Lysyl-phosphatidylglycerol Research Articles

Impact of the β-Lactam Resistance Modifier (-)-Epicatechin Gallate on the Non-Random Distribution of Phospholipids across the Cytoplasmic Membrane of Staphylococcus aureus.

The polyphenol (−)-epicatechin gallate (ECg) inserts into the cytoplasmic membrane (CM) of methicillin-resistant Staphylococcus aureus (MRSA) and reversibly abrogates resistance to β-lactam antibiotics. ECg elicits an increase in MRSA cell size and induces thickened cell walls. As ECg partially delocalizes penicillin-binding protein PBP2 from the septal division site, reduces PBP2 and PBP2a complexation and induces CM remodelling, we examined the impact of ECg membrane intercalation on phospholipid distribution across the CM and determined if ECg affects the equatorial, orthogonal mode of division. The major phospholipids of the staphylococcal CM, lysylphosphatidylglycerol (LPG), phosphatidylglycerol (PG), and cardiolipin (CL), were distributed in highly asymmetric fashion; 95%–97% of LPG was associated with the inner leaflet whereas PG (~90%) and CL (~80%) were found predominantly in the outer leaflet. ECg elicited small, significant changes in LPG distribution. Atomic force microscopy established that ECg-exposed cells divided in similar fashion to control bacteria, with a thickened band of encircling peptidoglycan representing the most recent plane of cell division, less distinct ribs indicative of previous sites of orthogonal division and concentric rings and “knobbles” representing stages of peptidoglycan remodelling during the cell cycle. Preservation of staphylococcal membrane lipid asymmetry and mode of division in sequential orthogonal planes appear key features of ECg-induced stress.

Untargeted metabolomics analysis revealed changes in the composition of glycerolipids and phospholipids in Bacillus subtilis under 1-butanol stress.

1-Butanol has been utilized widely in industry and can be produced or transformed by microbes. However, current knowledge about the mechanisms of 1-butanol tolerance in bacteria remains quite limited. Here, we applied untargeted metabolomics to study Bacillus subtilis cells under 1-butanol stress and identified 55 and 37 ions with significantly increased and decreased levels, respectively. Using accurate mass determination, tandem mass spectra, and synthetic standards, 86 % of these ions were characterized. The levels of phosphatidylethanolamine, diglucosyldiacylglycerol, and phosphatidylserine were found to be upregulated upon 1-butanol treatment, whereas those of diacylglycerol and lysyl phosphatidylglycerol were downregulated. Most lipids contained 15:0/15:0, 16:0/15:0, and 17:0/15:0 acyl chains, and all were mapped to membrane lipid biosynthetic pathways. Subsequent two-stage quantitative real-time reverse transcriptase PCR analyses of genes in the two principal membrane lipid biosynthesis pathways revealed elevated levels of ywiE transcripts in the presence of 1-butanol and reduced expression levels of cdsA, pgsA, mprF, clsA, and yfnI transcripts. Thus, the gene transcript levels showed agreement with the metabolomics data. Lastly, the cell morphology was investigated by scanning electron microscopy, which indicated that cells became almost twofold longer after 1.4 % (v/v) 1-butanol stress for 12 h. Overall, the studies uncovered changes in the composition of glycerolipids and phospholipids in B. subtilis under 1-butanol stress, emphasizing the power of untargeted metabolomics in the discovery of new biological insights.

Binding of Daptomycin to Lipid Bilayers is not Significantly Altered by the Inclusion of Lysyl-Phosphatidylglycerol

In many bacterial cytoplasmic membranes, including that of Staphylococcus aureus, approximately 20 mol% of the diacylphosphatidylglycerol fraction in the cytoplasmic membrane is esterified to the amino acid lysine. This secondary modification leads to the formation of a net-cationic membrane lipid, which reduces the negative surface charge of the bacterial membrane. Elevated levels of lysyl-phosphatidylglycerol (LPG) are associated with increased bacterial resistance to a number of cationic antibacterial peptides, including the clinically important calcium-dependent lipopeptide antibiotic daptomycin. Inclusion of lysylated phospholipids in mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) has been shown to effectively protect lipid vesicles against leakage of contents induced by the alpha-helical antimicrobial peptides RP-1, mastoparan X, and cecropin A. However, peptide binding to the lipid bilayer was only significantly reduced at LPG fractions exceeding 30 mol%. Here, we investigated whether binding of daptomycin to lipid vesicles containing mixtures of POPG, POPC and LPG is affected in ways similar to that of alpha-helical peptides. We find that daptomycin binds calcium ions in aqueous solution with an apparent dissociation constant on the order of 10 mM. In general, binding of daptomycin to lipid bilayers depends strongly on calcium and the fraction of POPG in the mixture. However, when 20 mol% of POPG are replaced by LPG, daptomycin binding is only minimally affected. We also show that daptomycin does not cross the lipid bilayer into the vesicle lumen in giant lipid vesicles. These data suggest that clinical daptomycin resistance in S. aureus strains is caused by mechanisms beyond an LPG-mediated reduction in daptomycin binding.

LysPGS formation in Listeria monocytogenes has broad roles in maintaining membrane integrity beyond antimicrobial peptide resistance

Listeria monocytogenes is an intracellular, foodborne gastrointestinal pathogen that is primarily responsible for causing listeriosis or food poisoning in otherwise healthy individuals. Infections that arise during pregnancy or within immune compromised individuals are much more serious resulting in the risk of fetal termination or fetal fatality postpartum in the former and septicemia or meningitis with a 20% fatality rate in the latter. While the roles of internalin proteins and listeriolysin-O in the infection process are well characterized, the specific roles of lysine-modified phospholipids in the membrane of L. monocytogenes are not. Investigation into the lipid bilayer composition of L. monocytogenes indicated that the overall proportions of lipids, including lysylcardiolipin and lysylphosphatidylglycerol (LysPG), vary with growth temperature and growth phase. In addition, we demonstrate that LysPG formation is essential for L. monocytogenes survival in the presence of increased osmolytic stress but has no effect on bacterial adherence, invasion or survival in the presence of physiologically relevant concentrations of human neutrophil peptide (HNP-1). In the absence of LysPG synthesis, L. monocytogenes unexpectedly retained flagellum-mediated motility at 37 °C. Taken together, these findings show that LysPG formation in L. monocytogenes has broader functions in virulence and survival beyond its known role in the modification of membrane potential previously observed in other bacteria.

Causal Role of Single Nucleotide Polymorphisms within the mprF Gene of Staphylococcus aureus in Daptomycin Resistance

Single nucleotide polymorphisms (SNPs) within the mprF open reading frame (ORF) have been commonly observed in daptomycin-resistant (DAP(r)) Staphylococcus aureus strains. Such SNPs are usually associated with a gain-in-function phenotype, in terms of either increased synthesis or enhanced translocation (flipping) of lysyl-phosphatidylglycerol (L-PG). However, it is unclear if such mprF SNPs are causal in DAP(r) strains or are merely a biomarker for this phenotype. In this study, we used an isogenic set of S. aureus strains: (i) Newman, (ii) its isogenic ΔmprF mutant, and (iii) several in trans plasmid complementation constructs, expressing either a wild-type or point-mutated form of the mprF ORF cloned from two isogenic DAP-susceptible (DAP(s))-DAP(r) strain pairs (616-701 and MRSA11/11-REF2145). Complementation of the ΔmprF strain with singly point-mutated mprF genes (mprFS295L or mprFT345A) revealed that (i) individual and distinct point mutations within the mprF ORF can recapitulate phenotypes observed in donor strains (i.e., changes in DAP MICs, positive surface charge, and cell membrane phospholipid profiles) and (ii) these gain-in-function SNPs (i.e., enhanced L-PG synthesis) likely promote reduced DAP binding to S. aureus by a charge repulsion mechanism. Thus, for these two DAP(r) strains, the defined mprF SNPs appear to be causally related to this phenotype.

Emergence of Daptomycin Resistance in Daptomycin-Naïve Rabbits with Methicillin-Resistant Staphylococcus aureus Prosthetic Joint Infection Is Associated with Resistance to Host Defense Cationic Peptides and mprF Polymorphisms

BackgroundPrevious studies of both clinically-derived and in vitro passage-derived daptomycin–resistant (DAP-R) Staphylococcus aureus strains demonstrated the coincident emergence of increased DAP MICs and resistance to host defense cationic peptides (HDP-R).MethodsIn the present investigation, we studied a parental DAP-susceptible (DAP-S) methicillin-resistant Staphylococcus aureus (MRSA) strain and three isogenic variants with increased DAP MICs which were isolated from both DAP-treated and DAP-untreated rabbits with prosthetic joint infections. These strains were compared for: in vitro susceptibility to distinct HDPs differing in size, structure, and origin; i.e.; thrombin-induced platelet microbicidal proteins [tPMPs] and human neutrophil peptide-1 [hNP-1]; cell membrane (CM) phospholipid and fatty acid content; CM order; envelope surface charge; cell wall thickness; and mprF single nucleotide polymorphisms (SNPs) and expression profiles.ResultsIn comparison with the parental strain, both DAP-exposed and DAP-naive strains exhibited: (i) significantly reduced susceptibility to each HDP (P<0.05); (ii) thicker cell walls (P<0.05); (iii) increased synthesis of CM lysyl-phosphatidylglycerol (L-PG); (iv) reduced content of CM phosphatidylglycerol (PG); and (v) SNPs within the mprF locus No significant differences were observed between parental or variant strains in outer CM content of L-PG, CM fluidity, CM fatty acid contents, surface charge, mprF expression profiles or MprF protein content. An isolate which underwent identical in vivo passage, but without evolving increased DAP MICs, retained parental phenotypes and genotype.ConclusionsThese results suggest: i) DAP MIC increases may occur in the absence of DAP exposures in vivo and may be triggered by organism exposure to endogenous HDPs: and ii) gain-in-function SNPs in mprF may contribute to such HDP-DAP cross-resistance phenotypes, although the mechanism of this relationship remains to be defined.

Daptomycin resistance in enterococci is associated with distinct alterations of cell membrane phospholipid content.

BackgroundThe lipopeptide antibiotic, daptomycin (DAP) interacts with the bacterial cell membrane (CM). Development of DAP resistance during therapy in a clinical strain of Enterococcus faecalis was associated with mutations in genes encoding enzymes involved in cell envelope homeostasis and phospholipid metabolism. Here we characterized changes in CM phospholipid profiles associated with development of DAP resistance in clinical enterococcal strains.MethodologyUsing two clinical strain-pairs of DAP-susceptible and DAP-resistant E. faecalis (S613 vs. R712) and E. faecium (S447 vs. R446) recovered before and after DAP therapy, we compared four distinct CM profiles: phospholipid content, fatty acid composition, membrane fluidity and capacity to be permeabilized and/or depolarized by DAP. Additionally, we characterized the cell envelope of the E. faecium strain-pair by transmission electron microscopy and determined the relative cell surface charge of both strain-pairs.Principal FindingsBoth E. faecalis and E. faecium mainly contained four major CM PLs: phosphatidylglycerol (PG), cardiolipin, lysyl-phosphatidylglycerol (L-PG) and glycerolphospho-diglycodiacylglycerol (GP-DGDAG). In addition, E. faecalis CMs (but not E. faecium) also contained: i) phosphatidic acid; and ii) two other unknown species of amino-containing PLs. Development of DAP resistance in both enterococcal species was associated with a significant decrease in CM fluidity and PG content, with a concomitant increase in GP-DGDAG. The strain-pairs did not differ in their outer CM translocation (flipping) of amino-containing PLs. Fatty acid content did not change in the E. faecalis strain-pair, whereas a significant decrease in unsaturated fatty acids was observed in the DAP-resistant E. faecium isolate R446 (vs S447). Resistance to DAP in E. faecium was associated with distinct structural alterations of the cell envelope and cell wall thickening, as well as a decreased ability of DAP to depolarize and permeabilize the CM.ConclusionDistinct alterations in PL content and fatty acid composition are associated with development of enterococcal DAP resistance.

Alanyl-Phosphatidylglycerol and Lysyl-Phosphatidylglycerol Are Translocated by the Same MprF Flippases and Have Similar Capacities To Protect against the Antibiotic Daptomycin in Staphylococcus aureus

The lysinylation of negatively charged phosphatidylglycerol by MprF proteins reduces the affinity of cationic antimicrobial peptides (CAMPs) for bacterial cytoplasmic membranes and reduces the susceptibility of several Gram-positive bacterial pathogens to CAMPs. MprF of Staphylococcus aureus encompasses a lysyl-phosphatidylglycerol (Lys-PG) synthase and a Lys-PG flippase domain. In contrast, Clostridium perfringens encodes two MprF homologs which specifically synthesize alanyl-phosphatidylglycerol (Ala-PG) or Lys-PG, while only the Lys-PG synthase is fused to a putative flippase domain. It remains unknown whether cationic Lys-PG and zwitterionic Ala-PG differ in their capacities to be translocated by MprF flippases and if both can reduce CAMP susceptibility in Gram-positive bacteria. By expressing the MprF proteins of C. perfringens in an S. aureus mprF deletion mutant, we found that both lipids can be efficiently produced in S. aureus. Simultaneous expression of the Lys-PG and Ala-PG synthases led to the production of both lipids and slightly increased the overall amounts of aminoacyl phospholipids. Ala-PG production by the corresponding C. perfringens enzyme did not affect susceptibility to CAMPs such as nisin and gallidermin or to the CAMP-like antibiotic daptomycin. However, coexpression of the Ala-PG synthase with flippase domains of Lys-PG synthesizing MprF proteins led to a wild-type level of daptomycin susceptibility, indicating that Ala-PG can also protect bacterial membranes against daptomycin and suggesting that Lys-PG flippases can also translocate the related lipid Ala-PG. Thus, bacterial aminoacyl phospholipid flippases exhibit more relaxed substrate specificity and Ala-PG and Lys-PG are more similar in their capacities to modulate membrane functions than anticipated.

LC-MS/MS characterization of phospholipid content in daptomycin-susceptible and -resistant isolates ofStaphylococcus aureuswith mutations inmprF

Daptomycin (DAP) is a cyclic lipopeptide antibiotic used for the treatment of certain Staphylococcus aureus infections. Although rare, strains have been isolated that are DAP resistant. These strains usually have mutations in mprF, a gene encoding a membrane protein with both lysylphosphatidylglycerol (LPG) synthase and flippase activities. Because ΔmprF strains have increased DAP susceptibility, the mechanism of resistance is not likely due to a loss of mprF function. In this study, we developed an LC-MS assay to examine the effect of different mprF mutations on the ratio of phosphatidylglycerol (PG) to LPG in the membrane. Our assay demonstrated that some, but not all, mutations in the flippase and synthase domains result in small but reproducible increases in the proportion of LPG relative to PG. Techniques described herein represent a higher throughput and more sensitive method for measuring relative phospholipids levels. These results offer guidance in the understanding of how mprF confers DAP resistance; namely, mprF-mediated resistance may be through more than one mechanism, including increased overall LPG synthesis and increased LPG present on the outer leaflet of the cytoplasmic membrane.

Resistance Phenotypes Mediated by Aminoacyl-Phosphatidylglycerol Synthases

The specific aminoacylation of the phospholipid phosphatidylglycerol (PG) with alanine or with lysine catalyzed by aminoacyl-phosphatidylglycerol synthases (aaPGS) was shown to render various organisms less susceptible to antibacterial agents. This study makes use of Pseudomonas aeruginosa chimeric mutant strains producing lysyl-phosphatidylglycerol (L-PG) instead of the naturally occurring alanyl-phosphatidylglycerol (A-PG) to study the resulting impact on bacterial resistance. Consequences of such artificial phospholipid composition were studied in the presence of an overall of seven antimicrobials (β-lactams, a lipopeptide antibiotic, cationic antimicrobial peptides [CAMPs]) to quantitatively assess the effect of A-PG substitution (with L-PG, L-PG and A-PG, increased A-PG levels). For the employed Gram-negative P. aeruginosa model system, an exclusive charge repulsion mechanism does not explain the attenuated antimicrobial susceptibility due to PG modification. Additionally, the specificity of nine orthologous aaPGS enzymes was experimentally determined. The newly characterized protein sequences allowed for the establishment of a significant group of A-PG synthase sequences which were bioinformatically compared to the related group of L-PG synthesizing enzymes. The analysis revealed a diverse origin for the evolution of A-PG and L-PG synthases, as the specificity of an individual enzyme is not reflected in terms of a characteristic sequence motif. This finding is relevant for future development of potential aaPGS inhibitors.

Differential Adaptations of Methicillin-ResistantStaphylococcus aureusto SerialIn VitroPassage in Daptomycin: Evolution of Daptomycin Resistance and Role of Membrane Carotenoid Content and Fluidity

Previous studies showed serial 20 d in vitro passage of MRSA strain MW2 in sublethal daptomycin (DAP) resulted in diverse perturbations in both cell membrane (CM) and cell wall (CW) characteristics, including increased CM rigidity; increased CW thickness; “gain-in-function” single nucleotide polymorphisms (SNPs) in the mprF locus (i.e., increased synthesis and translocation of lysyl-phosphatidylglycerol (L-PG)); progressive accumulation of SNPs in yyc and rpo locus genes; reduced carotenoid production; cross-resistance to innate host defense peptides. The current study was designed to characterize the reproducibility of these phenotypic and genotypic modifications following in vitro serial passages of the same parental strain. After a second 20d serial in vitro passage of parental MW2, emergence of DAP-R was associated with evolution of several phenotypes closely mirroring previous passage outcomes. However, in contrast to the initial serial passage strain set, we observed (i) only modest increase in L-PG synthesis and no increase in L-PG outer CM translocation; (ii) significantly increased carotenoid synthesis (P < 0.05); (iii) a different order of SNP accumulations (mprF ≫ rpoB ≫ yycG); (iv) a different cadre and locations of such SNPs. Thus, MRSA strains are not “pre-programmed” to phenotypically and/or genotypically adapt in an identical manner during induction of DAP resistance.

Comparative Genome Sequencing of an Isogenic Pair of USA800 Clinical Methicillin-Resistant Staphylococcus aureus Isolates Obtained before and after Daptomycin Treatment Failure

We describe here a clinical daptomycin treatment failure in a patient with recurrent methicillin-resistant Staphylococcus aureus (MRSA) bacteremia in whom daptomycin was administered after a failed empirical treatment course with vancomycin and piperacillin-tazobactam. We had the opportunity to compare the genome sequences of an isogenic pair of daptomycin-susceptible and -resistant MRSA isolates obtained before and after initiation of daptomycin therapy, respectively. The genotype of both isolates was USA800, ST5, SCCmec type IV, agr type II. There was no increase in cell wall thickness in the daptomycin-resistant strain despite having decreased susceptibility to both vancomycin and daptomycin. By comparing the genome sequences by pyrosequencing, we identified a polymorphism (S337L) in the tenth transmembrane segment of the multiple peptide resistance factor, MprF, encoding lysyl phosphatidylglycerol transferase. This enzyme has been shown previously to promote repulsion of daptomycin at the cell surface by addition of positively charged lysine to phosphatidylglycerol. Also, the hlb open reading frame (ORF) encoding the β-toxin was interrupted by a prophage in the daptomycin-susceptible strain; this phage was missing in the daptomycin-resistant isolate and the hlb ORF was restored. Loss of the phage in the resistant isolate also resulted in loss of the virulence factor genes clpP, scn, and sak. This is the first study to use pyrosequencing to compare the genomes of a daptomycin-susceptible/resistant MRSA isolate pair obtained during failed daptomycin therapy in humans.

Rapid direct lipid profiling of bacteria using desorption electrospray ionization mass spectrometry

Desorption electrospray ionization (DESI) was employed to measure lipids directly from sixteen bacterial samples without extraction or other sample preparation. Differentiation of different bacterial species and some sub-species was achieved using either the positive or the negative ion mode DESI mass spectra covering the mass/charge range up to m/ z 1000. The data were confirmed by electrospray mass spectrometry (ESI-MS) of lipid extracts from the same bacterial samples. Although the signals were lower, the quality of the direct ionization DESI spectra compared favorably with that of the ESI spectra extracts prepared using chloroform/methanol. The use of unit mass resolution in these experiments allows for overlaps of nominally isobaric and isomeric lipids at particular m/ z values. Tandem mass spectrometry was performed to validate the presence of particular lipids falling into several classes of phospholipids, including phosphatidylethanolamines (PE), phosphatidylglycerols (PG) and lysophospholipids. In addition, lysyl-phosphatidylglycerol (LPG) and lipopeptides were observed in the cases of gram-positive bacteria. DESI-MS lipid profiling was applied to the characterization of four different bacterial species including thirteen Salmonella strains. Two bacterial samples Escherichia coli K-12 and Salmonella typhimurium INSP24 were also grown in three different media. Data were compared using principal component analysis (PCA), which indicated that the different species are readily distinguished and that different growth media do not prevent bacterial species differentiation in the cases examined. Several different Salmonella strains are also distinguishable from each other based on the PCA results.

Changing the phospholipid composition of Staphylococcus aureus causes distinct changes in membrane proteome and membrane‐sensory regulators

The dynamic lipid composition of bacterial cytoplasmic membranes has a profound impact on vital bacterial fitness and susceptibility to membrane-damaging agents, temperature, or osmotic stress. However, it has remained largely unknown how changes in lipid patterns affect the abundance and expression of membrane proteins. Using recently developed gel-free proteomics technology, we explored the membrane proteome of the important human pathogen Staphylococcus aureus in the presence or absence of the cationic phospholipid lysyl-phosphatidylglycerol (Lys-PG). We were able to detect almost half of all theoretical integral membrane proteins and could reliably quantify more than 35% of them. It is worth noting that the deletion of the Lys-PG synthase MprF did not lead to a massive alteration but a very distinct up- or down-regulation of only 1.5 or 3.5% of the quantified proteins. Lys-PG deficiency had no major impact on the abundance of lipid-biosynthetic enzymes but significantly affected the amounts of the cell envelope stress-sensing regulatory proteins such as SaeS and MsrR, and of the SaeS-regulated proteins Sbi, Efb, and SaeP. These data indicate very critical interactions of membrane-sensory proteins with phospholipids and they demonstrate the power of membrane proteomics for the characterization of bacterial physiology and pathogenicity.

The Bacterial Defensin Resistance Protein MprF Consists of Separable Domains for Lipid Lysinylation and Antimicrobial Peptide Repulsion

Many bacterial pathogens achieve resistance to defensin-like cationic antimicrobial peptides (CAMPs) by the multiple peptide resistance factor (MprF) protein. MprF plays a crucial role in Staphylococcus aureus virulence and it is involved in resistance to the CAMP-like antibiotic daptomycin. MprF is a large membrane protein that modifies the anionic phospholipid phosphatidylglycerol with l-lysine, thereby diminishing the bacterial affinity for CAMPs. Its widespread occurrence recommends MprF as a target for novel antimicrobials, although the mode of action of MprF has remained incompletely understood. We demonstrate that the hydrophilic C-terminal domain and six of the fourteen proposed trans-membrane segments of MprF are sufficient for full-level lysyl-phosphatidylglycerol (Lys-PG) production and that several conserved amino acid positions in MprF are indispensable for Lys-PG production. Notably, Lys-PG production did not lead to efficient CAMP resistance and most of the Lys-PG remained in the inner leaflet of the cytoplasmic membrane when the large N-terminal hydrophobic domain of MprF was absent, indicating a crucial role of this protein part. The N-terminal domain alone did not confer CAMP resistance or repulsion of the cationic test protein cytochrome c. However, when the N-terminal domain was coexpressed with the Lys-PG synthase domain either in one protein or as two separate proteins, full-level CAMP resistance was achieved. Moreover, only coexpression of the two domains led to efficient Lys-PG translocation to the outer leaflet of the membrane and to full-level cytochrome c repulsion, indicating that the N-terminal domain facilitates the flipping of Lys-PG. Thus, MprF represents a new class of lipid-biosynthetic enzymes with two separable functional domains that synthesize Lys-PG and facilitate Lys-PG translocation. Our study unravels crucial details on the molecular basis of an important bacterial immune evasion mechanism and it may help to employ MprF as a target for new anti-virulence drugs.

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

Our previous studies of clinical daptomycin-resistant (Dap(r)) 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, Dap(r) 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 Dap(r) mutants. These phenotypic and genotypic changes correlated with reduced DAP surface binding, mirroring observations made in clinical Dap(r) isolates. In this strain, serial exposure to DAP induced an increase in vancomycin MICs into the vancomycin-intermediate S. aureus (VISA) range (4 microg/ml) in parallel with increasing DAP MICs. Also, this Dap(r) 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 Dap(r) phenotype. Importantly, despite the overexpression of mprF and dlt, the relative net positive surface charge was decreased in the Dap(r) mutants, suggesting that other factors contribute to the surface charge alterations and that a simple charge repulsion mechanism could not entirely explain the Dap(r) phenotype in these strains.

The Lipid Lysyl-Phosphatidylglycerol Is Present in Membranes of Rhizobium tropici CIAT899 and Confers Increased Resistance to Polymyxin B Under Acidic Growth Conditions

Lysyl-phosphatidylglycerol (LPG) is a well-known membrane lipid in several gram-positive bacteria but is almost unheard of in gram-negative bacteria. In Staphylococcus aureus, the gene product of mprF is responsible for LPG formation. Low pH-inducible genes, termed IpiA, have been identified in the gram-negative alpha-proteobacteria Rhizobium tropici and Sinorhizobium medicae in screens for acid-sensitive mutants and they encode homologs of MprF. An analysis of the sequenced bacterial genomes reveals that genes coding for homologs of MprF from S. aureus are present in several classes of organisms throughout the bacterial kingdom. In this study, we show that the expression of lpiA from R. tropici in the heterologous hosts Escherichia coli and Sinorhizobium meliloti causes formation of LPG. A wild-type strain of R. tropici forms LPG (about 1% of the total lipids) when the cells are grown in minimal medium at pH 4.5 but not when grown in minimal medium at neutral pH or in complex tryptone yeast (TY) medium at either pH. LPG biosynthesis does not occur when lpiA is deleted and is restored upon complementation of lpiA-deficient mutants with a functional copy of the lpiA gene. When grown in the low-pH medium, lpiA-deficient rhizobial mutants are over four times more susceptible to the cationic peptide polymyxin B than the wild type.

The MprF protein is required for lysinylation of phospholipids in listerial membranes and confers resistance to cationic antimicrobial peptides (CAMPs) on Listeria monocytogenes

Pathogenic bacteria have to cope with defence mechanisms mediated by adaptive and innate immunity of the host cells. Cationic antimicrobial peptides (CAMPs) represent one of the most effective components of the host innate immune response. Here we establish the function of Lmo1695, a member of the VirR-dependent virulence regulon, recently identified in Listeria monocytogenes. Lmo1695 encodes a membrane protein of 98 kDa with strong homology to the multiple peptide resistance factor (MprF) of Staphylococcus aureus. Like staphylococcal MprF, we found that Lmo1695 is involved in the synthesis of the membrane phospholipid lysylphosphatidylglycerol (L-PG). In addition, Lmo1695 is also essential for lysinylation of diphosphatidylglycerol (DPG), another phospholipid widely distributed in bacterial membranes. A Deltalmo1695 mutant lacking the lysinylated phospholipids was particularly susceptible to CAMPs of human and bacterial origin. The mutant strain infected both epithelial cells and macrophages only poorly and was attenuated for virulence when tested in a mouse model of infection. Lmo1695 is a member of a growing list of survival factors which enable growth of L. monocytogenes in different environments.

The Sinorhizobium medicae WSM419 lpiA gene is transcriptionally activated by FsrR and required to enhance survival in lethal acid conditions

Sinorhizobium medicae WR101 was identified as a mutant of WSM419 that contained a minitransposon-induced transcriptional gusA fusion activated at least 20-fold at pH 5.7. The expression of this fusion in moderately acid conditions was dependent on the calcium concentration; increasing the calcium concentration to enhance cell growth and survival in acid conditions decreased the expression of the fusion. A gene region containing the gusA fusion was sequenced, revealing five S. medicae genes: tcsA, tcrA, fsrR, lpiA and acvB. The gusA reporter in WR101 was fused to lpiA, which encodes a putative transmembrane protein also found in other Alphaproteobacteria such as Sinorhizobium meliloti, Rhizobium tropici and Agrobacterium tumefaciens. As LpiA has partial sequence similarity to the lysyl-phosphatidylglycerol (LPG) synthetase FmtC/MprF from Staphylococcus aureus, membrane lipid compositions of S. medicae strains were analysed. Cells cultured under neutral or acidic growth conditions did not induce any detectable LPG and therefore this lipid cannot be a major constituent of S. medicae membranes. Expression studies in S. medicae localized the acid-activated lpiA promoter within a 372 bp region upstream of the start codon. The acid-activated transcription of lpiA required the fused sensor-regulator product of the fsrR gene, because expression of lpiA was severely reduced in an S. medicae fsrR mutant. S. meliloti strain 1021 does not contain fsrR and acid-activated expression of the lpiA-gusA fusion did not occur in this species. Although acid-activated lpiA transcription was not required for cell growth, its expression was crucial in enhancing the viability of cells subsequently exposed to lethal acid (pH 4.5) conditions.

Characterization of the Staphylococcus aureus mprF gene, involved in lysinylation of phosphatidylglycerol.

Lysylphosphatidylglycerol (LPG) is a basic phospholipid in which L-lysine from lysyl-tRNA is transferred to phosphatidylglycerol (PG). This study examined whether the Staphylococcus aureus mprF gene encodes LPG synthetase. A crude membrane fraction prepared from wild-type S. aureus cells had LPG synthetase activity that depended on PG and lysyl-tRNA, whereas the membrane fraction from an mprF deletion mutant did not. When S. aureus MprF protein was trans-expressed in wild-type Escherichia coli cells, LPG synthesis was induced, whereas it was not observed in E. coli pgsA3 mutant cells in which the amount of PG is significantly reduced. In addition, LPG synthetase activity and a 93 kDa protein whose molecular size corresponded to that of MprF protein were co-induced in the crude membrane fraction prepared from E. coli cells expressing MprF protein. The Km values of the LPG synthetase activity for PG and for lysyl-tRNA were 56 microM and 6.9 microM, respectively, consistent with those of S. aureus membranes. These results suggest that the MprF protein is LPG synthetase.