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Daptomycin stimulon in Bacillus subtilis W168

ABSTRACT: 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.

ORGANISM(S): Bacillus subtilis

PROVIDER: GSE13900 | GEO | 2008/12/11

SECONDARY ACCESSION(S): PRJNA110309

REPOSITORIES: GEO

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Daptomycin stimulon in Bacillus subtilis W168

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

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

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

Increased membrane fluidity and cell wall thickening contribute to high-level daptomycin resistance in S. aureus with defective pgsA and yycG

Project description:Daptomycin is an important, last-resort antimicrobial therapeutic for the treatment of infections caused by Staphylococcus aureus that has acquired beta-lactam nonsusceptibility or reduced vancomycin susceptibility. The mechanism of action of daptomycin involves disruption of the cell membrane rather than cell wall synthesis, as with beta-lactams and vancomycin. In the rare event of failed daptomycin therapy, the source of resistance is often attributable to single-nucleotide polymorphisms 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 strain selected from methicillin-resistant S. aureus (MRSA) N315 with mutations in the most commonly reported SNPs 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 relative to its parental strain. Despite the enrichment of BCFAs, we found that the daptomycin-resistant strain successfully utilized isotope-labeled straight-chain fatty acids (SCFAs) in the synthesis of membrane lipids and that supplementation of the culture broth with SCFAs restored membrane fluidity in the daptomycin-resistant strain to the state of its parental strain. These results demonstrate that exogenous fatty acids can mitigate, in part, the phenotypes associated with daptomycin resistance when it is driven by mutations in yycG and pgsA.

2023-04-12 | MSV000091690 | MassIVE

Global profiling of B. subtilis comparing wild-type to mutants with altered cytoplasmic membrane composition

Project description:The Bacillus subtilis membrane contains diacylglycerol-based lipids with at least five distinct headgroups that impart distinct physical and chemical properties to the lipid bilayer. Here, we describe the phenotypic characterization of mutant strains lacking one or more of the following lipids: glycolipids (ugtP mutants), phosphatidylethanolamine (pssA and psd mutants), lysylphosphatidylglycerol (mprF), and cardiolipin (ywnE and ywjE). Alterations of membrane lipid headgroup composition are generally well tolerated by the cell and even severe alterations lead to only modest effects on growth proficiency. Mutants with decreased levels of positively charged lipids display an increased sensitivity to a cationic antimicrobial compounds and cells lacking glycolipids are sensitive to the lantibiotic sublancin and are defective in swarming motility. A quadruple mutant strain (ugtP pssA mprF ywnE), with a membrane comprised predominantly of phosphatidylglycerol, is viable and grows at near wild-type rates, although it forms long, coiled filaments. Transcriptome comparisons identify numerous regulons with altered expression in ugtP mutant cells, the pssA mprF ywnE triple mutant, and the uptP pssA mprF ywnE quadruple mutant. These effects include a general decrease in expression of the SigD and FapR regulons, and increased expression of cell envelope stress responses mediated by ?M and the YvrGHb two-component system.

2008-10-09 | GSE13036 | GEO

Global profiling of B. subtilis comparing wild-type to mutants with altered cytoplasmic membrane composition

2010-05-18 | E-GEOD-13036 | biostudies-arrayexpress

The antimicrobial peptide-sensing system aps of Staphylococcus aureus

Project description:Staphylococcus aureus is a leading cause of hospital-associated infections. In addition, highly virulent strains of methicillin-resistant S. aureus (MRSA) are currently spreading outside health care settings. Survival in the human host is largely defined by the ability of S. aureus to resist mechanisms of innate host defense, of which antimicrobial peptides form a key part especially on epithelia and in neutrophil phagosomes. Here we demonstrate that the antimicrobial-peptide sensing system aps of the standard community-associated MRSA strain MW2 controls resistance to cationic antimicrobial peptides. The core of aps-controlled resistance mechanisms comprised the D-alanylation of teichoic acids (dlt operon), the incorporation of cationic lysyl-phosphatidylglycerol (L-PG) in the bacterial membrane (mprF), and the vraF/vraG putative antimicrobial peptide transporter. Further, the observed increased production of L-PG under the influence of cationic antimicrobial peptides was accompanied by the up-regulation of lysine biosynthesis. In noticeable difference to the aps system of S. epidermidis, only selected antimicrobial peptides strongly induced the aps response. Heterologous complementation with the S. epidermidis apsS gene indicated that this is likely caused by differences in the short extracellular loop of ApsS that interacts with the inducing antimicrobial peptide. Our study shows that the antimicrobial peptide sensor system aps is functional in the important human pathogen S. aureus, significant interspecies differences exist in the induction of the aps gene regulatory response, and aps inducibility is clearly distinguishable from effectiveness towards a given antimicrobial peptide. Keywords: Wild type control vs treated vs mutant Wild type untreated in triplicate is compared to wild type treated in triplicate along with three mutants in triplicate with and without treatment of indolicidin, totalling 30 samples

2008-04-06 | E-GEOD-8400 | biostudies-arrayexpress

Bacillus subtilis

Project description:Reduction in membrane phosphatidylglycerol content leads to daptomycin resistance in Bacillus subtilis

| PRJNA140751 | ENA

Proteomic Investigation of Multiple Methicillin-Resistant Staphylococcus aureus strains with distinct Daptomycin Tolerance and Resistance Phenotypes

Project description:Several groups have shown that through evolution experiments, tolerance and resistance evolved rapidly under cyclic antibiotic treatment. In other words, intermittent antibiotic exposure performed in a typical adaptive laboratory evolution (ALE) experiments will “train” the bacteria to become tolerant/resistant to the drug. Using this experimental strategy, we performed in vitro laboratory evolution in MRSA using daptomycin, and mine novel daptomycin tolerance and resistance mutants, which were isolated at specific time points during the evolution experiments. Three daptomycin-tolerant isolates with different tolerance level were generated from our laboratory evolution (TOL2 and TOL5 with a mild-tolerance phenotype, and TOL6 with a high-tolerance phenotype). They all bear mutations at different genes, and have no increase in MIC towards daptomycin. Besides, we also isolated three daptomycin-resistant isolates (RES1, RES2, RES3) that have a single point mutation in the same gene, mprF, but at different locations, leading to an increased MIC towards daptomycin. Through proteomics, we uncovered the differential adaptation strategies of these daptomycin tolerant and resistant MRSA strains, and how they respond differently to antibiotics compared to the ancestral wild-type.

2022-07-14 | PXD026741 | Pride

The antimicrobial peptide-sensing system aps of Staphylococcus aureus

2008-04-07 | GSE8400 | GEO

A mutation in PP2C Phosphatase Gene in a Staphylococcus aureus USA300 Clinical Isolate with Reduced Susceptibility to Vancomycin and Daptomycin

Project description:Investigation of baseline transcription activity of two different clinical isolates of Staphylococcus aureus with two different susceptibility levels to the antibiotics Vancomycin and Daptomycin. Two different strains of Staphylococcus aureus, one that is fully Vancomycin and Daptomycin Sensitive and one with decreased Vancomycin and Daptomycin Sensitivity - grown to mid-log phase in rich broth.

2012-08-03 | E-GEOD-39868 | biostudies-arrayexpress

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