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Daptomycin-mediated reorganization of membrane architecture causes mislocalization of essential cell division proteins.

ABSTRACT: Daptomycin is a lipopeptide antibiotic used clinically for the treatment of certain types of Gram-positive infections, including those caused by methicillin-resistant Staphylococcus aureus (MRSA). Details of the mechanism of action of daptomycin continue to be elucidated, particularly the question of whether daptomycin acts on the cell membrane, the cell wall, or both. Here, we use fluorescence microscopy to directly visualize the interaction of daptomycin with the model Gram-positive bacterium Bacillus subtilis. We show that the first observable cellular effects are the formation of membrane distortions (patches of membrane) that precede cell death by more than 30 min. Membrane patches are able to recruit the essential cell division protein DivIVA. Recruitment of DivIVA correlates with membrane defects and changes in cell morphology, suggesting a localized alteration in the activity of enzymes involved in cell wall synthesis that could account for previously described effects of daptomycin on cell wall morphology and septation. Membrane defects colocalize with fluorescently labeled daptomycin, DivIVA, and fluorescent reporters of peptidoglycan biogenesis (Bocillin FL and BODIPY FL-vancomycin), suggesting that daptomycin plays a direct role in these events. Our results support a mechanism for daptomycin with a primary effect on cell membranes that in turn redirects the localization of proteins involved in cell division and cell wall synthesis, causing dramatic cell wall and membrane defects, which may ultimately lead to a breach in the cell membrane and cell death. These results help resolve the longstanding questions regarding the mechanism of action of this important class of antibiotics.

SUBMITTER: Pogliano J

PROVIDER: S-EPMC3415520 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Daptomycin-mediated reorganization of membrane architecture causes mislocalization of essential cell division proteins.

Pogliano Joe J Pogliano Nicolas N Silverman Jared A JA

Journal of bacteriology 20120601 17

Daptomycin is a lipopeptide antibiotic used clinically for the treatment of certain types of Gram-positive infections, including those caused by methicillin-resistant Staphylococcus aureus (MRSA). Details of the mechanism of action of daptomycin continue to be elucidated, particularly the question of whether daptomycin acts on the cell membrane, the cell wall, or both. Here, we use fluorescence microscopy to directly visualize the interaction of daptomycin with the model Gram-positive bacterium ...[more]

PMID: 22661688

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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.

Project description:The bacterial FtsZ-ring initiates division by recruiting a large repertoire of proteins (the divisome; Z-ring) needed for septation and separation of cells. Although FtsZ is essential and its role as the main orchestrator of cell division is conserved in most eubacteria, the regulators of Z-ring presence and positioning are not universal. This study characterizes factors that regulate divisome presence and placement in the ovoid-shaped pathogen, Streptococcus pneumoniae (Spn), focusing on FtsZ, EzrA, SepF, ZapA, and ZapJ, which is reported here as a partner of ZapA. Epi-fluorescence microscopy (EFm) and high-resolution microscopy experiments showed that FtsZ and EzrA co-localize during the entire Spn cell cycle, whereas ZapA and ZapJ are late-arriving divisome proteins. Depletion and conditional mutants demonstrate that EzrA is essential in Spn and required for normal cell growth, size, shape homeostasis, and chromosome segregation. Moreover, EzrA(Spn) is required for midcell placement of FtsZ-rings and PG synthesis. Notably, overexpression of EzrA leads to the appearance of extra Z-rings in Spn. Together, these observations support a role for EzrA as a positive regulator of FtsZ-ring formation in Spn. Conversely, FtsZ is required for EzrA recruitment to equatorial rings and for the organization of PG synthesis. In contrast to EzrA depletion, which causes a bacteriostatic phenotype in Spn, depletion of FtsZ results in enlarged spherical cells that are subject to LytA-dependent autolysis. Co-immunoprecipitation and bacterial two-hybrid assays show that EzrA(Spn) is in complexes with FtsZ, Z-ring regulators (FtsA, SepF, ZapA, MapZ), division proteins (FtsK, StkP), and proteins that mediate peptidoglycan synthesis (GpsB, aPBP1a), consistent with a role for EzrA at the interface of cell division and PG synthesis. In contrast to the essentiality of FtsZ and EzrA, ZapA and SepF have accessory roles in regulating pneumococcal physiology. We further show that ZapA interacts with a non-ZapB homolog, named here as ZapJ, which is conserved in Streptococcus species. The absence of the accessory proteins, ZapA, ZapJ, and SepF, exacerbates growth defects when EzrA is depleted or MapZ is deleted. Taken together, these results provide new information about the spatially and temporally distinct proteins that regulate FtsZ-ring organization and cell division in Spn.

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Daptomycin-mediated reorganization of membrane architecture causes mislocalization of essential cell division proteins.

Publications

Daptomycin-mediated reorganization of membrane architecture causes mislocalization of essential cell division proteins.

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