appears to link the stress response to biofilm formation through a number of pathways associated with biofilm development. Understanding this link may identify systems relevant to persistent and recurrent infections.