difficile, face a range of stresses in the environment and within the host. Our study thus provides new insight into the regulatory role of RsbW and the complexity of regulatory networks in C. Furthermore, the unexpected lower intracellular levels of σ B observed suggest post translational control mechanisms. Interestingly, sinRR’ locus that encodes a pleiotropic regulator, was highly upregulated in Δ rsbW indicating a potential indirect role for σ B or RsbW in control of sinRR’. A transcriptomic analysis to understand this unique phenotype showed a change in expression of some σ B -controlled genes along with several non-σ B controlled genes. Δ rsbW was defective in spore and biofilm formation, adhered better to human gut epithelia and was less virulent in a Galleria mellonella infection model. Δ rsbW did not have deleterious fitness defects but tolerated acidic environments and detoxified reactive oxygen and nitrogen species better. difficile physiology, a rsbW mutant (Δ rsbW ) where σ B is ‘always on’, was generated. To cope with environmental stresses, it uses the alternative sigma factor B (σ B ) to modulate gene transcription, which is regulated by an anti-sigma factor, RsbW. The anaerobic pathogen Clostridioides difficile, a primary cause of antibiotic-associated diarrhoea, faces a variety of stresses in the environment and in the mammalian gut. ![]() Our data demonstrate that HFHPD altered the community structure of lactase bacteria in the intestinal mucosa, decreased the abundance of the critical lactase bacteria, and promoted the occurrence of diarrhea. Compared to NM, the abundance of Bifidobacterium were lower in MD, while MD added sources for lactase bacteria of Rhizobium, Amycolatopsis, and Cedecea. At the genus level, Bifidobacterium was the dominant genus (over 90% of the total). Where Actinobacteria were higher in NM, and Proteobacteria were higher in MD. In taxonomic composition, lactase bacteria on the intestinal mucosa were sourced from Actinobacteria and Proteobacteria. Meanwhile, the Principal coordinate analysis (PCoA) and Adonis test showed that the community structures of lactase bacteria in NM and MD were significantly different (P < 0.05). The Chao1 and Observed specie indexes in the MD were higher than those in the NM, but this was not significant (P > 0.05). Operational Taxonomic Units (OTUs) were 23 and 31 in the normal group (NM) and model group (MD), respectively, and 11 of these were identical. Therefore, we reconnoiter the relationship between diarrhea induced by a high-fat and high-protein diet (HFHPD) and intestinal mucosal lactase bacteria from the perspective of functional genes. Lactase is a functional enzyme strongly associated with diarrhea, while lactase bacteria in the intestine are an important source of microbial lactase. Significant differentially expressed genes were those with a p <0.05 and fold change of <☒.Excessive fat and protein in food can cause diarrhea by disturbing the intestinal microecology. For DGE statistical significance was determined using the EDGE test in CLC Genomics Workbench. For RT-qPCRs, statistical significance was determined using Students’ t-test. (A-E) represent 3 biological replicates ± SD, n = 3. Downregulated genes are shown on top in yellow and upregulated genes are shown on the bottom in blue. (E) Ingenuity pathway analysis of gene expression changes in RAW 264.7 macrophages infected with R. (D) RT-qPCR validation of upregulated genes ( Lif, Nlrp3) and downregulated genes ( Mafb, S1pr1) in RAW 264.7 macrophages at 4 and 8h post infection with R. Blue genes are upregulated in infected cells, yellow genes are downregulated in infected cells. equi or Mtb compared to uninfected cells. (C) Heatmap showing gene expression analysis of RAW 264.7 macrophages infected for 4h with R. Upregulated genes are shown in the left Venn diagram and downregulated genes are shown in the right. (B) Venn diagram comparing differentially expressed genes in RAW 264.7 genes during R. Downregulated genes are plotted on the left and upregulated genes are on the right. ![]() x axis shows fold change of gene expression and y axis shows statistical significance. ![]() (A) Volcano plot showing gene expression analysis of RAW 264.7 macrophages infected with R. RNA-seq analysis reveals upregulation of pro-inflammatory cytokines and type I interferon response genes in R. The opportunistic intracellular bacterial pathogen Rhodococcus equi elicits type I interferon by engaging cytosolic DNA sensing in macrophages Fig 2
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