Identification of a new family of peptidoglycan transpeptidases reveals unusualcrosslinking is essential for viability inC. difficile
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ABSTRACT:
PROVIDER: PRJNA1125117 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR29444116_1.fastq.gz | Fastqsanger.gz | |||
| SRR29444116_2.fastq.gz | Fastqsanger.gz | |||
| SRR29444117_1.fastq.gz | Fastqsanger.gz | |||
| SRR29444117_2.fastq.gz | Fastqsanger.gz | |||
| SRR29444118_1.fastq.gz | Fastqsanger.gz |
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Project description:Most bacteria are surrounded by a peptidoglycan cell wall composed of glycan strands held together by short peptide crosslinks. There are two major types of crosslinks, termed 4-3 and 3-3 based on the amino acids involved. 4-3 crosslinks are created by penicillin-binding proteins (PBPs), while 3-3 crosslinks created byL,D-transpeptidases (LDTs). In well studied bacteria 3-3 crosslinks comprise only about 10% of the total and are not essential. However, in the opportunistic intestinal pathogenClostridioides difficile,about 70% of the crosslinks are 3-3. We show here that 3-3 crosslinks and LDTs are essential for viability inC. difficile. We also show thatC. difficilehas five LDTs, three with a YkuD catalytic domain as in all previously known LDTs and two with a VanW catalytic domain, whose function was until now unknown. The five LDTs exhibit extensive functional redundancy. VanW domain proteins are found in many Gram-positive bacteria but scarce in other lineages. We tested seven non-C. difficileVanW domain proteins and confirmed LDT activity in three cases. In summary, our findings uncover a new family of peptidoglycan crosslinking enzymes, assign a catalytic function to VanW domains, and demonstrate that 3-3 crosslinking is essential for viability inC. difficile, the first time this has been shown in any bacterial species.
2024-07-30 | GSE270128 | GEO
Project description:We compared transcriptomes of wild-type and ∆vanS strains of Clostridioides difficile 630 growing in the presence or absence of peptidoglycan-targeting antibiotics, vancomycin or ramoplanin. VanS is a histidine kinase of a two-component system that regulates expression of the vancomycin-induced vanG operon.
2024-04-30 | GSE262388 | GEO
Project description:Clostridioides difficile, the leading cause of antibiotic-associated diarrhea, relies primarily on 3-3 crosslinks created by L,D-transpeptidases (LDTs) to fortify its peptidoglycan (PG) cell wall. This is unusual, as in most bacteria the vast majority of PG crosslinks are 4-3 crosslinks, which are created by penicillin-binding proteins (PBPs). Here we report the unprecedented observation that 3-3 crosslinking is essential for viability in C. difficile. We also report the discovery of a new family of LDTs that use a VanW domain to catalyze 3-3 crosslinking rather than a YkuD domain as in all previously known LDTs. Bioinformatic analyses indicate VanW domain LDTs are less common than YkuD domain LDTs and are largely restricted to Gram-positive bacteria. Our findings suggest that LDTs might be exploited as targets for antibiotics that kill C. difficile without disrupting the intestinal microbiota that is important for keeping C. difficile in check.
| S-EPMC10980060 | biostudies-literature
Project description:Cell growth and division require a balance between synthesis and hydrolysis of the peptidoglycan (PG). Inhibition of PG synthesis or uncontrolled PG hydrolysis can be lethal for the cells, making it imperative to control peptidoglycan hydrolase (PGH) activity. The activity of several enzymes involved in PG synthesis is regulated by serine/threonine kinase (STKs). In firmicutes, inactivation of genes encoding STKs is associated with a range of phenotypes including cell division defects and changes in cell wall metabolism, but only a few kinase substrates have been identified. We previously demonstrated that the STK PrkC plays an important role in cell division, cell wall metabolism and drug resistance in Clostridioides difficile. In this work, we identified and characterized a PGH, CD1135 that is a PrkC substrate. We showed that CD1135 hydrolyses PG between daughter cells to allow cell separation. We demonstrated that CD1135 phosphorylation inhibits CD1135 its export, therefore controlling the hydrolytic activity in the cell wall. High level of CD1135 in the cell wall leads to cell elongation, whereas low level causes cell separation defects. We provided evidences that the STK signaling pathway regulates PGHs homeostasis to control PG hydrolysis during growth and cell division.
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Project description:Clostridioides difficile is one of the most common nosocomial pathogens and a global public health threat. Upon colonization of the gastrointestinal tract, C. difficile is exposed to a rapidly changing polymicrobial environment and a dynamic metabolic milieu. Despite the link between the gut microbiota and susceptibility to C. difficile, the impact of synergistic interactions between the microbiota and pathogens on the outcome of infection is largely unknown. Here, we show that microbial cooperation between C. difficile and Enterococcus has a profound impact on the growth, metabolism, and pathogenesis of C. difficile.. Through a process of nutrient restriction and metabolite cross-feeding, E. faecalis shapes the metabolic environment in the gut to enhance C. difficile fitness and increase toxin production. These findings demonstrate that members of the microbiota, such as Enterococcus, have a previously unappreciated impact on C. difficile behavior and virulence.
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Project description:The intestines house a diverse microbiota that must compete for nutrients to survive, but the specific limiting nutrients that control pathogen colonization are not clearly defined. Clostridioides difficile colonization typically requires prior disruption of the microbiota, suggesting that outcompeting commensals for resources is key in establishing C. difficile infection (CDI). The immune protein calprotectin (CP) is released into the gut lumen during CDI to chelate zinc (Zn) and other essential nutrient metals. Yet, the impact of Zn limitation on C. difficile colonization is unknown. To define C. difficile responses to Zn limitation, we performed RNA sequencing on C. difficile exposed to CP. In media with CP, C. difficile upregulated genes involved in metal homeostasis and amino acid metabolism.
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Project description:INC-Seq: Accurate single molecule reads using nanopore sequencing
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Project description:Clostridioides difficile is 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 treat C. difficile infections. Daptomycin interacts with both phosphatidylglycerol and Lipid II to disrupt the membrane and halt peptidoglycan synthesis. C. difficile has an unusual lipid membrane composition as it has no phosphatidylserine or phosphatidylethanolamine, and ~50% of its membrane is composed of glycolipids, including the unique C. difficile lipid aminohexosyl-hexosyldiradylglycerol (HNHDRG). We identified a two-component system (TCS) HexRK that is required for C. difficile resistance to daptomycin. Using RNAseq we found that HexRK regulates a three gene operon of unknown function hexSDF. Based on bioinformatic predictions, hexS encodes a monogalactosyldiacylglycerol synthase, hexD encodes a polysaccharide deacetylase, and hexF encodes an MprF-like flippase. We find that deletion of hexRK leads to a 4-fold decrease in daptomycin MIC, and that deletion of hexSDF leads to an 8-16-fold decrease in daptomycin MIC. The ∆hexSDF mutant 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 WT C. difficile the unique glycolipid, HNHDRG makes up ~17% of the lipids in the membrane. However, in a ∆hexSDF mutant, 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.
2022-12-10 | GSE220119 | GEO
Project description:The strict anaerobe Clostridium difficile is an important nosocomial enteropathogen that has become the most common cause of antibiotic-associated diarrhea. The oxygen-resistant C. difficile spores play a central role in the infectious cycle, contributing to transmission, infection and recurrence. The spore surface layers, coat and exosporium, enable resistance of the spores to extreme physical and chemical stresses. Despite the critical importance of the spore in C. difficile infection, little is known about the mechanisms that orchestrate the assembly of its external layers. In this study, we identified and characterized a new C. difficile spore protein, named CotL, which is required for the assembly of the spore surface layers. The cotL gene was expressed in the mother cell compartment under the dual control of σE and σK. The CotL protein was localized in the C. difficile spore coat and cotL mutant spores had a major morphologic defect at the level of the coat/exosporium layers, as observed by transmission electron microscopy. Accordingly, spores of the cotL mutant contained a reduced amount of several proteins of the coat and exosporium, including CotB, CotE and CdeC. Additionally, cotL inactivation resulted in a defect in localization of late spore coat proteins in sporulating cells. Finally, spores of the cotL mutant were more sensitive to lysozyme and were impaired in germination. These defects are most likely associated with the structurally altered coat. Collectively, these results strongly suggest that CotL is a morphogenetic protein essential for the assembly of the spore coat in C. difficile.
2019-10-16 | PXD010359 | Pride
Project description:Gene expression level of Clostridioides difficile (C. difficile) strain R20291 comparing control C. difficile carring pMTL84151 as vector plasmid with C. difficile conjugated with a pMTL84151-03890 gene. Goal was to determine the effects of 03890 gene conjugation on C. difficile strain R20291 gene expression.
2023-11-30 | GSE219066 | GEO