The WalRK Two-Component System Is Essential for Proper Cell Envelope Biogenesis in Clostridioides difficile

Müh, Ute; Ellermeier, Craig D.; Weiss, David S.

doi:10.1128/jb.00121-22

Cited by 15 publications

(21 citation statements)

References 75 publications

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“…YrkQ is a relatively unstudied TCS and B. subtilis is not known to produce HNHDRG nor is it known what signals activate YrkPQ. To date we have been unable to identify a signal to which HexRK responds, neither daptomycin (Figure 2C) nor bacitracin (Supplemental Figure 4C) lead to activation of the P hexS - sLuc opt reporter, and our RNA-seq data suggests hexRK has a high basal expression (Müh et al, 2022). While the signals are not known both the HexRK and YrkPQ regulons have been identified (Kobayashi et al, 2001).…”

Section: Discussionmentioning

confidence: 86%

“…Thus, we performed RNA-seq, comparing WT with Δ hexRK to identify any genes that were differentially regulated in the absence of hexRK . Previous RNA-seq experiments showed hexRK was expressed at relatively high levels in log phase growth (Müh et al, 2022) thus we performed RNA-seq experiments on cultures grown to mid-log (OD 600 0.7-0.8)(Table S2).…”

Section: Resultsmentioning

confidence: 99%

“…Samples were stored at -80 ⁰C until ready for harvest. Extraction of RNA was performed using the RNeasy extraction kit (Qiagen) as previously described (Müh et al, 2022). RIN was determined using the Agilent BioAnalyzer 2000 at the University of Iowa.…”

Section: Rna Extractionmentioning

confidence: 99%

“…The response regulator Spo0A is a major regulator of sporulation, however its cognate histidine kinase has not been identified, and the manner in which Spo0A becomes activated is still unclear (Deakin et al, 2012; Mackin et al, 2013; Rosenbusch et al, 2012). The WalRK TCS is essential and required for proper cell wall morphology in C. difficile (Müh et al, 2022). The genes encoding the CmrST TCS, are phase variable and consists of two RRs and a single HK.…”

Section: Introductionmentioning

confidence: 99%

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HexSDF are Required for Synthesis of a Novel Glycolipid that Mediates Daptomycin and Bacitracin Resistance inC. difficile

Pannullo

Guan

Goldfine

et al. 2022

Preprint

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Clostridioides difficileis a Gram-positive opportunistic pathogen that results in 250,000 infections, 12,000 deaths, and $1 billion in medical costs in the US each year. There has been recent interest in using a daptomycin analog, Surotomycin, to treatC. difficileinfections. Daptomycin interacts with both phosphatidylglycerol and Lipid II to disrupt the membrane and halt peptidoglycan synthesis.C. difficilehas an unusual lipid membrane composition as it has no phosphatidylserine or phosphatidylethanolamine, and ∼50% of its membrane is composed of glycolipids, including the uniqueC. difficilelipid aminohexosyl-hexosyldiradylglycerol (HNHDRG). We identified a two-component system (TCS) HexRK that is required forC. difficileresistance to daptomycin. Using RNAseq we found that HexRK regulates a three gene operon of unknown functionhexSDF. Based on bioinformatic predictions,hexSencodes a monogalactosyldiacylglycerol synthase,hexDencodes a polysaccharide deacetylase, andhexFencodes an MprF-like flippase. We find that deletion ofhexRKleads to a 4-fold decrease in daptomycin MIC, and that deletion ofhexSDFleads to an 8-16-fold decrease in daptomycin MIC. The ΔhexSDFmutant is also 4-fold less resistant to bacitracin but no other cell wall active antibiotics. Our data indicate that in the absence of HexSDF the phospholipid membrane composition is altered. In WTC. difficilethe unique glycolipid, HNHDRG makes up ∼17% of the lipids in the membrane. However, in a ΔhexSDFmutant, HNHDRG is completely absent. While it is unclear how HNHDRG contributes daptomycin resistance, the requirement for bacitracin resistance suggests it has a general role in cell membrane biogenesis.ImportanceClostridioides difficileis 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. Little is understood aboutC. difficilemembrane lipids, but a unique glycolipid, HNHDRG, has been previously identified inC. difficileand, currently, has not been identified in other organisms. Here we show that HexSDF and HexRK are required for synthesis of HNHDRG, and that production of HNHDRG impacts resistance to daptomycin and bacitracin.

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Section: Discussionmentioning

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Section: Rna Extractionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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HexSDF are Required for Synthesis of a Novel Glycolipid that Mediates Daptomycin and Bacitracin Resistance inC. difficile

Pannullo

Guan

Goldfine

et al. 2022

Preprint

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“…The response regulator Spo0A is a major regulator of sporulation; however, its cognate histidine kinase has not been identified, and the manner in which Spo0A becomes activated is still unclear ( 51 – 53 ). The WalRK TCS is essential and required for proper cell wall morphology in C. difficile ( 54 ). The genes encoding the CmrRST TCS are phase variable and consist of two response regulators (RRs) and a single histidine kinase (HK).…”

Section: Introductionmentioning

confidence: 99%

HexSDF Is Required for Synthesis of a Novel Glycolipid That Mediates Daptomycin and Bacitracin Resistance in C. difficile

Pannullo

Guan

Goldfine

et al. 2023

mBio

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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. Little is understood about C. difficile membrane lipids, but a unique glycolipid, HNHDRG, has been previously identified in C. difficile and, currently, has not been identified in other organisms.

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Crystal structure of the catalytic ATP‐binding domain of the PhoR sensor histidine kinase

2023

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The two-component regulatory system (TCS) is a major regulatory system in bacteria that occurs in response to environmental changes and involves the sensor histidine kinase (HK) protein and response regulator (RR) protein. Among the TCSs, PhoR/ PhoB is crucial for bacteria to adapt to changes in environmental phosphate concentrations. In addition, recent studies have shown that PhoR binding to the MgtC virulence factor activates phosphate transport for normal pathogenesis. In this work, we determined the crystal structure of the catalytic ATP binding domain of the PhoR sensor histidine kinase from Vibrio cholera, compared the structure with the known HK protein structures and discussed the potential binding interface with MgtC.

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The WalRK Two-Component System Is Essential for Proper Cell Envelope Biogenesis in Clostridioides difficile

Abstract: The WalRK two-component system (TCS) is essential for coordinating synthesis and turnover of peptidoglycan in Firmicutes . We investigated the WalRK TCS in Clostridioides difficile , an important bacterial pathogen with an atypical cell envelope.

Cited by 15 publications

References 75 publications

HexSDF are Required for Synthesis of a Novel Glycolipid that Mediates Daptomycin and Bacitracin Resistance inC. difficile

HexSDF are Required for Synthesis of a Novel Glycolipid that Mediates Daptomycin and Bacitracin Resistance inC. difficile

HexSDF Is Required for Synthesis of a Novel Glycolipid That Mediates Daptomycin and Bacitracin Resistance in C. difficile

Crystal structure of the catalytic ATP‐binding domain of the PhoR sensor histidine kinase

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