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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-resistant Enterococcus 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 Nterminal 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 the Caenorhabditis elegans model, 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. [ABSTRACT FROM AUTHOR]

Daptomycin resistance in enterococci is often mediated by the LiaFSR system, which orchestrates the cell membrane stress response. Activation of LiaFSR through the response regulator LiaR generates major changes in cell membrane function and architecture (membrane adaptive response), permitting the organism to survive the antibiotic attack. Here, using a laboratory strain of Enterococcus faecalis, we developed a novel Caenorhabditis elegans model of daptomycin therapy and showed that disrupting LiaR-mediated cell membrane adaptation restores the in vivo activity of daptomycin. The LiaR effect was also seen in a clinical strain of daptomycin-resistant Enterococcus faecium, using a murine model of peritonitis. Furthermore, alteration of the cell membrane response increased the ability of human polymorphonuclear neutrophils to readily clear both E. faecalis and multidrug-resistant E. faecium. Our results provide proof of concept that targeting the cell membrane adaptive response restores the in vivo activity of antibiotics, prevents resistance, and enhances the ability of the innate immune system to kill infecting bacteria. [ABSTRACT FROM AUTHOR]

Inactivating mutations in the ontrol f irulence two-component regulatory system (covRS) often account for the hypervirulent phenotype in severe, invasive group A streptococcal (GAS) infections. As CovR represses production of the anti-phagocytic hyaluronic acid capsule, high level capsule production is generally considered critical to the hypervirulent phenotype induced by CovRS inactivation. There have recently been large outbreaks of GAS strains lacking capsule, but there are currently no data on the virulence of covRS-mutated, acapsular strains in vivo. We investigated the impact of CovRS inactivation in acapsular serotype M4 strains using a wild-type (M4-SC-1) and a naturally-occurring CovS-inactivated strain (M4-LC-1) that contains an 11bp covS insertion. M4-LC-1 was significantly more virulent in a mouse bacteremia model but caused smaller lesions in a subcutaneous mouse model. Over 10% of the genome showed significantly different transcript levels in M4-LC-1 vs. M4-SC-1 strain. Notably, the Mga regulon and multiple cell surface protein-encoding genes were strongly upregulated–a finding not observed for CovS-inactivated, encapsulated M1 or M3 GAS strains. Consistent with the transcriptomic data, transmission electron microscopy revealed markedly altered cell surface morphology of M4-LC-1 compared to M4-SC-1. Insertional inactivation of covS in M4-SC-1 recapitulated the transcriptome and cell surface morphology. Analysis of the cell surface following CovS-inactivation revealed that the upregulated proteins were part of the Mga regulon. Inactivation of mga in M4-LC-1 reduced transcript levels of multiple cell surface proteins and reversed the cell surface alterations consistent with the effect of CovS inactivation on cell surface composition being mediated by Mga. CovRS-inactivating mutations were detected in 20% of current invasive serotype M4 strains in the United States. Thus, we discovered that hypervirulent M4 GAS strains with covRS mutations can arise in an acapsular background and that such hypervirulence is associated with profound alteration of the cell surface. [ABSTRACT FROM AUTHOR]

Emergence of antibiotic resistance is an example of the incredible plasticity of bacteria to survive in all environments. The search for new antibiotics active against traditional targets is more challenging due not only to the lack of novel natural products to fulfill the current clinical needs against multidrug-resistant (MDR) bacteria, but also for the possible ‘collateral’ effects on the human microbiota. Thus, non-traditional approaches to combat MDR bacteria have been proposed. Here, we discuss the possibility of targeting the membrane response to the antibiotic attack (cell membrane adaptation) as a viable strategy to increase the activity of current antimicrobials, enhance the activity of the innate immune system and prevent development of resistance during therapy using the three-component regulatory system LiaFSR of enterococci as a model. [ABSTRACT FROM AUTHOR]

Daptomycin (DAP) is a cyclic lipopeptide with in vitro activity against a variety of Gram-positive pathogens, including multidrug-resistant organisms. Since its introduction into clinical practice in 2003, DAP has become an important key frontline antibiotic for severe or deep-seated infections caused by Gram-positive organisms. Unfortunately, DAP resistance (DAP-R) has been extensively documented in clinically important organisms such as Staphylococcus aureus, Enterococcus spp., and Streptococcus spp. Studies on the mechanisms of DAP-R in Bacillus subtilis and other Gram-positive bacteria indicate that the genetic pathways of DAP-R are diverse and complex. However, a common phenomenon emerging from these mechanistic studies is that DAP-R is associated with important adaptive changes in cell wall and cell membrane homeostasis with critical changes in cell physiology. Findings related to these adaptive changes have provided novel insights into the genetics and molecular mechanisms of bacterial cell envelope stress response and the manner in which Gram-positive bacteria cope with the antimicrobial peptide attack and protect vital structures of the cell envelope, such as the cell membrane. In this review, we will examine the most recent findings related to the molecular mechanisms of resistance to DAP in relevant Gram-positive pathogens and discuss the clinical implications for therapy against these important bacteria. [ABSTRACT FROM AUTHOR]

Background: The lipopeptide antibiotic, daptomycin (DAP) interacts with the bacterial cell membrane (CM). Development of DAP resistance during therapy in a clinical strain of Enterococcus faecalis was associated with mutations in genes encoding enzymes involved in cell envelope homeostasis and phospholipid metabolism. Here we characterized changes in CM phospholipid profiles associated with development of DAP resistance in clinical enterococcal strains. Methodology: Using two clinical strain-pairs of DAP-susceptible and DAP- resistant E. faecalis (S613 vs. R712) and E. faecium (S447 vs. R446) recovered before and after DAP therapy, we compared four distinct CM profiles: phospholipid content, fatty acid composition, membrane fluidity and capacity to be permeabilized and/or depolarized by DAP. Additionally, we characterized the cell envelope of the E. faecium strain-pair by transmission electron microscopy and determined the relative cell surface charge of both strain-pairs. Principal Findings: Both E. faecalis and E. faecium mainly contained four major CM PLs: phosphatidylglycerol (PG), cardiolipin, lysyl-phosphatidylglycerol (L-PG) and glycerolphospho-diglycodiacylglycerol (GP-DGDAG). In addition, E. faecalis CMs (but not E. faecium) also contained: i) phosphatidic acid; and ii) two other unknown species of amino-containing PLs. Development of DAP resistance in both enterococcal species was associated with a significant decrease in CM fluidity and PG content, with a concomitant increase in GP-DGDAG. The strain-pairs did not differ in their outer CM translocation (flipping) of amino-containing PLs. Fatty acid content did not change in the E. faecalis strain-pair, whereas a significant decrease in unsaturated fatty acids was observed in the DAP- resistant E. faecium isolate R446 (vs S447). Resistance to DAP in E. faecium was associated with distinct structural alterations of the cell envelope and cell wall thickening, as well as a decreased ability of DAP to depolarize and permeabilize the CM. Conclusion: Distinct alterations in PL content and fatty acid composition are associated with development of enterococcal DAP resistance. [ABSTRACT FROM AUTHOR]

Background DAP disrupts bacterial CM by binding to septal anionic phospholipids (APLs). LiaX, an effector of the LiaFSR stress system, modulates DAP-R by diverting APLs away from the septum. Enterococci are intrinsically resistant to β-lactams due to the presence of PBPs (e.g. PBP5) with low affinity to these drugs. However, emergence of DAP-R leads to increased susceptibility to β-lactams, a phenomenon designated as the see-saw effect. Here, we dissect the molecular mechanism of this phenomenon. Methods We studied a clinical strain pair of DAP-S (S613) and DAP-R (R712) E. faecalis strains recovered from a patient before and after DAP therapy. We generated deletions of liaX and PBPs (ponA and pbp5) in DAP-susceptible (DAP-S) E. faecalis OG1RF and JH2-2. APLs and membrane structures were visualized with NAO and/or FM4-64. PBPs and LiaX localization were evaluated with bocillin-FL or immunofluorescence. PBP transcripts and PBP5 protein levels were measured by qRT-PCR or immunoblotting, respectively. β-Lactam binding affinity of PBPs was assessed by SDS-PAGE of bocillin-FL stained membranes and a LiaX–PBP5 interaction was evaluated by the bacterial two-hybrid (BACTH) system. MICs were determined via E -test. Results Deletion of liaX led to DAP-R and redistribution of APL microdomains (nonseptal foci with CM aberrations; Figure 1A) in all strains, with a marked decrease in ceftriaxone (CRO) MICs. Only PBP5 was essential for β-lactam resistance but not for DAP-R. DAP-R was associated with mislocalization of PBPs to the sites of CM aberrations (Figure 2). Notably, LiaX and PBP5 were localized to the septum in DAP-S strains but redistributed away from septal areas upon development of DAP-R (Figure 3). An interaction of LiaX and PBP5 was confirmed by the BACTH system. Mislocalized PBPs, most notably PonA and PBP5, had increased affinity for β-lactams in all DAP-R strains. The increased affinity of PBPs to β-lactams was not associated with increased transcripts or PBP5 levels. Conclusion LiaX regulates CM adaptation and cell wall synthesis via membrane remodeling and direct interactions with key PBPs. Changes in LiaX that cause DAP-R results in mislocalization of PBPs to nonseptal areas and likely increases access of β-lactam to the active site, explaining the see-saw effect. Disclosures All Authors: No reported Disclosures. [ABSTRACT FROM AUTHOR]

Background Daptomycin (DAP) is a lipopeptide antibiotic that targets the cell membrane (CM) at the division septum. DAP resistance (DAP-R) in E. faecalis (Efs) has been linked to mutations in genes encoding the LiaFSR stress response system and lipid biosynthetic enzymes, including cardiolipin synthase (Cls). The signature phenotype of DAP-R is redistribution of CM anionic phospholipid (APL) microdomains. Using a genetic approach, we have identified a transmembrane protein (LiaY) as a major mediator of cell membrane APL redistribution associated with DAP-R. Here, we explore the mechanism of LiaY-mediated changes in the CM under the hypothesis that CM remodeling occurs through interactions with Cls. Methods Efs encodes two cls genes (cls1 and cls2). Deletion mutants of both cls genes were generated using the Crispr/cas9 system in the daptomycin-sensitive strain Efs OG117 and Efs OG117∆ liaX (a DAP-R derivative of OG117). DAP minimum inhibitory concentration (MIC) was determined using E-test on Mueller–Hinton II agar. Visualization of APL microdomains was performed by staining mid-logarithmic phase cells with 1 µM of 10-N-nonyl-acridine orange (NAO) and fluorescence microscopy. Bacterial two-hybrid system was used to study interactions between LiaY with Cls1 or Cls2. Results Single or double deletion of cls1 or cls2 in Efs OG117 did not affect DAP MIC, and no changes in CM architecture were seen by NAO staining. In contrast,deletion of cls1 (alone or in conjunction with a deletion of cls2) in a DAP-R derivative of OG117 OG117∆ liaX , resulted in a marked decrease in DAP MIC, and NAO staining of Efs OG117∆ liaX∆cls1∆cls2 shows a restoration of septal APL microdomain localization.In the same DAP-R background, deletion of cls2 alone did not have any effect on DAP MIC or APL microdomain distribution. Additionally, bacterial two-hybrid assays showed a positive interaction of LiaY with Cls1 but not with Cls2. Conclusion We have identified the biochemical basis for DAP-R associated CM remodeling. In a proposed model, the LiaR-mediated activation of the LiaY triggers specific interactions with Cls1 displacing the protein away from the septum, resulting in local generation of APL microdomains that prevents DAP-mediated damage to the CM. Disclosures All authors: No reported disclosures. [ABSTRACT FROM AUTHOR]

Background The YycFG system is an essential two-component regulatory system involved in cell wall homeostasis associated with the development of daptomycin (DAP) resistance in E. faecium. Importantly, the standard combination of DAP plus β-lactam is ineffective against strains harboring mutations in yycFG. Transcriptional profiling identified a cluster of two genes (xpaC and telA) that is upregulated in the presence of a YycGS333L substitution. xpaC and telA are annotated as 5-bromo-4-chloroindolyl phosphate hydrolysis and tellurite resistance proteins, respectively. Here, we aimed to determine the contribution of xpaC and telA in DAP resistance. Methods Non-polar in-frame deletions of xpaC / telA and complementation of xpaC were performed in clinical strain E. faecium R446RIF. All mutants were characterized by PFGE and sequencing of the open reading frames to confirm the deletion. DAP MIC determination was performed by Etest on Mueller–Hinton agar. Binding of DAP was evaluated using BODIPY-labeled DAP (BDP-DAP). Cell membrane phospholipid microdomains were visualized using 10- N -nonyl acridine orange. All assays were compared with a DAP-susceptible clinical E. faecium strain S447. Results R446RIFΔ  telA and R446RIFΔ  xpaCtelA did not alter DAP MICs in R446RIF (24–32 μg/mL). However, deletion of xpaC alone (R446RIFΔ  xpaC ) markedly decreased DAP MIC 8 fold (to 4 μg/mL). R446RIFΔ  telA and R446RIFΔ  xpaCtelA exhibited similar binding of BDP-DAP compared with parental R446RIF. In contrast, R446RIFΔ  xpaC exhibited increased binding of the antibiotic molecule to the cell membrane, similar to that of DAP-susceptible S447. Complementation of xpaC restored DAP MIC to 32–48 µg/mL and decrease binding of DAP. NAO staining of S447, R446RIF, R446RIFΔ  telA , R446RIFΔ  xpaCtelA , and R446RIFΔ  xpaC :: xpaC displayed septal and polar distribution. In stark contrast, R446RIFΔ  xpaC showed a redistribution of phospholipid microdomains away from the septa. Conclusion XpaC is a key contributor to DAP binding and phospholipid architecture of E. faecium but only in the presence of an intact TelA. The xpaC and telA gene cluster is a novel mediator of DAP-resistance in E. faecium via theYycFG system and independent of the LiaFSR system Disclosures All authors: No reported disclosures. [ABSTRACT FROM AUTHOR]