scholarly journals Binding of Daptomycin to Anionic Lipid Vesicles Is Reduced in the Presence of Lysyl-Phosphatidylglycerol

2016 ◽  
Vol 60 (8) ◽  
pp. 5051-5053 ◽  
Author(s):  
Tala O. Khatib ◽  
Heather Stevenson ◽  
Michael R. Yeaman ◽  
Arnold S. Bayer ◽  
Antje Pokorny
Keyword(s):  
Staphylococcus Aureus ◽  
Surface Charge ◽  
Cytoplasmic Membrane ◽  
Cationic Lipid ◽  
Lipid Vesicles ◽  
Cationic Peptides ◽  
Surface Binding ◽  
Anionic Lipid ◽  
Content Type

ABSTRACTThe cytoplasmic membrane ofStaphylococcus aureuscontains ∼20 mol% of the net cationic lipid lysyl-phosphatidylglycerol (LPG). Elevated fractions of LPG are associated with increased resistance to cationic antibiotics, including the lipopeptide daptomycin (DAP). Although the surface charge of the bacterial cytoplasmic membrane is altered by LPG, surface binding of DAP was found to be only moderately affected in anionic vesicles containing 20 mol% LPG. These results suggest that charge repulsion cannot fully explain LPG-mediated resistance to cationic peptides.

scholarly journals The Mechanism behind Bacterial Lipoprotein Release: Phenol-Soluble Modulins Mediate Toll-Like Receptor 2 Activation via Extracellular Vesicle Release from Staphylococcus aureus

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Katja Schlatterer ◽  
Christian Beck ◽  
Dennis Hanzelmann ◽  
Marco Lebtig ◽  
Birgit Fehrenbacher ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cytoplasmic Membrane ◽  
Cell Envelope ◽  
Membrane Vesicles ◽  
Extracellular Vesicle ◽  
Host Cells ◽  
Bacterial Cells ◽  
Toll Like Receptor ◽  
Vesicle Release ◽  
Content Type

ABSTRACT The innate immune system uses Toll-like receptor (TLR) 2 to detect conserved bacterial lipoproteins of invading pathogens. The lipid anchor attaches lipoproteins to the cytoplasmic membrane and prevents their release from the bacterial cell envelope. How bacteria release lipoproteins and how these molecules reach TLR2 remain unknown. Staphylococcus aureus has been described to liberate membrane vesicles. The composition, mode of release, and relevance for microbe-host interaction of such membrane vesicles have remained ambiguous. We recently reported that S. aureus can release lipoproteins only when surfactant-like small peptides, the phenol-soluble modulins (PSMs), are expressed. Here we demonstrate that PSM peptides promote the release of membrane vesicles from the cytoplasmic membrane of S. aureus via an increase in membrane fluidity, and we provide evidence that the bacterial turgor is the driving force for vesicle budding under hypotonic osmotic conditions. Intriguingly, the majority of lipoproteins are released by S. aureus as components of membrane vesicles, and this process depends on surfactant-like molecules such as PSMs. Vesicle disruption at high detergent concentrations promotes the capacity of lipoproteins to activate TLR2. These results reveal that vesicle release by bacterium-derived surfactants is required for TLR2-mediated inflammation. IMPORTANCE Our study highlights the roles of surfactant-like molecules in bacterial inflammation with important implications for the prevention and therapy of inflammatory disorders. It describes a potential pathway for the transfer of hydrophobic bacterial lipoproteins, the major TLR2 agonists, from the cytoplasmic membrane of Gram-positive bacteria to the TLR2 receptor at the surface of host cells. Moreover, our study reveals a molecular mechanism that explains how cytoplasmic and membrane-embedded bacterial proteins can be released by bacterial cells without using any of the typical protein secretion routes, thereby contributing to our understanding of the processes used by bacteria to communicate with host organisms and the environment.

scholarly journals Failures in Clinical Treatment of Staphylococcus aureus Infection with Daptomycin Are Associated with Alterations in Surface Charge, Membrane Phospholipid Asymmetry, and Drug Binding

2007 ◽  
Vol 52 (1) ◽  
pp. 269-278 ◽  
Author(s):  
Tiffanny Jones ◽  
Michael R. Yeaman ◽  
George Sakoulas ◽  
Soo-Jin Yang ◽  
Richard A. Proctor ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Surface Charge ◽  
Parental Strain ◽  
Clinical Treatment ◽  
Drug Binding ◽  
Cross Resistance ◽  
Surface Binding ◽  
Blood Isolate ◽  
Membrane Structure And Function

ABSTRACT Increasingly frequent reports have described the in vivo loss of daptomycin susceptibility in association with clinical treatment failures. The mechanism(s) of daptomycin resistance is not well understood. We studied an isogenic set of Staphylococcus aureus isolates from the bloodstream of a daptomycin-treated patient with recalcitrant endocarditis in which serial strains exhibited decreasing susceptibility to daptomycin. Since daptomycin is a membrane-targeting lipopeptide, we compared a number of membrane parameters in the initial blood isolate (parental) with those in subsequent daptomycin-resistant strains obtained during treatment. In comparison to the parental strain, resistant isolates demonstrated (i) enhanced membrane fluidity, (ii) increased translocation of the positively charged phospholipid lysyl-phosphotidylglycerol to the outer membrane leaflet, (iii) increased net positive surface charge (P < 0.05 versus the parental strain), (iv) reduced susceptibility to daptomycin-induced depolarization, permeabilization, and autolysis (P < 0.05 versus the parental strain), (v) significantly lower surface binding of daptomycin (P < 0.05 versus the parental strain), and (vi) increased cross-resistance to the cationic antimicrobial host defense peptides human neutrophil peptide 1 (hNP-1) and thrombin-induced platelet microbicidal protein 1 (tPMP-1). These data link distinct changes in membrane structure and function with in vivo development of daptomycin resistance in S. aureus. Moreover, the cross-resistance to hNP-1 and tPMP-1 may also impact the capacity of these daptomycin-resistant organisms to be cleared from sites of infection, particularly endovascular foci.

scholarly journals A Membrane-Bound Transcription Factor is Proteolytically Regulated by the AAA+ Protease FtsH in Staphylococcus aureus

2020 ◽  
Vol 202 (9) ◽  
Author(s):  
Won-Sik Yeo ◽  
Chiamara Anokwute ◽  
Philip Marcadis ◽  
Marcus Levitan ◽  
Mahmoud Ahmed ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Dna Binding ◽  
Cytoplasmic Membrane ◽  
Agar Plate ◽  
Repeat Sequence ◽  
Chromosomal Dna ◽  
Content Type ◽  
Membrane Bound

ABSTRACT In bacteria, chromosomal DNA resides in the cytoplasm, and most transcription factors are also found in the cytoplasm. However, some transcription factors, called membrane-bound transcription factors (MTFs), reside in the cytoplasmic membrane. Here, we report the identification of a new MTF in the Gram-positive pathogen Staphylococcus aureus and its regulation by the protease FtsH. The MTF, named MbtS (membrane-bound transcription factor of Staphylococcus aureus), is encoded by SAUSA300_2640 and predicted to have an N-terminal DNA binding domain and three transmembrane helices. The MbtS protein was degraded by membrane vesicles containing FtsH or by the purified FtsH. MbtS bound to an inverted repeat sequence in its promoter region, and the DNA binding was essential for its transcription. Transcriptional comparison between the ftsH deletion mutant and the ftsH mbtS double mutant showed that MbtS could alter the transcription of over 200 genes. Although the MbtS protein was not detected in wild-type (WT) cells grown in a liquid medium, the protein was detected in some isolated colonies on an agar plate. In a murine model of a skin infection, the disruption of mbtS increased the lesion size. Based on these results, we concluded that MbtS is a new S. aureus MTF whose activity is proteolytically regulated by FtsH. IMPORTANCE Staphylococcus aureus is an important pathogenic bacterium causing various diseases in humans. In the bacterium, transcription is typically regulated by the transcription factors located in the cytoplasm. In this study, we report an atypical transcription factor identified in S. aureus. Unlike most other transcription factors, the newly identified transcription factor is located in the cytoplasmic membrane, and its activity is proteolytically controlled by the membrane-bound AAA+ protease FtsH. The newly identified MTF, named MbtS, has the potential to regulate the transcription of over 200 genes. This study provides a molecular mechanism by which a protease affects bacterial transcription and illustrates the diversity of the bacterial transcriptional regulation.

scholarly journals Evaluation of Ultrasound-Induced Damage to Escherichia coli and Staphylococcus aureus by Flow Cytometry and Transmission Electron Microscopy

2016 ◽  
Vol 82 (6) ◽  
pp. 1828-1837 ◽  
Author(s):  
Jiao Li ◽  
Juhee Ahn ◽  
Donghong Liu ◽  
Shiguo Chen ◽  
Xingqian Ye ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Transmission Electron Microscopy ◽  
Flow Cytometry ◽  
Cytoplasmic Membrane ◽  
Ultrasound Treatment ◽  
Cellular Damage ◽  
Content Type ◽  
Transmission Electron

ABSTRACTAs a nonthermal sterilization technique, ultrasound has attracted great interest in the field of food preservation. In this study, flow cytometry and transmission electron microscopy were employed to investigate ultrasound-induced damage toEscherichia coliandStaphylococcus aureus. For flow cytometry studies, single staining with propidium iodide (PI) or carboxyfluorescein diacetate (cFDA) revealed that ultrasound treatment caused cell death by compromising membrane integrity, inactivating intracellular esterases, and inhibiting metabolic performance. The results showed that ultrasound damage was independent of initial bacterial concentrations, while the mechanism of cellular damage differed according to the bacterial species. For the Gram-negative bacteriumE. coli, ultrasound worked first on the outer membrane rather than the cytoplasmic membrane. Based on the double-staining results, we inferred that ultrasound treatment might be an all-or-nothing process: cells ruptured and disintegrated by ultrasound cannot be revived, which can be considered an advantage of ultrasound over other nonthermal techniques. Transmission electron microscopy studies revealed that the mechanism of ultrasound-induced damage was multitarget inactivation, involving the cell wall, cytoplasmic membrane, and inner structure. Understanding of the irreversible antibacterial action of ultrasound has great significance for its further utilization in the food industry.

scholarly journals DaptomycinIn VitroActivity against Methicillin-Resistant Staphylococcus aureus Is Enhanced by d-Cycloserine in a Mechanism Associated with a Decrease in Cell Surface Charge

2013 ◽  
Vol 57 (9) ◽  
pp. 4537-4539 ◽  
Author(s):  
O. Gasch ◽  
S. K. Pillai ◽  
J. Dakos ◽  
S. Miyakis ◽  
R. C. Moellering ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Surface ◽  
Surface Charge ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Binding Assays ◽  
Methicillin Resistant ◽  
Content Type ◽  
Killing Activity ◽  
Cell Surface Charge ◽  
Mrsa Strains

ABSTRACTThe killing activity of daptomycin against an isogenic pair of daptomycin-susceptible and daptomycin-nonsusceptible (DNS) methicillin-resistantStaphylococcus aureus(MRSA) strains was enhanced by the addition of certain cell wall agents at 1× MIC. However, when high inocula of the DNS strain were used, no significant killing was observed in our experiments. Cytochromecbinding assays revealedd-cycloserine as the only agent associated with a reduction in the cell surface charge for both strains at the concentrations used.

scholarly journals Frequency and Distribution of Single-Nucleotide Polymorphisms withinmprFin Methicillin-Resistant Staphylococcus aureus Clinical Isolates and Their Role in Cross-Resistance to Daptomycin and Host Defense Antimicrobial Peptides

2015 ◽  
Vol 59 (8) ◽  
pp. 4930-4937 ◽  
Author(s):  
Arnold S. Bayer ◽  
Nagendra N. Mishra ◽  
Liang Chen ◽  
Barry N. Kreiswirth ◽  
Aileen Rubio ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Single Nucleotide Polymorphisms ◽  
Surface Charge ◽  
Host Defense ◽  
Cross Resistance ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Methicillin Resistant ◽  
Content Type ◽  
Positive Surface Charge

ABSTRACTMprF is responsible for the lysinylation of phosphatidylglycerol (PG) to synthesize the positively charged phospholipid (PL) species, lysyl-PG (L-PG). It has been proposed that the single-nucleotide polymorphisms (SNPs) within themprFopen reading frame (ORF) are associated with a gain-in-function phenotype in terms of daptomycin resistance inStaphylococcus aureus. (Note that although the official term is daptomycin nonsusceptibility, we use the term daptomycin resistance in this paper for ease of presentation.) Using 22 daptomycin-susceptible (DAPs)/daptomycin-resistant (DAPr) clinical methicillin-resistantS. aureus(MRSA) strain pairs, we assessed (i) the frequencies and distribution of putativemprFgain-in-function SNPs, (ii) the relationships of the SNPs to both daptomycin resistance and cross-resistance to the prototypical endovascular host defense peptide (HDP) thrombin-induced platelet microbicidal protein (tPMP), and (iii) the impact ofmprFSNPs on positive surface charge phenotype and modifications of membrane PL profiles. Most of themprFSNPs identified in our DAPrstrains were clustered within the two MprF loci, (i) the central bifunctional domain and (ii) the C-terminal synthase domain. Moreover, we were able to correlate the presence and location ofmprFSNPs in DAPrstrains with HDP cross-resistance, positive surface charge, and L-PG profiles. Although DAPrstrains withmprFSNPs in the bifunctional domain showed higher resistance to tPMPs than DAPrstrains with SNPs in the synthase domain, this relationship was not observed in positive surface charge assays. These results demonstrated that both charge-mediated and -unrelated mechanisms are involved in DAP resistance and HDP cross-resistance inS. aureus.

scholarly journals In VitroActivities of Antibiotics and Antimicrobial Cationic Peptides Alone and in Combination against Methicillin-Resistant Staphylococcus aureus Biofilms

2012 ◽  
Vol 56 (12) ◽  
pp. 6366-6371 ◽  
Author(s):  
Emel Mataraci ◽  
Sibel Dosler
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Agents ◽  
Antibiofilm Activity ◽  
Cationic Peptides ◽  
Planktonic Cells ◽  
Methicillin Resistant ◽  
Content Type ◽  
Mrsa Strains ◽  
Almost All

ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) strains are most often found as hospital- and community-acquired infections. The danger of MRSA infections results from not only the emergence of multidrug resistance but also the occurrence of bacteria that form strong biofilms. We investigated thein vitroactivities of antibiotics (daptomycin, linezolid, teichoplanine, azithromycin, and ciprofloxacin) and antimicrobial cationic peptides {AMPs; indolicidin, CAMA [cecropin (1-7)–melittin A (2-9) amide], and nisin} alone or in combination against MRSA ATCC 43300 biofilms. The MICs and minimum biofilm eradication concentrations (MBECs) were determined by the broth microdilution technique. Antibiotic and AMP combinations were assessed using the checkerboard technique. For MRSA planktonic cells, MICs of antibiotics and AMPs ranged between 0.125 and 512 and 8 and 16 mg/liter, respectively, and the MBEC values were between 512 and 5,120 and 640 mg/liter, respectively. With a fractional inhibitory concentration of ≤0.5 as the borderline, synergistic interactions against MRSA biofilms were frequent with almost all antibiotic-antibiotic and antibiotic-AMP combinations. Against planktonic cells, they generally had an additive effect. No antagonism was observed. All of the antibiotics, AMPs, and their combinations were able to inhibit the attachment of bacteria at 1/10 MIC and biofilm formation at 1× MIC. Biofilm-associated MRSA was not affected by therapeutically achievable concentrations of antimicrobial agents. Use of a combination of antimicrobial agents can provide a synergistic effect, which rapidly enhances antibiofilm activity and may help prevent or delay the emergence of resistance. AMPs seem to be good candidates for further investigations in the treatment of MRSA biofilms, alone or in combination with antibiotics.

scholarly journals Prolonged Exposure to β-Lactam Antibiotics Reestablishes Susceptibility of Daptomycin-Nonsusceptible Staphylococcus aureus to Daptomycin

2020 ◽  
Vol 64 (9) ◽  
Author(s):  
Rachel E. Jenson ◽  
Sarah L. Baines ◽  
Benjamin P. Howden ◽  
Nagendra N. Mishra ◽  
Sabrina Farah ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Surface Charge ◽  
Prolonged Exposure ◽  
Cell Envelope ◽  
Serial Passage ◽  
Single Nucleotide ◽  
Genetic Changes ◽  
Content Type ◽  
Host Defense Peptide ◽  
Time Kill

ABSTRACT Daptomycin-nonsusceptible (DAP-NS) Staphylococcus aureus often exhibits gain-in-function mutations in the mprF gene (involved in positive surface charge maintenance). Standard β-lactams, although relatively inactive against methicillin-resistant S. aureus (MRSA), may prevent the emergence of mprF mutations and DAP-NS. We determined if β-lactams might also impact DAP-NS isolates already possessing an mprF mutation to revert them to DAP-susceptible (DAP-S) phenotypes and, if so, whether this is associated with specific penicillin-binding protein (PBP) targeting. This study included 25 DAP-S/DAP-NS isogenic, clinically derived MRSA bloodstream isolates. MICs were performed for DAP, nafcillin (NAF; PBP-promiscuous), cloxacillin (LOX; PBP-1), ceftriaxone (CRO; PBP-2), and cefoxitin (FOX; PBP-4). Three DAP-NS isolates were selected for a 28-day serial passage in subinhibitory β-lactams. DAP MICs and time-kill assays, host defense peptide (LL-37) susceptibilities, and whole-genome sequencing were performed to associate genetic changes with key phenotypic profiles. Pronounced decreases in baseline MICs were observed for NAF and LOX (but not for CRO or FOX) among DAP-NS versus DAP-S isolates (“seesaw” effect). Prolonged (28-d) β-lactam passage of three DAP-NS isolates significantly reduced DAP MICs. LOX was most impactful (∼16-fold decrease in DAP MIC; 2 to 0.125 mg/liter). In these DAP-NS isolates with preexisting mprF polymorphisms, accumulation of additional mprF mutations occurred with prolonged LOX exposures. This was associated with enhanced LL-37 killing activity and reduced surface charge (both mprF-dependent phenotypes). β-lactams that either promiscuously or specifically target PBP-1 have significant DAP “resensitizing” effects against DAP-NS S. aureus strains. This may relate to the acquisition of multiple mprF single nucleotide polymorphism (SNPs), which, in turn, affect cell envelope function and metabolism.

scholarly journals Maculatin 1.1 Disrupts Staphylococcus aureus Lipid Membranes via a Pore Mechanism

2013 ◽  
Vol 57 (8) ◽  
pp. 3593-3600 ◽  
Author(s):  
M.-A. Sani ◽  
T. C. Whitwell ◽  
J. D. Gehman ◽  
R. M. Robins-Browne ◽  
N. Pantarat ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Flow Cytometry ◽  
Lipid Membranes ◽  
Finite Size ◽  
Lipid Vesicles ◽  
Random Coil ◽  
Isotropic Phase ◽  
Membrane Composition ◽  
Content Type ◽  
Α Helix

ABSTRACTMaculatin 1.1 (Mac1) showed potent activity againstStaphylococcus aureuswith an MIC of 7 μM. The mode of action of Mac1 was investigated by combining assays withS. aureuscells and lipid vesicles mimicking their membrane composition. A change in Mac1 conformation was monitored by circular dichroism from random coil to ca. 70% α-helix structure in contact with vesicles. Electron micrographs ofS. aureusincubated with Mac1 showed rough and rippled cell surfaces. An uptake of 65% of small (FD, 4 kDa [FD-4]) and 35% of large (RD, 40 kDa [RD-40]) fluorescent dextrans byS. aureuswas observed by flow cytometry and indicate that Mac1 formed a pore of finite size. In model membranes with both dyes encapsulated together, the full release of FD-4 occurred, but only 40% of RD-40 was reached, supporting the flow cytometry results, and indicating a pore size between 1.4 and 4.5 nm. Finally, solid-state nuclear magnetic resonance showed formation of an isotropic phase signifying highly mobile lipids such as encountered in a toroidal pore structure. Overall, Mac1 is a promising antimicrobial peptide with the potent capacity to form pores inS. aureusmembranes.

scholarly journals Analysis of Cell Membrane Characteristics of In Vitro-Selected Daptomycin-Resistant Strains of Methicillin-Resistant Staphylococcus aureus

2009 ◽  
Vol 53 (6) ◽  
pp. 2312-2318 ◽  
Author(s):  
Nagendra N. Mishra ◽  
Soo-Jin Yang ◽  
Ayumi Sawa ◽  
Aileen Rubio ◽  
Cynthia C. Nast ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Membrane ◽  
Surface Charge ◽  
Parental Strain ◽  
Phospholipid Content ◽  
Surface Binding ◽  
Methicillin Resistant ◽  
Positive Surface Charge ◽  
Vancomycin Mics

ABSTRACT Our previous studies of clinical daptomycin-resistant (Dapr) Staphylococcus aureus strains suggested that resistance is linked to the perturbations of several key cell membrane (CM) characteristics, including the CM order (fluidity), phospholipid content and asymmetry, and relative surface charge. In the present study, we examined the CM profiles of a well-known methicillin-resistant Staphylococcus aureus (MRSA) strain (MW2) after in vitro selection for DAP resistance by a 20-day serial passage in sublethal concentrations of DAP. Compared to levels for the parental strain, Dapr strains exhibited (i) decreased CM fluidity, (ii) the increased synthesis of total lysyl-phosphatidylglycerol (LPG), (iii) the increased flipping of LPG to the CM outer bilayer, and (iv) the increased expression of mprF, the gene responsible for the latter two phenotypes. In addition, we found that the expression of the dlt operon, which also increases positive surface charge, was enhanced in the Dapr mutants. These phenotypic and genotypic changes correlated with reduced DAP surface binding, mirroring observations made in clinical Dapr isolates. In this strain, serial exposure to DAP induced an increase in vancomycin MICs into the vancomycin-intermediate S. aureus (VISA) range (4 μg/ml) in parallel with increasing DAP MICs. Also, this Dapr strain exhibited significantly thicker cell walls than the parental strain, potentially correlating with the coevolution of the VISA phenotype and implicating cell wall structure and/or function in the Dapr phenotype. Importantly, despite the overexpression of mprF and dlt, the relative net positive surface charge was decreased in the Dapr mutants, suggesting that other factors contribute to the surface charge alterations and that a simple charge repulsion mechanism could not entirely explain the Dapr phenotype in these strains.

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