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LiaR-independent pathways to daptomycin resistance in Enterococcus faecalis reveal a multilayer defense against cell envelope antibiotics.

ABSTRACT: The lipopeptide antibiotic daptomycin (DAP) is a key drug against serious enterococcal infections, but the emergence of resistance in the clinical setting is a major concern. The LiaFSR system plays a prominent role in the development of DAP resistance (DAP-R) in enterococci, and blocking this stress response system has been proposed as a novel therapeutic strategy. In this work, we identify LiaR-independent pathways in Enterococcus faecalis that regulate cell membrane adaptation in response to antibiotics. We adapted E. faecalis OG1RF (a laboratory strain) and S613TM (a clinical strain) lacking liaR to increasing concentrations of DAP, leading to the development of DAP-R and elevated MICs to bacitracin and ceftriaxone. Whole genome sequencing identified changes in the YxdJK two-component regulatory system and a putative fatty acid kinase (dak) in both DAP-R strains. Deletion of the gene encoding the YxdJ response regulator in both the DAP-R mutant and wild-type OG1RF decreased MICs to DAP, even when a functional LiaFSR system was present. Mutations in dak were associated with slower growth, decreased membrane fluidity and alterations of cell morphology. These findings suggest that overlapping stress response pathways can provide protection against antimicrobial peptides in E. faecalis at a significant cost in bacterial fitness.

SUBMITTER: Miller WR

PROVIDER: S-EPMC6417935 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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LiaR-independent pathways to daptomycin resistance in Enterococcus faecalis reveal a multilayer defense against cell envelope antibiotics.

Miller William R WR Tran Truc T TT Diaz Lorena L Rios Rafael R Khan Ayesha A Reyes Jinnethe J Prater Amy G AG Panesso Diana D Shamoo Yousif Y Arias Cesar A CA

Molecular microbiology 20190124 3

The lipopeptide antibiotic daptomycin (DAP) is a key drug against serious enterococcal infections, but the emergence of resistance in the clinical setting is a major concern. The LiaFSR system plays a prominent role in the development of DAP resistance (DAP-R) in enterococci, and blocking this stress response system has been proposed as a novel therapeutic strategy. In this work, we identify LiaR-independent pathways in Enterococcus faecalis that regulate cell membrane adaptation in response to ...[more]

PMID: 30582877

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Project description:UnlabelledEnterococcus faecalis is a commensal bacterium of the gastrointestinal tract that can cause nosocomial infections in immunocompromised humans. The hallmarks of this organism are its ability to survive in a variety of stressful habitats and, in particular, its ability to withstand membrane damage. One strategy used by E. faecalis to protect itself from membrane-damaging agents, including the antibiotic daptomycin, involves incorporation of exogenous fatty acids from bile or serum into the cell membrane. Additionally, the response regulator LiaR (a member of the LiaFSR [lipid II-interacting antibiotic response regulator and sensor] system associated with cell envelope stress responses) is required for the basal level of resistance E. faecalis has to daptomycin-induced membrane damage. This study aimed to determine if membrane fatty acid changes could provide protection against membrane stressors in a LiaR-deficient strain of E. faecalis We noted that despite the loss of LiaR, the organism readily incorporated exogenous fatty acids into its membrane, and indeed growth in the presence of exogenous fatty acids increased the survival of LiaR-deficient cells when challenged with a variety of membrane stressors, including daptomycin. Combined, our results suggest that E. faecalis can utilize both LiaR-dependent and -independent mechanisms to protect itself from membrane damage.ImportanceEnterococcus faecalis is responsible for a significant number of nosocomial infections. Worse, many of the antibiotics used to treat E. faecalis infection are no longer effective, as this organism has developed resistance to them. The drug daptomycin has been successfully used to treat some of these resistant strains; however, daptomycin-resistant isolates have been identified in hospitals. Many daptomycin-resistant isolates are found to harbor mutations in the genetic locus liaFSR, which is involved in membrane stress responses. Another mechanism shown to increase tolerance to daptomycin involves the incorporation of exogenous fatty acids from host fluids like serum or bile. This improved tolerance was found to be independent of liaFSR and suggests that there are additional ways to impact sensitivity to daptomycin. Thus, further studies are needed to understand how host fatty acid sources can influence antibiotic susceptibility.

Project description:Enterococcus faecalis is a Gram-positive bacterium that normally exists as an intestinal commensal in humans but is also a leading cause of nosocomial infections. Previous work noted that growth supplementation with serum induced tolerance to membrane-damaging agents, including the antibiotic daptomycin. Specific fatty acids found within serum could independently provide tolerance to daptomycin (protective fatty acids), yet some fatty acids found in serum did not and had negative effects on enterococcal physiology (nonprotective fatty acids). Here, we measured a wide array of physiological responses after supplementation with combinations of protective and nonprotective fatty acids to better understand how serum induces daptomycin tolerance. When cells were supplemented with either nonprotective fatty acid, palmitic acid, or stearic acid, there were marked defects in growth and morphology, but these defects were rescued upon supplementation with either protective fatty acid, oleic acid, or linoleic acid. Membrane fluidity decreased with growth in either palmitic or stearic acid alone but returned to basal levels when a protective fatty acid was supplied. Daptomycin tolerance could be induced if a protective fatty acid was provided with a nonprotective fatty acid, and some specific combinations protected as well as serum supplementation. While cell envelope charge has been associated with tolerance to daptomycin in other Gram-positive bacteria, we concluded that it does not correlate with the fatty acid-induced protection we observed. Based on these observations, we conclude that daptomycin tolerance by serum is driven by specific, protective fatty acids found within the fluid.IMPORTANCE With an increasing prevalence of antibiotic resistance in the clinic, we strive to understand more about microbial defensive mechanisms. A nongenetic tolerance to the antibiotic daptomycin was discovered in Enterococcus faecalis that results in the increased survival of bacterial populations after treatment with the drug. This tolerance mechanism likely synergizes with antibiotic resistance in the clinic. Given that this tolerance phenotype is induced by incorporation of fatty acids present in the host, it can be assumed that infections by this organism require a higher dose of antibiotic for successful eradication. The mixture of fatty acids in human fluids is quite diverse, with little understanding between the interplay of fatty acid combinations and the tolerance phenotype we observe. It is crucial to understand the effects of fatty acid combinations on E. faecalis physiology if we are to suppress the tolerance physiology in the clinic.

Project description:Treatment of multidrug-resistant enterococci has become a challenging clinical problem in hospitals around the world due to the lack of reliable therapeutic options. Daptomycin (DAP), a cell membrane-targeting cationic antimicrobial lipopeptide, is the only antibiotic with in vitro bactericidal activity against vancomycin-resistant enterococci (VRE). However, the clinical use of DAP against VRE is threatened by emergence of resistance during therapy, but the mechanisms leading to DAP resistance are not fully understood. The mechanism of action of DAP involves interactions with the cell membrane in a calcium-dependent manner, mainly at the level of the bacterial septum. Previously, we demonstrated that development of DAP resistance in vancomycin-resistant Enterococcus faecalis is associated with mutations in genes encoding proteins with two main functions, (i) control of the cell envelope stress response to antibiotics and antimicrobial peptides (LiaFSR system) and (ii) cell membrane phospholipid metabolism (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase). In this work, we show that these VRE can resist DAP-elicited cell membrane damage by diverting the antibiotic away from its principal target (division septum) to other distinct cell membrane regions. DAP septal diversion by DAP-resistant E. faecalis is mediated by initial redistribution of cell membrane cardiolipin-rich microdomains associated with a single amino acid deletion within the transmembrane protein LiaF (a member of a three-component regulatory system [LiaFSR] involved in cell envelope homeostasis). Full expression of DAP resistance requires additional mutations in enzymes (glycerophosphoryl diester phosphodiesterase and cardiolipin synthase) that alter cell membrane phospholipid content. Our findings describe a novel mechanism of bacterial resistance to cationic antimicrobial peptides.The emergence of antibiotic resistance in bacterial pathogens is a threat to public health. Understanding the mechanisms of resistance is of crucial importance to develop new strategies to combat multidrug-resistant microorganisms. Vancomycin-resistant enterococci (VRE) are one of the most recalcitrant hospital-associated pathogens against which new therapies are urgently needed. Daptomycin (DAP) is a calcium-decorated antimicrobial lipopeptide whose target is the bacterial cell membrane. A current paradigm suggests that Gram-positive bacteria become resistant to cationic antimicrobial peptides via an electrostatic repulsion of the antibiotic molecule from a more positively charged cell surface. In this work, we provide evidence that VRE use a novel strategy to avoid DAP-elicited killing. Instead of "repelling" the antibiotic from the cell surface, VRE diverts the antibiotic molecule from the septum and "traps" it in distinct membrane regions. We provide genetic and biochemical bases responsible for the mechanism of resistance and disclose new targets for potential antimicrobial development.

Dataset Information

LiaR-independent pathways to daptomycin resistance in Enterococcus faecalis reveal a multilayer defense against cell envelope antibiotics.

Publications

LiaR-independent pathways to daptomycin resistance in Enterococcus faecalis reveal a multilayer defense against cell envelope antibiotics.

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