Identification of DraRS in Clostridioides difficile, a Two-Component Regulatory System That Responds to Lipid II-Interacting Antibiotics

Pannullo, Anthony G.; Zbylicki, Brianne R; Ellermeier, Craig D.

doi:10.1128/jb.00164-23

Cited by 3 publications

(12 citation statements)

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“…Increased lysozyme resistance was discovered in the draS 605* strain, in which a premature stop codon has excised the catalytic portion of DraS ( 35 ). Deletion of DraR, the cognate RR of DraS, restored wild-type lysozyme susceptibility in the draS 605* strain, suggesting that increased lysozyme resistance was due to an excess of active DraR rather than its absence.…”

Section: Commentarymentioning

confidence: 99%

“…Deletion of DraR, the cognate RR of DraS, restored wild-type lysozyme susceptibility in the draS 605* strain, suggesting that increased lysozyme resistance was due to an excess of active DraR rather than its absence. This indicated that DraS might function mainly as a phosphatase for DraR, such that deletion of DraS catalytic activity could lead to an excess of phosphorylated DraR ( 35 ). The authors confirmed this by mutating the phospho-accepting aspartic acid residue of DraR into a glutamic acid—which, as it is longer than an aspartic acid and still contains a negative charge at the end, is a structural mimic of phosphoaspartate and can serve to constitutively activate an RR.…”

Section: Commentarymentioning

confidence: 99%

“…The authors confirmed this by mutating the phospho-accepting aspartic acid residue of DraR into a glutamic acid—which, as it is longer than an aspartic acid and still contains a negative charge at the end, is a structural mimic of phosphoaspartate and can serve to constitutively activate an RR. Induction of the DraR D54E protein increased lysozyme resistance and upregulated draR transcription in a luciferase transcriptional reporter strain ( 35 ). Overproduction of wild-type DraR had no effect, confirming that phosphorylation is necessary for DraR activity.…”

Section: Commentarymentioning

confidence: 99%

“…Overproduction of wild-type DraR had no effect, confirming that phosphorylation is necessary for DraR activity. The authors used their draR luciferase reporter to monitor the transcriptional response of draRS to cell envelope stresses and found that lysozyme, as well as a suite of cell-wall active antibiotics, had no effect but that a subset of antibiotics (vancomycin, bacitracin, ramoplanin, and daptomycin), which each bind lipid-II, did upregulate draRS transcription ( 35 ). Notably, the lipid-II binding antibiotic nisin had no effect, so draRS does not respond broadly to all lipid-II binding compounds ( 35 ).…”

Section: Commentarymentioning

confidence: 99%

“…The authors used their draR luciferase reporter to monitor the transcriptional response of draRS to cell envelope stresses and found that lysozyme, as well as a suite of cell-wall active antibiotics, had no effect but that a subset of antibiotics (vancomycin, bacitracin, ramoplanin, and daptomycin), which each bind lipid-II, did upregulate draRS transcription ( 35 ). Notably, the lipid-II binding antibiotic nisin had no effect, so draRS does not respond broadly to all lipid-II binding compounds ( 35 ). The authors tested several C. difficile strains and found that most, but not all, increased draRS transcription in response to daptomycin exposure ( 35 ).…”

Section: Commentarymentioning

confidence: 99%

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Expanding our grasp of two-component signaling in Clostridioides difficile

Berumen Alvarez,

Purcell

2023

J Bacteriol

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The intestinal pathogen Clostridioides difficile encodes roughly 50 TCS, but very few have been characterized in terms of their activating signals or their regulatory roles. A. G. Pannullo, B. R. Zbylicki, and C. D. Ellermeier (J Bacteriol 205:e00164-23, 2023, https://doi.org/10.1128/jb.00164-23 ) have identified both for the novel C. difficile TCD DraRS. DraRS responds to antibiotics that target lipid-II molecules in the bacterial cell envelope, and regulates the production of a novel glycolipid necessary for bacitracin and daptomycin resistance in C. difficile .

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Section: Commentarymentioning

confidence: 99%

Section: Commentarymentioning

confidence: 99%

Section: Commentarymentioning

confidence: 99%

Section: Commentarymentioning

confidence: 99%

Section: Commentarymentioning

confidence: 99%

See 3 more Smart Citations

Expanding our grasp of two-component signaling in Clostridioides difficile

Berumen Alvarez,

Purcell

2023

J Bacteriol

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Histidine kinase‐mediated cross‐regulation of the vancomycin‐resistance operon in Clostridioides difficile

Belitsky

2024

Molecular Microbiology

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The dipeptide D‐Ala‐D‐Ala is an essential component of peptidoglycan and the target of vancomycin. Most Clostridioides difficile strains possess the vanG operon responsible for the synthesis of D‐Ala‐D‐Ser, which can replace D‐Ala‐D‐Ala in peptidoglycan. The C. difficile vanG operon is regulated by a two‐component system, VanRS, but is not induced sufficiently by vancomycin to confer resistance to this antibiotic. Surprisingly, in the absence of the VanS histidine kinase (HK), the vanG operon is still induced by vancomycin and also by another antibiotic, ramoplanin, in a VanR‐dependent manner. This suggested the cross‐regulation of VanR by another HK or kinases that are activated in the presence of certain lipid II‐targeting antibiotics. We identified these HKs as CD35990 and CD22880. However, mutations in either or both HKs did not affect the regulation of the vanG operon in wild‐type cells suggesting that intact VanS prevents the cross‐activation of VanR by non‐cognate HKs. Overproduction of VanR in the absence of VanS, CD35990, and CD22880 led to high expression of the vanG operon indicating that VanR can potentially utilize at least one more phosphate donor for its activation. Candidate targets of CD35990‐ and CD22880‐mediated regulation in the presence of vancomycin or ramoplanin were identified by RNA‐Seq.

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Identification of Clostridioides difficile mutants with increased daptomycin resistance

Zbylicki,

Murphy,

Petsche

et al. 2024

J Bacteriol

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Daptomycin is a cyclic lipopeptide antibiotic used to treat infections caused by some Gram-positive bacteria. Daptomycin disrupts synthesis of the peptidoglycan (PG) cell wall by inserting into the cytoplasmic membrane and binding multiple forms of the undecaprenyl carrier lipid required for PG synthesis. Membrane insertion requires phosphatidylglycerol, so studies of daptomycin can provide insight into assembly and maintenance of the cytoplasmic membrane. Here, we studied the effects of daptomycin on Clostridioides difficile , the leading cause of healthcare-associated diarrhea. We observed that growth of C. difficile strain R20291 in the presence of sub-MIC levels of daptomycin resulted in a chaining phenotype, minicell formation, and lysis—phenotypes broadly consistent with perturbation of membranes and PG synthesis. We also selected for and characterized eight mutants with elevated daptomycin resistance. The mutations in these mutants were mapped to four genes: cdsA ( cdr20291_2041 ), ftsH2 ( cdr20291_3396 ), esrR ( cdr20291_1187 ), and draS ( cdr20291_2456 ). Of these four genes, only draS has been characterized previously. Follow-up studies indicate these mutations confer daptomycin resistance by two general mechanisms: reducing the amount of phosphatidylglycerol in the cytoplasmic membrane ( cdsA ) or altering the regulation of membrane processes ( ftsH2 , esrR , and draS ). Thus, the mutants described here provide insights into phospholipid synthesis and identify signal transduction systems involved in cell envelope biogenesis and stress response in C. difficile . IMPORTANCE C. difficile is the leading cause of healthcare-associated diarrhea and is a threat to public health due to the risk of recurrent infections. Understanding biosynthesis of the atypical cell envelope of C. difficile may provide insight into novel drug targets to selectively inhibit C. difficile . Here, we identified mutations that increased daptomycin resistance and allowed us to better understand phospholipid synthesis, cell envelope biogenesis, and stress response in C. difficile .

show abstract

Identification of DraRS in Clostridioides difficile, a Two-Component Regulatory System That Responds to Lipid II-Interacting Antibiotics

Cited by 3 publications

References 86 publications

Expanding our grasp of two-component signaling in Clostridioides difficile

Expanding our grasp of two-component signaling in Clostridioides difficile

Histidine kinase‐mediated cross‐regulation of the vancomycin‐resistance operon in Clostridioides difficile

Identification of Clostridioides difficile mutants with increased daptomycin resistance

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