scholarly journals Antimicrobial sensing coupled with cell membrane remodeling mediates antibiotic resistance and virulence inEnterococcus faecalis

2019 ◽  
Vol 116 (52) ◽  
pp. 26925-26932 ◽  
Author(s):  
Ayesha Khan ◽  
Milya Davlieva ◽  
Diana Panesso ◽  
Sandra Rincon ◽  
William R. Miller ◽  
...  
Keyword(s):  
Stress Response ◽  
Response Regulator ◽  
Cell Envelope ◽  
Multidrug Resistant ◽  
Innate Immune ◽  
Evolutionary Strategies ◽  
Phospholipid Content ◽  
Anionic Phospholipid ◽  
Extracellular Milieu ◽  
Terminal Domain

Bacteria have developed several evolutionary strategies to protect their cell membranes (CMs) from the attack of antibiotics and antimicrobial peptides (AMPs) produced by the innate immune system, including remodeling of phospholipid content and localization. Multidrug-resistantEnterococcus faecalis,an opportunistic human pathogen, evolves resistance to the lipopeptide daptomycin and AMPs by diverting the antibiotic away from critical septal targets using CM anionic phospholipid redistribution. The LiaFSR stress response system regulates this CM remodeling via the LiaR response regulator by a previously unknown mechanism. Here, we characterize a LiaR-regulated protein, LiaX, that senses daptomycin or AMPs and triggers protective CM remodeling. LiaX is surface exposed, and in daptomycin-resistant clinical strains, both LiaX and the N-terminal domain alone are released into the extracellular milieu. The N-terminal domain of LiaX binds daptomycin and AMPs (such as human LL-37) and functions as an extracellular sentinel that activates the cell envelope stress response. The C-terminal domain of LiaX plays a role in inhibiting the LiaFSR system, and when this domain is absent, it leads to activation of anionic phospholipid redistribution. Strains that exhibit LiaX-mediated CM remodeling and AMP resistance show enhanced virulence in theCaenorhabditis elegansmodel, an effect that is abolished in animals lacking an innate immune pathway crucial for producing AMPs. In conclusion, we report a mechanism of antibiotic and AMP resistance that couples bacterial stress sensing to major changes in CM architecture, ultimately also affecting host–pathogen interactions.

scholarly journals Alternative Sigma Factor RpoE Is Important for Vibrio parahaemolyticus Cell Envelope Stress Response and Intestinal Colonization

2014 ◽  
Vol 82 (9) ◽  
pp. 3667-3677 ◽  
Author(s):  
Brandy Haines-Menges ◽  
W. Brian Whitaker ◽  
E. Fidelma Boyd
Keyword(s):  
Stress Response ◽  
Vibrio Parahaemolyticus ◽  
Response Regulator ◽  
Cell Envelope ◽  
Polymyxin B ◽  
Content Type ◽  
Alternative Sigma Factors ◽  
Envelope Stress

ABSTRACTVibrio parahaemolyticusis a halophile that inhabits brackish waters and a wide range of hosts, including crustaceans, fish, mollusks, and humans. In humans, it is the leading cause of bacterial seafood-borne gastroenteritis. The focus of this work was to determine the role of alternative sigma factors in the stress response ofV. parahaemolyticusRIMD2210633, an O3:K6 pandemic isolate. Bioinformatics identified five putative extracytoplasmic function (ECF) family of alternative sigma factors: VP0055, VP2210, VP2358, VP2578, and VPA1690. ECF factors typically respond to cell wall/cell envelope stress, iron levels, and the oxidation state of the cell. We have demonstrated here that one such sigma factor, VP2578, a homologue of RpoE fromEscherichia coli, is important for survival under a number of cell envelope stress conditions and in gastrointestinal colonization of a streptomycin-treated adult mouse. In this study, we determined that anrpoEdeletion mutant strain BHM2578 compared to the wild type (WT) was significantly more sensitive to polymyxin B, ethanol, and high-temperature stresses. We demonstrated that inin vivocompetition assays between therpoEmutant and the WT marked with the β-galactosidase genelacZ(WBWlacZ), the mutant strain was defective in colonization compared to the WT. In contrast, deletion of therpoSstress response regulator did not affectin vivosurvival. In addition, we examined the role of the outer membrane protein, OmpU, which inV. choleraeis proposed to be the sole activator of RpoE. We found that anompUdeletion mutant was sensitive to bile salt stress but resistant to polymyxin B stress, indicating OmpU is not essential for the cell envelope stress responses or RpoE function. Overall, these data demonstrate that RpoE is a key cell envelope stress response regulator and, similar toE. coli, RpoE may have several factors that stimulate its function.

scholarly journals Characterization of the CpxRA Regulon in Haemophilus ducreyi

2010 ◽  
Vol 78 (11) ◽  
pp. 4779-4791 ◽  
Author(s):  
Maria Labandeira-Rey ◽  
Chad A. Brautigam ◽  
Eric J. Hansen
Keyword(s):  
Stress Response ◽  
Response Regulator ◽  
Cell Envelope ◽  
Bacterial Species ◽  
Regulatory System ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Envelope Stress ◽  
Two Component ◽  
Genes Encoding

ABSTRACT The H aemophilus ducreyi 35000HP genome encodes a homolog of the CpxRA two-component cell envelope stress response system originally characterized in E scherichia coli. CpxR, the cytoplasmic response regulator, was shown previously to be involved in repression of the expression of the lspB-lspA2 operon (M. Labandeira-Rey, J. R. Mock, and E. J. Hansen, Infect. Immun. 77:3402-3411, 2009). In the present study, the H. ducreyi CpxR and CpxA proteins were shown to closely resemble those of other well-studied bacterial species. A cpxA deletion mutant and a CpxR-overexpressing strain were used to explore the extent of the CpxRA regulon. DNA microarray and real-time reverse transcriptase (RT) PCR analyses indicated several potential regulatory targets for the H. ducreyi CpxRA two-component regulatory system. Electrophoretic mobility shift assays (EMSAs) were used to prove that H. ducreyi CpxR interacted with the promoter regions of genes encoding both known and putative virulence factors of H. ducreyi, including the lspB-lspA2 operon, the flp operon, and dsrA. Interestingly, the use of EMSAs also indicated that H. ducreyi CpxR did not bind to the promoter regions of several genes predicted to encode factors involved in the cell envelope stress response. Taken together, these data suggest that the CpxRA system in H. ducreyi, in contrast to that in E. coli, may be involved primarily in controlling expression of genes not involved in the cell envelope stress response.

A key regulatory mechanism of antimicrobial resistance in pathogenic Acinetobacter baumannii

2017 ◽  
Vol 38 (3) ◽  
pp. 122
Author(s):  
Felise G Adams
Keyword(s):  
Acinetobacter Baumannii ◽  
Histidine Kinase ◽  
Bacterial Infections ◽  
Regulatory Mechanism ◽  
Response Regulator ◽  
Bacterial Pathogen ◽  
Multidrug Resistant ◽  
Hospital Environment ◽  
Extracellular Milieu ◽  
Global Health Issue

Acinetobacter baumannii is a Gram-negative bacterial pathogen that has become a pressing global health issue in recent decades. Although virulence factors for this pathogen have been identified, details of how they are regulated are largely unknown. One widely employed regulatory mechanism that bacteria, such as A. baumannii, have adopted is through two component signal transduction systems (TCS). TCS consist of two proteins; a histidine kinase and response regulator. The histidine kinase allows the bacterium to sense alterations in the extracellular milieu, transmitting the information to the response regulator which prompts the cell to modify gene expression levels accordingly. Bacteria can encode multiple TCS, where each system can mediate specific responses to particular conditions or stressors. Identifying those conditions in which these TCS are expressed, and the genes they regulate known as their ‘regulon', is vital for understanding how A. baumannii survives and persists within the hospital environment or the human host during infection. As we enter the post-antibiotic era, knowledge of TCS could prove to be invaluable, as they offer an alternative target for the treatment of multidrug resistant bacterial infections.

scholarly journals The Positive Regulator, TraJ, of the Escherichia coli F Plasmid Is Unstable in a cpxA* Background

2002 ◽  
Vol 184 (20) ◽  
pp. 5781-5788 ◽  
Author(s):  
Michael J. Gubbins ◽  
Isabella Lau ◽  
William R. Will ◽  
Janet M. Manchak ◽  
Tracy L. Raivio ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Response Regulator ◽  
Cell Envelope ◽  
Conjugative Plasmid ◽  
F Plasmid ◽  
Gain Of Function ◽  
Positive Regulator ◽  
Envelope Stress ◽  
Detection Of Stress

ABSTRACT The Cpx (conjugative plasmid expression) stress response of Escherichia coli is induced in response to extracytoplasmic signals generated in the cell envelope, such as misfolded proteins in the periplasm. Detection of stress is mediated by the membrane-bound histidine kinase, CpxA. Signaling of the response regulator CpxR by activated CpxA results in the expression of several factors required for responding to cell envelope stress. CpxA was originally thought to be required for the expression of the positive regulator of the F plasmid transfer (tra) operon, TraJ. It was later determined that constitutive gain-of-function mutations in cpxA led to activation of the Cpx envelope stress response and decreased TraJ expression. In order to determine the nature of the downregulation of TraJ, the level of expression of TraJ, TraM, and TraY, the F-encoded regulatory proteins of the F tra region, was determined both in a cpxA* background and in a wild-type background in which the Cpx stress response was induced by overexpression of the outer membrane lipoprotein, NlpE. Our results suggest that TraJ downregulation is controlled by a posttranscriptional mechanism that operates in the cytoplasm in response to upregulation of the Cpx stress response by both the cpxA* gain-of-function mutation and the overexpression of NlpE.

scholarly journals The σE Cell Envelope Stress Response of Streptomyces coelicolor Is Influenced by a Novel Lipoprotein, CseA

2006 ◽  
Vol 188 (20) ◽  
pp. 7222-7229 ◽  
Author(s):  
Matthew I. Hutchings ◽  
Hee-Jeon Hong ◽  
Emmanuelle Leibovitz ◽  
Iain C. Sutcliffe ◽  
Mark J. Buttner
Keyword(s):  
Stress Response ◽  
Response Regulator ◽  
Cell Envelope ◽  
Streptomyces Coelicolor ◽  
Signal Transduction System ◽  
Two Component System ◽  
Positive Bacterium ◽  
Sensor Histidine Kinase ◽  
Envelope Stress

ABSTRACT We have investigated the role of CseA in the σE cell envelope stress response of the gram-positive bacterium Streptomyces coelicolor. σE is an extracytoplasmic function RNA polymerase sigma factor required for normal cell envelope integrity in S. coelicolor. σE is encoded within a four-gene operon that also encodes CseA, a protein of unknown function, CseB, a response regulator and CseC, a transmembrane sensor histidine kinase (Cse represents control of sigma E). Previous work has shown that transcription of the sigE gene is completely dependent on the CseBC two-component system and that the CseBC-σE signal transduction system is induced by a wide variety of cell-wall-damaging agents. Here we address the role of CseA, a protein with no homologues outside the streptomycetes. We show that CseA is a novel lipoprotein localized to the extracytoplasmic face of the cell membrane and that loss of CseA results in upregulation of the sigE promoter.

scholarly journals 599. LiaF is an Activator of the LiaR-Mediated Response Against Daptomycin and Antimicrobial Peptides in Multidrug-Resistant Enterococcus faecalis (Efs)

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S281-S281
Author(s):  
Laura C Ortiz-Velez ◽  
Sandra L Rincon ◽  
Jesse Degani ◽  
Yousif Shamoo ◽  
Truc T Tran ◽  
...  
Keyword(s):  
Stress Response ◽  
Stop Codon ◽  
Response Regulator ◽  
Transmembrane Protein ◽  
Premature Stop Codon ◽  
Regulatory System ◽  
Laboratory Strain ◽  
Positive Interaction ◽  
Anionic Phospholipid ◽  
Membrane Architecture

Abstract Background Daptomycin (DAP) is a key first-line agent for the treatment of vancomycin-resistant enterococcal infections. Resistance to DAP in enterococci is regulated by the liaFSR three-component regulatory system that consists of a histidine kinase sensor (LiaS), a response regulator (LiaR) and a transmembrane protein of unknown function (LiaF). Previous studies indicate that deletion of isoleucine in position 177 of LiaF results in DAP tolerance and is sufficient to change membrane architecture. Here, we dissect the role of LiaF in DAP resistance Methods We generated three liaF mutants in OG1RF, a DAP-susceptible laboratory strain of Efs (DAP MIC = 2 µg/mL): (i) a non-polar, C-terminal truncation of liaF (OG1RFliaF∆152), (ii) a null liaF mutant with a premature stop-codon (OG1RFliaF*11), and (iii) an isoleucine deletion at position 177 (OG1RFliaF177). We determined DAP MIC by Etest and characterized the localization of anionic phospholipids microdomains using 10-nonyl-acridine-orange (NAO). The expression of the liaXYZ (the main target of LiaR) and liaFSR clusters were evaluated by qRT-PCR and relative expression ratios (Log2 fold change) were calculated by normalizing to gyrB expression. We assessed activation of LiaFSR by evaluating surface exposure of LiaX by ELISA. We used the bacterial adenylate cyclase two-hybrid system (BACTH) to evaluate the protein-protein interaction between LiaF and LiaS. Results Full deletion of liaF or the C-terminal truncation of LiaF did not have any effect on DAP MICs, membrane architecture or a significant increase in LiaX surface exposure compared with parental strain OG1RF. In contrast, deletion of the codon encoding isoleucine in position 177 of LiaF caused a major increase (8-fold) in LiaX exposure and redistribution of anionic phospholipid microdomains away from the septum without changes in the actual DAP MIC. Transcriptional analyses indicated upregulation (>2 log2-fold) in the liaXYZ gene cluster indicating activation of the stress response. We also observed a positive interaction between LiaF and LiaS. Conclusion LiaF is likely a key activator of the LiaFSR stress response and the critical regulatory domain appears to be located in a stretch of four isoleucines toward the C-terminal of the protein. Disclosures All authors: No reported disclosures.

scholarly journals Environment Shapes the Accessible Daptomycin Resistance Mechanisms inEnterococcus faecium

2019 ◽  
Author(s):  
Amy G. Prater ◽  
Heer Mehtaa ◽  
Abigael J. Kosgei ◽  
William R. Miller ◽  
Truc T. Tran ◽  
...  
Keyword(s):  
Cell Envelope ◽  
Multidrug Resistant ◽  
Resistance Mechanisms ◽  
Clinical Strain ◽  
Anionic Phospholipid ◽  
Component Cell ◽  
Evolutionary Trajectories ◽  
Stress Response System ◽  
High Level ◽  
Cell Envelopes

AbstractDaptomycin binds to bacterial cell membranes and disrupts essential cell envelope processes leading to cell death. Bacteria respond to daptomycin by altering their cell envelopes to either decrease antibiotic binding to the membrane or by diverting binding away from vulnerable septal targets to remodeled anionic phospholipid membrane patches. InEnterococcus faecalis, daptomycin resistance is typically coordinated by the three-component cell-envelope-stress-response system, LiaFSR. Here, studying a clinical strain of multidrug-resistantEnterococcus faeciumcontaining alleles associated with activation of the LiaFSR signaling pathway, we found that specific environments selected for different evolutionary trajectories leading to high-level daptomycin resistance. Planktonic environments favored pathways that increased cell surface charge viayvcRSupregulation ofdltABCDandmprF, causing a reduction in daptomycin binding. Alternatively, environments favoring complex structured communities, including biofilms, evolved both diversion and repulsion strategies viadivIVAandoatAmutations, respectively. Both environments subsequently converged on cardiolipin synthase (cls) mutations, suggesting the importance of membrane modification across strategies. Our findings indicate thatE. faeciumcan evolve diverse evolutionary trajectories to daptomycin resistance that are shaped by the environment to produce a combination of resistance strategies. The accessibility of multiple and different biochemical pathways simultaneously suggests that the outcome of daptomycin exposure results in a polymorphic population of resistant phenotypes makingE. faeciuma recalcitrant pathogen.

scholarly journals Genetic Analysis of Factors Affecting Susceptibility of Bacillus subtilis to Daptomycin

2009 ◽  
Vol 53 (4) ◽  
pp. 1598-1609 ◽  
Author(s):  
Anna-Barbara Hachmann ◽  
Esther R. Angert ◽  
John D. Helmann
Keyword(s):  
Bacillus Subtilis ◽  
Cell Envelope ◽  
Transcriptional Profiling ◽  
Protein A ◽  
Multidrug Resistant ◽  
Dependent Manner ◽  
Membrane Composition ◽  
Factors Affecting ◽  
Anionic Phospholipids ◽  
Cyclic Lipopeptide

ABSTRACT 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 threefold 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 the 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.

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