Browsing by Author "Munita, Jose M."

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    Daptomycin-Resistant Enterococcus faecalis Diverts the Antibiotic Molecule from the Division Septum and Remodels Cell Membrane Phospholipids
    (American Society for Microbiology, 2013) Tran, Truc T.; Panesso, Diana; Mishra, Nagendra N.; Mileykovskaya, Eugenia; Guan, Ziqiang; Munita, Jose M.; Reyes, Jinnethe; Diaz, Lorena; Weinstock, George M.; Murray, Barbara E.; Shamoo, Yousif; Dowhan, William; Bayer, Arnold S.; Arias, Cesar A.
    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 calciumdependent 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.
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    Deletion of liaR Reverses Daptomycin Resistance in Enterococcus faecium Independent of the Genetic Background
    (American Society for Microbiology, 2015) Panesso, Diana; Reyes, Jinnethe; Gaston, Elizabeth; Deal, Morgan; Londoño, Alejandra; Nigo, Masayuki; Munita, Jose M.; Miller, William R.; Shamoo, Yousif; Tran, Truc T.; Arias, Cesar A.
    We have shown previously that changes in LiaFSR, a three-component regulatory system predicted to orchestrate the cell membrane stress response, are important mediators of daptomycin (DAP) resistance in enterococci. Indeed, deletion of the gene encoding the response regulator LiaR in a clinical strain of Enterococcus faecalis reversed DAP resistance (DAP-R) and produced a strain hypersusceptible to antimicrobial peptides. Since LiaFSR is conserved in Enterococcus faecium, we investigated the role of LiaR in a variety of clinical E. faecium strains representing the most common DAP-R genetic backgrounds. Deletion of liaR in DAP-R E. faecium R446F (DAP MIC of 16 μg/ml) and R497F (MIC of 24 μg/ml; harboring changes in LiaRS) strains fully reversed resistance (DAP MICs decreasing to 0.25 and 0.094 μg/ml, respectively). Moreover, DAP at concentrations of 13 μg/ml (achieved with human doses of 12 mg/kg body weight) retained bactericidal activity against the mutants. Furthermore, the liaR deletion derivatives of these two DAP-R strains exhibited increased binding of boron-dipyrromethene difluoride (BODIPY)-daptomycin, suggesting that high-level DAP-R mediated by LiaR in E. faecium involves repulsion of the calcium-DAP complex from the cell surface. In DAP-tolerant strains HOU503F and HOU515F (DAP MICs within the susceptible range but bacteria not killed by DAP concentrations of 5× the MIC), deletion of liaRnot only markedly decreased the DAP MICs (0.064 and 0.047 μg/ml, respectively) but also restored the bactericidal activity of DAP at concentrations as low as 4 μg/ml (achieved with human doses of 4 mg/kg). Our results suggest that LiaR plays a relevant role in the enterococcal cell membrane adaptive response to antimicrobial peptides independent of the genetic background and emerges as an attractive target to restore the activity of DAP against multidrug-resistant strains.