Reduction in Membrane Phosphatidylglycerol Content Leads to Daptomycin Resistance in Bacillus subtilis

Abstract

Daptomycin (DAP) is a cyclic lipopeptide that disrupts the functional integrity of the cell membranes of Gram-positive bacteria in a Ca2؉ -dependent manner. Here we present genetic, genomic, and phenotypic analyses of an evolved DAP-resistant isolate, Dap R 1, from the model bacterium Bacillus subtilis 168. Dap R 1 was obtained by serial passages with increasing DAP concentrations, is 30-fold more resistant than the parent strain, and displays cross-resistance to vancomycin, moenomycin, and bacitracin. Dap R 1 is characterized by aberrant septum placement, notably thickened peptidoglycan at the cell poles, and pleiotropic alterations at both the transcriptome and proteome levels. Genome sequencing of Dap R 1 revealed 44 point mutations, 31 of which change protein sequences. An intermediate isolate that was 20-fold more resistant to DAP than the wild type had only three of these point mutations: mutations affecting the cell shape modulator gene mreB, the stringent response gene relA, and the phosphatidylglycerol synthase gene pgsA. Genetic reconstruction studies indicated that the pgsA(A64V) allele is primarily responsible for DAP resistance. Allelic replacement with wild-type pgsA restored DAP sensitivity to wild-type levels. The additional point mutations in the evolved strain may contribute further to DAP resistance, serve to compensate for the deleterious effects of altered membrane composition, or represent neutral changes. These results suggest a resistance mechanism by which reduced levels of phosphatidylglycerol decrease the net negative charge of the membrane, thereby weakening interaction with the positively charged Ca 2؉ -DAP complex.Daptomycin (DAP) is a cyclic lipopeptide antibiotic used to treat complicated skin and skin structure infections caused by Staphylococcus aureus or enterococci. In addition, it has been approved to treat S. aureus-induced bacteremia and infective endocarditis (21), and animal model studies suggest that it may be a useful alternative for treatment of inhalational anthrax (26). The mechanism of action involves the calcium-dependent insertion of DAP into the bacterial membrane, followed by depolarization of the membrane and extrusion of potassium ions, leading to arrest of macromolecular synthesis and to cell death (49, 51).The introduction of new antibacterial compounds seems to be followed inevitably by the emergence of resistant isolates. It is estimated that over 1 million patients have been treated with DAP (J. Silverman, personal communication). According to the SENTRY Antimicrobial Surveillance Program in the United States for the years 2002 to 2010, 99.9% of methicillin-resistant S. aureus (MRSA) isolates treated with DAP had an MIC of 1.0 g/ml or lower, with only a slight increase of MIC over time (47; http://www.gp-pathogens.com/data/default.cfm).Previous studies to define mechanisms of resistance to DAP were performed on clinical isolates and by in vitro selection (22,31). After serial passages with increasing DAP concentrations, Friedman et al. characterized three...

Daptomycin-Mediated Reorganization of Membrane Architecture Causes Mislocalization of Essential Cell Division Proteins

“…The role of the membrane as a central target is further supported by studies of daptomycin-resistant mutants in multiple species (2,15,18,38). Many of the mutations that alter susceptibility to daptomycin have been shown to directly affect membrane lipid composition.…”

“…In S. aureus, mutations that eliminate the expression of the mprF gene sensitize cells to daptomycin, and mutations that confer resistance are believed to be "gain-of-function" alleles that increase the amount of lysylphosphatidylglycerol (15,47,57). Interestingly, the specific membrane modifications that confer resistance vary by species: in enterococci, changes in cardiolipin synthesis are associated with resistance (2,38), while in Bacillus subtilis, resistance may be linked to the overall phosphatidylglycerol content due to mutations in pgsA (18).…”

The action mechanism of daptomycin

“…Mutations that reduce its activity reduce membrane PG levels and daptomycin susceptibility in Bacillus subtilis and Staphylococcus aureus. 64,90,91 …”

Resistance Phenotypes Mediated by Aminoacyl-Phosphatidylglycerol Synthases

“…Besides the well-described effect of L-PG formation, also the reduction of the cellular PG content was described as a mechanism lowering the overall negative surface charge, thereby rendering Bacillus subtilis and P. aeruginosa more resistant to daptomycin and polymyxin, respectively (8,24,25).…”