A two-component system signaling hub controls enterococcal membrane remodeling in response to daptomycin
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ABSTRACT:
ORGANISM(S): Enterococcus faecalis
PROVIDER: GSE309958 | GEO | 2025/11/19
REPOSITORIES: GEO
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Project description:Abstract of associated manuscript: Daptomycin is the first of a new class of cyclic lipopeptide antibiotics used against multidrug-resistant Gram-positive pathogens. The proposed mechanism of action involves disruption of the functional integrity of the bacterial membrane in a Ca2+-dependent manner. We have used transcriptional profiling to demonstrate that treatment of Bacillus subtilis with daptomycin strongly induces the lia operon including the autoregulatory LiaRS two-component system (homologous to Staphylococcus aureus VraSR). The lia operon protects against daptomycin and deletion of liaH, encoding a phage shock protein A (PspA)-like protein, leads to 3-fold increased susceptibility. Since daptomycin interacts with the membrane, we tested mutants with altered membrane composition for effects on susceptibility. Deletion mutations of mprF (lacking lysyl-phosphatidylglycerol) or des (lipid desaturase) increased daptomycin susceptibility, whereas overexpression of MprF decreased susceptibility. Conversely, depletion of the cell for the anionic lipid phosphatidylglycerol led to increased resistance. Fluorescently-labeled daptomycin localized to the septa and in a helical pattern around the cell envelope and was delocalized upon depletion of phosphatidylglycerol. Together, these results indicate that the daptomycin-Ca2+ complex interacts preferentially with regions enriched in anionic phospholipids and leads to membrane stresses that can be ameliorated by PspA family proteins.
2008-12-11 | GSE13900 | GEO
Project description:Abstract of associated manuscript: Daptomycin is the first of a new class of cyclic lipopeptide antibiotics used against multidrug-resistant Gram-positive pathogens. The proposed mechanism of action involves disruption of the functional integrity of the bacterial membrane in a Ca2+-dependent manner. We have used transcriptional profiling to demonstrate that treatment of Bacillus subtilis with daptomycin strongly induces the lia operon including the autoregulatory LiaRS two-component system (homologous to Staphylococcus aureus VraSR). The lia operon protects against daptomycin and deletion of liaH, encoding a phage shock protein A (PspA)-like protein, leads to 3-fold increased susceptibility. Since daptomycin interacts with the membrane, we tested mutants with altered membrane composition for effects on susceptibility. Deletion mutations of mprF (lacking lysyl-phosphatidylglycerol) or des (lipid desaturase) increased daptomycin susceptibility, whereas overexpression of MprF decreased susceptibility. Conversely, depletion of the cell for the anionic lipid phosphatidylglycerol led to increased resistance. Fluorescently-labeled daptomycin localized to the septa and in a helical pattern around the cell envelope and was delocalized upon depletion of phosphatidylglycerol. Together, these results indicate that the daptomycin-Ca2+ complex interacts preferentially with regions enriched in anionic phospholipids and leads to membrane stresses that can be ameliorated by PspA family proteins. Bacillus subtilis W168, WT (+DAP) vs. WT (-DAP). The experiment was conducted in triplicate using three independent total RNA preparations. For WT-rep1 and WT-rep2, daptomycin treated samples were labeled with Alexa Fluor 647 and untreated samples with Alexa Fluor 555. For WT-rep3, the daptomycin treated sample was labeled with Alexa Fluor 555 and the untreated sample with Alexa Fluor 647.
2008-12-10 | E-GEOD-13900 | biostudies-arrayexpress
Project description:Daptomycin represents a reserve antibiotic effective against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Daptomycin resistance (Dap-R) and clinical treatment failure has been associated with adaptive chromosomal mutations, but so far not with acquisition of specific resistance genes. We previously isolated Staphylococcus/Mammaliicoccus sciuri strain TS92, which displays high-level Dap-R but lacks reported adaptive mutations. To identify the underlying resistance mechanism, we performed transcription profiling upon daptomycin exposure of TS92, with subsequent subcloning and mutational analysis of candidate genes. In TS92, Dap-R is mediated by a novel two-gene operon (named drcAB), controlled by an adjacent two-component regulatory system (drcRS). Heterologous drc expression demonstrated drcAB to be required and sufficient to mediate Dap-R in S. aureus (including clinically relevant MRSA) and Bacillus subtilis via inactivation of the antibiotic. The drc genes and proteins show homologies to membrane-associated antimicrobial peptide (AMP) transporters of Gram-positive bacteria but are distinct from currently known systems. In TS92, drc is flanked by insertion sequences (IS) and integrated near a staphylococcal cassette chromosome (SCC) element, suggesting mobility and acquisition of the locus from another species. Consistent with this hypothesis, IS-flanked drc was identified in various bacterial backgrounds in database searches. Here we report for the first time a horizontally acquired Dap-R mechanism that appears to circulate among Gram-positive bacteria, including staphylococci and enterococci. Increasing clinical daptomycin usage increases the likelihood that drc may enter S. aureus and other pathogens, which calls for vigilant monitoring of drc spread and prudent use of the antibiotic.
2025-04-29 | GSE255509 | GEO
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:Daptomycin is a lipopeptide antibiotic that has recently been approved for treatment of Gram-positive bacterial infections. The mode of action of daptomycin is not yet entirely clear. To further understand the mechanism transcriptomic analysis of changes in gene expression in daptomycin-treated Staphylococcus aureus was carried out. The expression profile indicated that cell wall stress stimulon member genes (B. J. Wilkinson, A. Muthaiyan, and R. K. Jayaswal. 2005. Curr. Med. Chem. Anti-Infective Agents 4: 259-276) were significantly induced by daptomycin, and by the cell wall-active antibiotics vancomycin and oxacillin. Comparison of the daptomycin response of a two-component cell wall stress stimulon regulator VraSR mutant, S. aureus KVR, to its parent N315 showed diminished expression of the cell wall stress stimulon in the mutant. Daptomycin has been proposed to cause membrane depolarization, and the transcriptional responses to carbonyl cyanide m-chlorophenylhydrazone (CCCP) and nisin were determined. Transcriptional profiles of the responses to these antimicrobial agents showed significantly different patterns compared to those of the cell wall-active antibiotics, including little or no induction of the cell wall stress stimulon. However, there were a significant number of genes induced by both CCCP and daptomycin that were not induced by oxacillin or vancomycin, such that the daptomycin transcriptome was probably reflecting a membrane depolarizing activity of this antimicrobial also. The results indicate that inhibition of peptidoglycan biosynthesis, either directly or indirectly, and membrane depolarization are parts of the mode of action of daptomycin. Keywords: mode of action, transcriptional profiling
2007-12-25 | GSE9494 | GEO
Project description:Sortase-assembled pili contribute to virulence in many Gram-positive bacteria. In Enterococcus faecalis, the endocarditis and biofilm-associated pilus (Ebp) is polymerized on the membrane by sortase C (SrtC) and attached to the cell wall by sortase A (SrtA). In the absence of SrtA, polymerized pili remain anchored to the membrane (i.e. off-pathway). Here we show that the high temperature requirement A (HtrA) bifunctional chaperone/protease of E. faecalis is a quality control system that clears aberrant off-pathway pili from the cell membrane. In the absence of HtrA and SrtA, accumulation of membrane-bound pili leads to cell envelope stress and partially induces the regulon of the ceftriaxone resistance-associated CroRS two-component system, which in turn causes hyper-piliation and cell morphology alterations. Inactivation of croR in the ∆srtA∆htrA background partially restores the observed defects of the ∆srtA∆htrA strain, supporting a role for CroRS in the response to membrane perturbations. Moreover, absence of SrtA and HtrA decreases basal tolerance of E. faecalis against cephalosporins and daptomycin. The link between HtrA, pilus biogenesis and the CroRS two-component system provides new insights into the E. faecalis response to endogenous membrane perturbations.
2024-05-31 | GSE261583 | GEO
Project description:Daptomycin (DAP) is the last-resort treatment for heterogeneous Vancomycin-Intermediate-Staphylococcus aureus (hVISA) and Vancomycin-Intermediate-S.aureus (VISA), and DAP-resistance onset which is also linked to reduced vancomycin susceptibility, is an increasing public health problem. To have more insight into the mechanisms of daptomycin resistance, the comparative transcriptomes of two DAP-R (1C-3B) clinical isogenic isolates vs their DAP-S (1A-3A) counterparts were investigated by Illumina RNA-seq, the Rockhopper tool, computational filtering analyses and bioinformatic tools.
2020-08-07 | GSE121797 | GEO
Project description:Acquisition of daptomycin resistance results in decreased virulence
| PRJEB72842 | ENA
Project description:Daptomycin is a membrane-targeting last-resort antimicrobial therapeutic for the treatment of infections caused by methicillin- and/or vancomycin-resistant Staphylococcus aureus. In the rare event of failed daptomycin therapy, the source of resistance is often attributable to mutations directly within the membrane phospholipid biosynthetic pathway of S. aureus or in the regulatory systems that control cell envelope response and membrane homeostasis. Here we describe the structural changes to the cell envelope in a daptomycin-resistant isolate of S. aureus strain N315 that has acquired mutations in the genes most commonly reported associated with daptomycin resistance: mprF, yycG, and pgsA. In addition to the decreased phosphatidylglycerol (PG) levels that are the hallmark of daptomycin resistance, the mutant with high-level daptomycin resistance had increased branched-chain fatty acids (BCFAs) in its membrane lipids, increased membrane fluidity, and increased cell wall thickness. However, the successful utilization of isotope-labeled straight-chain fatty acids (SCFAs) in lipid synthesis suggested that the aberrant BCFA:SCFA ratio arose from upstream alteration in fatty acid synthesis rather than a structural preference in PgsA. RT-qPCR studies revealed that expression of pyruvate dehydrogenase (pdhB) was suppressed in the daptomycin-resistant isolate, which is known to increase BCFA levels. While complementation with an additional copy of pdhB had no effect, complementation of the pgsA mutation resulted in increased PG formation, reduction in cell wall thickness, restoration of normal BCFA levels, and increased daptomycin susceptibility. Collectively, these results demonstrate that pgsA contributes to daptomycin resistance through its influence on membrane fluidity and cell wall thickness, in addition to phosphatidylglycerol levels.
2023-04-12 | MSV000091690 | MassIVE
Project description:The study aims to identify genes associated with daptomycin resistance.
2011-04-08 | GSE26249 | GEO