Project description:Clostridioides difficile is a Gram-positive opportunistic pathogen that results in 220,000 infections, 12,000 deaths, and upwards of $1 billion in medical costs in the United States each year. C. difficile is highly resistant to a variety of antibiotics, but we have a poor understanding of how C. difficile senses and responds to antibiotic stress and how such sensory systems affect clinical outcomes. We have identified a spontaneous C. difficile mutant that displays increased daptomycin resistance. We performed whole-genome sequencing and found a nonsense mutation, S605*, in draS, which encodes a putative sensor histidine kinase of a two-component system (TCS). The draSS605* mutant has an ~4- to 8-fold increase in the daptomycin MIC compared to the wild type (WT). We found that the expression of constitutively active DraRD54E in the WT increases daptomycin resistance 8- to 16-fold and increases bacitracin resistance ~4-fold. We found that a selection of lipid II-inhibiting compounds leads to the increased activity of the luciferase-based reporter PdraR-slucopt, including vancomycin, bacitracin, ramoplanin, and daptomycin. Using RNA sequencing (RNA-seq), we identified the DraRS regulon. Interestingly, we found that DraRS can induce the expression of the previously identified hex locus required for the synthesis of a novel glycolipid produced in C. difficile. Our data suggest that the induction of the hex locus by DraR explains some, but not all, of the DraR-induced daptomycin and bacitracin resistance. IMPORTANCE Clostridioides difficile is a major cause of hospital-acquired diarrhea and represents an urgent concern due to the prevalence of antibiotic resistance and the rate of recurrent infections. C. difficile encodes ~50 annotated two-component systems (TCSs); however, only a few have been studied. The function of these unstudied TCSs is not known. Here, we show that the TCS DraRS plays a role in responding to a subset of lipid II-inhibiting antibiotics and mediates resistance to daptomycin and bacitracin in part by inducing the expression of the recently identified hex locus, which encodes enzymes required for the production of a novel glycolipid in C. difficile.

Project description:Two-component systems (TCS) are often used by bacteria to rapidly assess and respond to environmental changes. ChvG/ChvI (ChvGI) is a TCS conserved in γ-proteobacteria and is known for regulating expression of genes related to exopolysaccharide production, virulence and growth. The sensor kinase ChvG autophosphorylates upon yet unknown signals and phosphorylates the response regulator ChvI to activate transcription. Recent studies in Caulobacter crescentus showed that chv mutants are sensitive to vancomycin treatment and fail to grow in synthetic minimal media. In this work, we identified the osmotic imbalance as the main cause of growth impairment in synthetic minimal media. We also determined the ChvI regulon and confirmed that ChvI regulates cell envelope architecture at different levels by controlling outer membrane, peptidoglycan assembly/recycling and inner membrane proteins. Furthermore, we identified genes with osmoregulatory properties and confirmed that osmotic upshift is a signal triggering ChvG-dependent phosphorylation of ChvI. In addition, we challenged chv mutants with other cell envelope related stress and found that targeting with antibiotics the transpeptidation of peptidoglycan during cell elongation impairs growth of the mutant. Moreover, these antibiotics activate expression of the chvIG-hprK operon in ChvI-dependent and independent ways. ChvI phosphorylation is also shown to be activated upon antibiotic treatment with vancomycin. Finally, we observed that the sensor kinase ChvG fused to a fluorescent protein relocates from a patchy-spotty distribution to distinctive foci after transition from complex to synthetic minimal media. Interestingly, this pattern of (re)location has been described for proteins involved in cell growth control and peptidoglycan synthesis upon osmotic shock. Overall, our data support that the ChvGI TCS is mainly used to monitor and respond to osmotic imbalances and damages in the peptidoglycan layer.

			Action	DRS
	SRR24937804_1.fastq.gz	Fastqsanger.gz
	SRR24937804_2.fastq.gz	Fastqsanger.gz
	SRR24937805_1.fastq.gz	Fastqsanger.gz
	SRR24937805_2.fastq.gz	Fastqsanger.gz
	SRR24937806_1.fastq.gz	Fastqsanger.gz

Dataset Information

Identification of DraRS in C. difficile, a two-component regulatory system that responds to lipid II interacting antibiotics

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