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Find video protocols related to scientific articles indexed in Pubmed.
Production of an attenuated phenol-soluble modulin variant unique to the MRSA clonal complex 30 increases severity of bloodstream infection.
PLoS Pathog.
PUBLISHED: 08-21-2014
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Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of morbidity and death. Phenol-soluble modulins (PSMs) are recently-discovered toxins with a key impact on the development of Staphylococcus aureus infections. Allelic variants of PSMs and their potential impact on pathogen success during infection have not yet been described. Here we show that the clonal complex (CC) 30 lineage, a major cause of hospital-associated sepsis and hematogenous complications, expresses an allelic variant of the PSM?3 peptide. We found that this variant, PSM?3N22Y, is characteristic of CC30 strains and has significantly reduced cytolytic and pro-inflammatory potential. Notably, CC30 strains showed reduced cytolytic and chemotactic potential toward human neutrophils, and increased hematogenous seeding in a bacteremia model, compared to strains in which the genome was altered to express non-CC30 PSM?3. Our findings describe a molecular mechanism contributing to attenuated pro-inflammatory potential in a main MRSA lineage. They suggest that reduced pathogen recognition via PSMs allows the bacteria to evade elimination by innate host defenses during bloodstream infections. Furthermore, they underscore the role of point mutations in key S. aureus toxin genes in that adaptation and the pivotal importance PSMs have in defining key S. aureus immune evasion and virulence mechanisms.
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Bacteriophage-based latex agglutination test for rapid identification of Staphylococcus aureus.
J. Clin. Microbiol.
PUBLISHED: 07-16-2014
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Rapid diagnosis is essential for the management of Staphylococcus aureus infections. A host recognition protein from S. aureus bacteriophage phiSLT was recombinantly produced and used to coat streptavidin latex beads to develop a latex agglutination test (LAT). The diagnostic accuracy of this bacteriophage-based test was compared with that of a conventional LAT, Pastorex Staph-Plus, by investigating a clinical collection of 86 S. aureus isolates and 128 coagulase-negative staphylococci (CoNS) from deep tissue infections. All of the clinical S. aureus isolates were correctly identified by the bacteriophage-based test. While most of the CoNS were correctly identified as non-S. aureus isolates, 7.9% of the CoNS caused agglutination. Thus, the sensitivity of the bacteriophage-based LAT for identification of S. aureus among clinical isolates was 100%, its specificity was 92.1%, its positive predictive value (PPV) was 89.6%, and its negative predictive value (NPV) was 100%. The sensitivity, specificity, PPV, and NPV of the Pastorex LAT for the identification of S. aureus were 100%, 99.2%, 98.9%, and 100%, respectively. Among the additionally tested 35 S. aureus and 91 non-S. aureus staphylococcal reference and type strains, 1 isolate was false negative by each system; 13 and 8 isolates were false positive by the bacteriophage-based and Pastorex LATs, respectively. The ability of the phiSLT protein to detect S. aureus was dependent on the presence of wall teichoic acid (WTA) and corresponded to the production of ribitol phosphate WTA, which is found in most S. aureus clones but only a small minority of CoNS. Bacteriophage-based LAT identification is a promising strategy for rapid pathogen identification. Finding more specific bacteriophage proteins would increase the specificity of this novel diagnostic approach.
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Translational research strategy: an essential approach to fight the spread of antimicrobial resistance.
J. Antimicrob. Chemother.
PUBLISHED: 07-09-2014
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Translation research strategy in infectious diseases, combining the results from basic research with patient-orientated research, aims to bridge the gap between laboratory findings and clinical infectious disease practice to improve disease management. In an era of increasing antimicrobial resistance, there are four main areas of clinical and scientific uncertainty that need to be urgently addressed by translational research: (i) early diagnosis of antibiotic-resistant infections and the appropriateness of empirical antibiotic therapy; (ii) the identification of reservoirs of antibiotic-resistant pathogens; (iii) the development of new antibiotics with lower propensities to evoke resistance; and (iv) the development of new non-antibiotic drugs to be used in the prevention of the spread of resistant bacterial strains. Strict European collaboration among major stakeholders is therefore essential. Appropriate educational tools to train a new generation of scientists with regard to a multifaceted approach to antimicrobial resistance research should be developed. Key areas include the support and implementation of European networks focused on translational research and related education activities, making potential therapeutics more attractive to investors and helping academic investigators to determine whether new molecules can be developed with clinical applicability.
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Biosynthesis of the unique wall teichoic acid of Staphylococcus aureus lineage ST395.
MBio
PUBLISHED: 04-10-2014
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ABSTRACT The major clonal lineages of the human pathogen Staphylococcus aureus produce cell wall-anchored anionic poly-ribitol-phosphate (RboP) wall teichoic acids (WTA) substituted with d-Alanine and N-acetyl-d-glucosamine. The phylogenetically isolated S. aureus ST395 lineage has recently been found to produce a unique poly-glycerol-phosphate (GroP) WTA glycosylated with N-acetyl-d-galactosamine (GalNAc). ST395 clones bear putative WTA biosynthesis genes on a novel genetic element probably acquired from coagulase-negative staphylococci (CoNS). We elucidated the ST395 WTA biosynthesis pathway and identified three novel WTA biosynthetic genes, including those encoding an ?-O-GalNAc transferase TagN, a nucleotide sugar epimerase TagV probably required for generation of the activated sugar donor substrate for TagN, and an unusually short GroP WTA polymerase TagF. By using a panel of mutants derived from ST395, the GalNAc residues carried by GroP WTA were found to be required for infection by the ST395-specific bacteriophage ?187 and to play a crucial role in horizontal gene transfer of S. aureus pathogenicity islands (SaPIs). Notably, ectopic expression of ST395 WTA biosynthesis genes rendered normal S. aureus susceptible to ?187 and enabled ?187-mediated SaPI transfer from ST395 to regular S. aureus. We provide evidence that exchange of WTA genes and their combination in variable, mosaic-like gene clusters have shaped the evolution of staphylococci and their capacities to undergo horizontal gene transfer events. IMPORTANCE The structural highly diverse wall teichoic acids (WTA) are cell wall-anchored glycopolymers produced by most Gram-positive bacteria. While most of the dominant Staphylococcus aureus lineages produce poly-ribitol-phosphate WTA, the recently described ST395 lineage produces a distinct poly-glycerol-phosphate WTA type resembling the WTA backbone of coagulase-negative staphylococci (CoNS). Here, we analyzed the ST395 WTA biosynthesis pathway and found new types of WTA biosynthesis genes along with an evolutionary link between ST395 and CoNS, from which the ST395 WTA genes probably originate. The elucidation of ST395 WTA biosynthesis will help to understand how Gram-positive bacteria produce highly variable WTA types and elucidate functional consequences of WTA variation.
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Nutrient limitation governs Staphylococcus aureus metabolism and niche adaptation in the human nose.
PLoS Pathog.
PUBLISHED: 01-01-2014
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Colonization of the human nose by Staphylococcus aureus in one-third of the population represents a major risk factor for invasive infections. The basis for adaptation of S. aureus to this specific habitat and reasons for the human predisposition to become colonized have remained largely unknown. Human nasal secretions were analyzed by metabolomics and found to contain potential nutrients in rather low amounts. No significant differences were found between S. aureus carriers and non-carriers, indicating that carriage is not associated with individual differences in nutrient supply. A synthetic nasal medium (SNM3) was composed based on the metabolomics data that permits consistent growth of S. aureus isolates. Key genes were expressed in SNM3 in a similar way as in the human nose, indicating that SNM3 represents a suitable surrogate environment for in vitro simulation studies. While the majority of S. aureus strains grew well in SNM3, most of the tested coagulase-negative staphylococci (CoNS) had major problems to multiply in SNM3 supporting the notion that CoNS are less well adapted to the nose and colonize preferentially the human skin. Global gene expression analysis revealed that, during growth in SNM3, S. aureus depends heavily on de novo synthesis of methionine. Accordingly, the methionine-biosynthesis enzyme cysteine-?-synthase (MetI) was indispensable for growth in SNM3, and the MetI inhibitor DL-propargylglycine inhibited S. aureus growth in SNM3 but not in the presence of methionine. Of note, metI was strongly up-regulated by S. aureus in human noses, and metI mutants were strongly abrogated in their capacity to colonize the noses of cotton rats. These findings indicate that the methionine biosynthetic pathway may include promising antimicrobial targets that have previously remained unrecognized. Hence, exploring the environmental conditions facultative pathogens are exposed to during colonization can be useful for understanding niche adaptation and identifying targets for new antimicrobial strategies.
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Glycoepitopes of staphylococcal wall teichoic acid govern complement-mediated opsonophagocytosis via human serum antibody and mannose-binding lectin.
J. Biol. Chem.
PUBLISHED: 09-17-2013
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Serum antibodies and mannose-binding lectin (MBL) are important host defense factors for host adaptive and innate immunity, respectively. Antibodies and MBL also initiate the classical and lectin complement pathways, respectively, leading to opsonophagocytosis. We have shown previously that Staphylococcus aureus wall teichoic acid (WTA), a cell wall glycopolymer consisting of ribitol phosphate substituted with ?- or ?-O-N-acetyl-d-glucosamine (GlcNAc) and d-alanine, is recognized by MBL and serum anti-WTA IgG. However, the exact antigenic determinants to which anti-WTA antibodies or MBL bind have not been determined. To answer this question, several S. aureus mutants, such as ?-GlcNAc glycosyltransferase-deficient S. aureus ?tarM, ?-GlcNAc glycosyltransferase-deficient ?tarS, and ?tarMS double mutant cells, were prepared from a laboratory and a community-associated methicillin-resistant S. aureus strain. Here, we describe the unexpected finding that ?-GlcNAc WTA-deficient ?tarS mutant cells (which have intact ?-GlcNAc) escape from anti-WTA antibody-mediated opsonophagocytosis, whereas ?-GlcNAc WTA-deficient ?tarM mutant cells (which have intact ?-GlcNAc) are efficiently engulfed by human leukocytes via anti-WTA IgG. Likewise, MBL binding in S. aureus cells was lost in the ?tarMS double mutant but not in either single mutant. When we determined the serum concentrations of the anti-?- or anti-?-GlcNAc-specific WTA IgGs, anti-?-GlcNAc WTA-IgG was dominant in pooled human IgG fractions and in the intact sera of healthy adults and infants. These data demonstrate the importance of the WTA sugar conformation for human innate and adaptive immunity against S. aureus infection.
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Phenol-soluble modulins and staphylococcal infection.
Nat. Rev. Microbiol.
PUBLISHED: 09-10-2013
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Staphylococcus aureus is an important human pathogen and a leading cause of death worldwide. Phenol-soluble modulins (PSMs) have recently emerged as a novel toxin family defining the virulence potential of highly aggressive S. aureus isolates. PSMs have multiple roles in staphylococcal pathogenesis, causing lysis of red and white blood cells, stimulating inflammatory responses and contributing to biofilm development and the dissemination of biofilm-associated infections. Moreover, the pronounced capacity of PSMs to kill human neutrophils after phagocytosis might explain failures in the development of anti-staphylococcal vaccines. Here, we discuss recent progress made in our understanding of the biochemical and genetic properties of PSMs and their role in S. aureus pathogenesis, and suggest potential avenues to target PSMs for the development of anti-staphylococcal drugs.
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Insight into structure-function relationship in phenol-soluble modulins using an alanine screen of the phenol-soluble modulin (PSM) ?3 peptide.
FASEB J.
PUBLISHED: 09-05-2013
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Phenol-soluble modulins (PSMs) are a family of peptides with multiple functions in staphylococcal pathogenesis. To gain insight into the structural features affecting PSM functions, we analyzed an alanine substitution library of PSM?3, a strongly cytolytic and proinflammatory PSM of Staphylococcus aureus with a significant contribution to S. aureus virulence. Lysine residues were essential for both receptor-dependent proinflammatory and receptor-independent cytolytic activities. Both phenotypes also required additional structural features, with the C terminus being crucial for receptor activation. Biofilm formation was affected mostly by hydrophobic amino acid positions, suggesting that the capacity to disrupt hydrophobic interactions is responsible for the effect of PSMs on biofilm structure. Antimicrobial activity, absent from natural PSM?3, could be created by the exchange of large hydrophobic side chains, indicating that PSM?3 has evolved to exhibit cytolytic rather than antimicrobial activity. In addition to gaining insight into the structure-function relationship in PSMs, our study identifies nontoxic PSM?3 derivatives for active vaccination strategies and lays the foundation for future efforts aimed to understand the biological role of PSM recognition by innate host defense.-Cheung, G. Y., Kretschmer, D., Queck, S. Y., Joo, H.-S., Wang, R., Duong, A. C., Nguyen, T. H., Bach, T.-H., Porter, A. R., DeLeo, F. R., Peschel, A., Otto, M. Insight into structure-function relationship in phenol-soluble modulins using an alanine screen of the phenol-soluble modulin (PSM) ?3 peptide.
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Pathways and roles of wall teichoic acid glycosylation in Staphylococcus aureus.
Int. J. Med. Microbiol.
PUBLISHED: 08-08-2013
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The thick peptidoglycan layers of Gram-positive bacteria are connected to polyanionic glycopolymers called wall teichoic acids (WTA). Pathogens such as Staphylococcus aureus, Listeria monocytogenes, or Enterococcus faecalis produce WTA with diverse, usually strain-specific structure. Extensive studies on S. aureus WTA mutants revealed important functions of WTA in cell division, growth, morphogenesis, resistance to antimicrobials, and interaction with host or phages. While most of the S. aureus WTA-biosynthetic genes have been identified it remained unclear for long how and why S. aureus glycosylates WTA with ?- or ?-linked N-acetylglucosamine (GlcNAc). Only recently the discovery of two WTA glycosyltransferases, TarM and TarS, yielded fundamental insights into the roles of S. aureus WTA glycosylation. Mutants lacking WTA GlcNAc are resistant towards most of the S. aureus phages and, surprisingly, TarS-mediated WTA ?-O-GlcNAc modification is essential for ?-lactam resistance in methicillin-resistant S. aureus. Notably, S. aureus WTA GlcNAc residues are major antigens and activate the complement system contributing to opsonophagocytosis. WTA glycosylation with a variety of sugars and corresponding glycosyltransferases were also identified in other Gram-positive bacteria, which paves the way for detailed investigations on the diverse roles of WTA modification with sugar residues.
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Hypoxia-inducible factor 1-regulated lysyl oxidase is involved in Staphylococcus aureus abscess formation.
Infect. Immun.
PUBLISHED: 05-06-2013
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Hypoxia-inducible factor 1 (HIF-1) is the key transcription factor involved in the adaptation of mammals to hypoxia and plays a crucial role in cancer angiogenesis. Recent evidence suggests a leading role for HIF-1 in various inflammatory and infectious diseases. Here we describe the role of HIF-1 in Staphylococcus aureus infections by investigating the HIF-1-dependent host cell response. For this purpose, transcriptional profiling of HIF-1?-deficient HepG2 and control cells, both infected with Staphylococcus aureus, was performed. Four hours after infection, the expression of 190 genes, 24 of which were regulated via HIF-1, was influenced. LOX (encoding lysyl oxidase) was one of the upregulated genes with a potential impact on the course of S. aureus infection. LOX is an amine oxidase required for biosynthetic cross-linking of extracellular matrix components. LOX was upregulated in vitro in different cell cultures infected with S. aureus and also in vivo, in kidney abscesses of mice intravenously infected with S. aureus and in clinical skin samples from patients with S. aureus infections. Inhibition of LOX by ?-aminopropionitrile (BAPN) did not affect the bacterial load in kidneys or blood but significantly influenced abscess morphology and collagenization. Our data provide evidence for a crucial role of HIF-1-regulated LOX in abscess formation.
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Antibacterial potency of V.A.C. GranuFoam Silver(®) Dressing.
Injury
PUBLISHED: 04-16-2013
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V.A.C.(®) GranuFoam™ therapy is regularly used in the surgical therapy of infected wounds and soft tissue injuries. Silver nanoparticles can destroy bacterial cell walls and inhibit enzymes for cell replication. Silver dressings are therefore successfully used for many indications in wound therapy. In this study, we investigated the antimicrobial potency of ionic silver released from the silver-coated V.A.C.(®) GranuFoam™ during vacuum therapy. Silver dressing was exposed to agar plates populated with bacteria to measure silver release.
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Staphylococcus aureus phenol-soluble modulin peptides modulate dendritic cell functions and increase in vitro priming of regulatory T cells.
J. Immunol.
PUBLISHED: 03-04-2013
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The major human pathogen Staphylococcus aureus has very efficient strategies to subvert the human immune system. Virulence of the emerging community-associated methicillin-resistant S. aureus depends on phenol-soluble modulin (PSM) peptide toxins, which are known to attract and lyse neutrophils. However, their influences on other immune cells remain elusive. In this study, we analyzed the impact of PSMs on dendritic cells (DCs) playing an essential role in linking innate and adaptive immunity. In human neutrophils, PSMs exert their function by binding to the formyl peptide receptor (FPR) 2. We show that mouse DCs express the FPR2 homolog mFPR2 as well as its paralog mFPR1 and that PSMs are chemoattractants for DCs at noncytotoxic concentrations. PSMs reduced clathrin-mediated endocytosis and inhibited TLR2 ligand-induced secretion of the proinflammatory cytokines TNF, IL-12, and IL-6, while inducing IL-10 secretion by DCs. As a consequence, treatment with PSMs impaired the capacity of DCs to induce activation and proliferation of CD4(+) T cells, characterized by reduced Th1 but increased frequency of FOXP3(+) regulatory T cells. These regulatory T cells secreted high amounts of IL-10, and their suppression capacity was dependent on IL-10 and TGF-?. Interestingly, the induction of tolerogenic DCs by PSMs appeared to be independent of mFPRs, as shown by experiments with mice lacking mFPR2 (mFPR2(-/-)) and the cognate G protein (p110?(-/-)). Thus, PSMs from highly virulent pathogens affect DC functions, thereby modulating the adaptive immune response and probably increasing the tolerance toward the pathogen.
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Staphylococcus aureus subverts cutaneous defense by D-alanylation of teichoic acids.
Exp. Dermatol.
PUBLISHED: 02-07-2013
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The Gram-positive bacterium Staphylococcus aureus is a frequent skin colonizer that often causes severe skin infections. It has been reported that neutralizing the negatively charged bacterial surface through the incorporation of d-alanine in its teichoic acids confers reduced susceptibility of S. aureus towards cationic antimicrobial peptides (AMPs). Using a S. aureus strain deficient in d-alanylated teichoic acids (dltA mutant), we demonstrate that d-alanylation of its surface reduces the susceptibility of S. aureus to skin-derived AMPs such as RNase 7 and human beta-defensins. This is accompanied by a higher killing activity of skin extracts towards the S. aureus dltA mutant as well as towards clinical isolates expressing lower levels of dltA. We conclude that modulation of cell envelope d-alanylation may help S. aureus to persist on human skin through evasion of cutaneous innate defense provided by cationic skin-derived AMPs.
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Wall teichoic acid structure governs horizontal gene transfer between major bacterial pathogens.
Nat Commun
PUBLISHED: 01-28-2013
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Mobile genetic elements (MGEs) encoding virulence and resistance genes are widespread in bacterial pathogens, but it has remained unclear how they occasionally jump to new host species. Staphylococcus aureus clones exchange MGEs such as S. aureus pathogenicity islands (SaPIs) with high frequency via helper phages. Here we report that the S. aureus ST395 lineage is refractory to horizontal gene transfer (HGT) with typical S. aureus but exchanges SaPIs with other species and genera including Staphylococcus epidermidis and Listeria monocytogenes. ST395 produces an unusual wall teichoic acid (WTA) resembling that of its HGT partner species. Notably, distantly related bacterial species and genera undergo efficient HGT with typical S. aureus upon ectopic expression of S. aureus WTA. Combined with genomic analyses, these results indicate that a glycocode of WTA structures and WTA-binding helper phages permits HGT even across long phylogenetic distances thereby shaping the evolution of Gram-positive pathogens.
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Role of N-terminal protein formylation in central metabolic processes in Staphylococcus aureus.
BMC Microbiol.
PUBLISHED: 01-11-2013
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Bacterial protein biosynthesis usually depends on a formylated methionyl start tRNA but Staphylococcus aureus is viable in the absence of Fmt, the tRNAMet formyl transferase. fmt mutants exhibit reduced growth rates indicating that the function of certain proteins depends on formylated N-termini but it has remained unclear, which cellular processes are abrogated by the lack of formylation.
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Staphylococcus aureus skin colonization is promoted by barrier disruption and leads to local inflammation.
Exp. Dermatol.
PUBLISHED: 01-03-2013
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Experimental mouse models of bacterial skin infections that have been described show that pathogenic microorganisms can readily invade the epidermis and dermis to produce localized infections. We used an epicutaneous mouse skin infection model to determine how the level of barrier disruption by tape-stripping correlates with persistence of Staphylococcus aureus skin colonization, concomitant induction of cutaneous inflammation and infection. Furthermore, we investigated how murine skin responds to S. aureus colonization in a physiologic setting by analysing proinflammatory cytokines and antimicrobial peptides in mouse skin. We show that previous cutaneous damage allows skin inflammation to develop and favours S. aureus persistence leading to cutaneous colonization, suggesting an interdependence of cutaneous bacteria and skin. Our study suggests that skin barrier defects favour S. aureus skin colonization, which is associated with profound cutaneous inflammation.
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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.
PLoS ONE
PUBLISHED: 01-01-2013
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Previous 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).
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Highly efficient Staphylococcus carnosus mutant selection system based on suicidal bacteriocin activation.
Appl. Environ. Microbiol.
PUBLISHED: 12-16-2011
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Strains from various staphylococcal species produce bacteriocin peptides, which are thought to play important roles in bacterial competition and offer interesting biotechnological avenues. Many bacteriocins are secreted as inactive prepeptides with subsequent activation by specific proteolytic cleavage. By deletion of the protease gene gdmP in Staphylococcus gallinarum Tü3928, which produces the highly active lanthionine-containing bacteriocin gallidermin (lantibiotic), a strain was created producing inactive pregallidermin. On this basis, a new suicidal mutant selection system in the food-grade bacterium Staphylococcus carnosus was developed. Whereas pregallidermin was inactive against S. carnosus, it exerted potent bactericidal activity toward GdmP-secreting S. carnosus strains. To take advantage of this effect, gdmP was cloned in plasmid vectors used for random transposon mutagenesis or targeted allelic replacement of chromosomal genes. Both mutagenesis strategies rely on rare recombination events, and it has remained difficult and laborious to identify mutants among a vast majority of bacterial clones that still contain the delivery vectors. The gdmP-expressing plasmids pGS1 and pGS2 enabled very fast, easy, and reliable identification of transposon and gene replacement mutants, respectively. Mutant selection in the presence of pregallidermin caused suicidal inactivation of all clones that had retained the plasmids and allowed growth of only plasmid-cured mutants. Efficiency of mutant identification was several magnitudes higher than standard screening for the absence of plasmid-encoded antibiotic resistance markers and reached 100% specificity. Thus, the new pregallidermin-based mutant selection system represents a substantial improvement of staphylococcal mutagenesis methodology.
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Exometabolome analysis identifies pyruvate dehydrogenase as a target for the antibiotic triphenylbismuthdichloride in multiresistant bacterial pathogens.
J. Biol. Chem.
PUBLISHED: 12-05-2011
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The desperate need for new therapeutics against notoriously antibiotic-resistant bacteria has led to a quest for novel antibacterial target structures and compounds. Moreover, defining targets and modes of action of new antimicrobial compounds remains a major challenge with standard technologies. Here we characterize the antibacterial properties of triphenylbismuthdichloride (TPBC), which has recently been successfully used against device-associated infections. We demonstrate that TPBC has potent antimicrobial activity against many bacterial pathogens. Using an exometabolome profiling approach, a unique TPBC-mediated change in the metabolites of Staphylococcus aureus was identified, indicating that TPBC blocks bacterial pyruvate catabolism. Enzymatic studies showed that TPBC is a highly efficient, uncompetitive inhibitor of the bacterial pyruvate dehydrogenase complex. Our study demonstrates that metabolomics approaches can offer new avenues for studying the modes of action of antimicrobial compounds, and it indicates that inhibition of the bacterial pyruvate dehydrogenase complex may represent a promising strategy for combating multidrug-resistant bacteria.
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Formylated peptides are important virulence factors in Staphylococcus aureus arthritis in mice.
J. Infect. Dis.
PUBLISHED: 11-18-2011
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Staphylococcus aureus is the most common pathogen causing septic arthritis in humans. The affected joints are often rapidly and permanently damaged despite antibiotic treatment, indicating that the elicited host immune response contributes substantially to joint destruction. Bacterial formylated peptides are important chemotactic molecules mediating neutrophil recruitment into infected tissues as an important first step of host defense against invading bacteria. The role of formylated peptides in S. aureus infections has been unknown.
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New role of the disulfide stress effector YjbH in ?-lactam susceptibility of Staphylococcus aureus.
Antimicrob. Agents Chemother.
PUBLISHED: 09-26-2011
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Staphylococcus aureus is exposed to multiple antimicrobial compounds, including oxidative burst products and antibiotics. The various mechanisms and regulatory pathways governing susceptibility or resistance are complex and only superficially understood. Bacillus subtilis recently has been shown to control disulfide stress responses by the thioredoxin-related YjbH protein, which binds to the transcriptional regulator Spx and controls its degradation via the proteasome-like ClpXP protease. We show that the S. aureus YjbH homolog has a role in susceptibility to the disulfide stress-inducing agent diamide that is similar to that in B. subtilis, and we demonstrate that the four cysteine residues in YjbH are required for this activity. In addition, the inactivation of YjbH led to moderate resistance to oxacillin and other ?-lactam antibiotics, and this phenotypic change was associated with higher penicillin-binding protein 4 levels and increased peptidoglycan cross-linking. Of note, the impact of YjbH on ?-lactam susceptibility still was observed when the four cysteines of YjbH were mutated, indicating that the roles of YjbH in disulfide stress and ?-lactam resistance rely on different types of interactions. These data suggest that the ClpXP adaptor YjbH has more target proteins than previously thought, and that oxidative burst and ?-lactam resistance mechanisms of S. aureus are closely linked.
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In vitro activity against Staphylococcus aureus of a novel antimicrobial agent, PRF-119, a recombinant chimeric bacteriophage endolysin.
Antimicrob. Agents Chemother.
PUBLISHED: 07-11-2011
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Antistaphylococcal activity of the novel chimeric endolysin PRF-119 was evaluated with the microdilution method. The MIC(50) and MIC(90) of 398 methicillin-susceptible Staphylococcus aureus isolates were 0.098 ?g/ml and 0.391 ?g/ml, respectively (range, 0.024 to 0.780 ?g/ml). Both the MIC(50) and MIC(90) values of 776 methicillin-resistant S. aureus isolates were 0.391 ?g/ml (range, 0.024 to 1.563 ?g/ml). All 192 clinical isolates of coagulase-negative staphylococci exhibited MIC values of >50 ?g/ml. In conclusion, PRF-119 exhibited very good activity specifically against S. aureus.
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The virulence regulator Agr controls the staphylococcal capacity to activate human neutrophils via the formyl peptide receptor 2.
J Innate Immun
PUBLISHED: 07-06-2011
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The Agr quorum-sensing system represents the master regulator for staphylococcal virulence factors and is known to have a strong impact on the release of pathogen-associated molecular pattern (PAMP) molecules. Among the various staphylococcal PAMPs, phenol-soluble modulin (PSM) peptides have attracted increasing interest because they are crucial for staphylococcal virulence and have neutrophil-recruiting properties. The latter depend on recognition of PSMs by the neutrophil formyl peptide receptor 2 (FPR2/ALX), for which PSMs are highly efficient agonists. We demonstrate that Agr inactivation in Staphylococcus aureus or S. epidermidis leads to strongly reduced neutrophil responses, which is in agreement with the previously reported strict control of PSM expression by Agr. Agr had a distinct and profound impact on activation of FPR2/ALX but not of the related FPR1 receptor that senses bacterial formylated peptides. S. epidermidis PSMs had similar FPR2/ALX-activating properties but differed in their dependence on N-terminal formylation compared to S. aureus PSMs. Moreover, S. aureus and S. epidermidis PSMs upregulated the neutrophil complement receptor CD11b via FPR2/ALX stimulation in an Agr-dependent fashion. Hence, Agr controls the capacity of staphylococcal pathogens to activate FPR2/ALX-dependent neutrophil responses, underscoring the crucial role of FPR2/ALX and PSMs in staphylococcus-host interaction.
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Wall teichoic Acid-dependent adsorption of staphylococcal siphovirus and myovirus.
J. Bacteriol.
PUBLISHED: 06-03-2011
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The molecular interactions between staphylococcal phages and host cell surfaces are poorly understood. Employing Staphylococcus aureus teichoic acid mutants, we demonstrate that wall teichoic acid (WTA), but not lipoteichoic acid, serves as a receptor for staphylococcal siphovirus and myovirus, while only the siphovirus requires glycosylated WTA.
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Does Staphylococcus aureus nasal colonization involve biofilm formation?
Future Microbiol
PUBLISHED: 05-19-2011
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The human anterior nares are used by Staphylococcus aureus as the major colonization site in 20-30% of the human population. Eradication of S. aureus carriage can significantly reduce the numbers of nosocomial infections. However, the interactions governing the colonization process have remained elusive and it has been debated whether S. aureus adopts a biofilm-like state in the nose. We summarize recent studies on staphylococcal living conditions during nasal colonization, which favour a dispersed rather than a biofilm-related mode of growth during S. aureus nasal colonization. This notion is of major importance for future directions in the development of new decolonization strategies.
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Bacterial resistance mechanisms against host defense peptides.
Cell. Mol. Life Sci.
PUBLISHED: 04-19-2011
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Host defense peptides and proteins are important components of the innate host defense against pathogenic microorganisms. They target negatively charged bacterial surfaces and disrupt microbial cytoplasmic membranes, which ultimately leads to bacterial destruction. Throughout evolution, pathogens devised several mechanisms to protect themselves from deleterious damage of host defense peptides. These strategies include (a) inactivation and cleavage of host defense peptides by production of host defense binding proteins and proteases, (b) repulsion of the peptides by alteration of pathogens surface charge employing modifications by amino acids or amino sugars of anionic molecules (e.g., teichoic acids, lipid A and phospholipids), (c) alteration of bacterial membrane fluidity, and (d) expulsion of the peptides using multi drug pumps. Together with bacterial regulatory network(s) that regulate expression and activity of these mechanisms, they represent attractive targets for development of novel antibacterials.
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Multiple peptide resistance factor (MprF)-mediated Resistance of Staphylococcus aureus against antimicrobial peptides coincides with a modulated peptide interaction with artificial membranes comprising lysyl-phosphatidylglycerol.
J. Biol. Chem.
PUBLISHED: 04-07-2011
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Modification of the membrane lipid phosphatidylglycerol (PG) of Staphylococcus aureus by enzymatic transfer of a l-lysine residue leading to lysyl-PG converts the net charge of PG from -1 to +1 and is thought to confer resistance to cationic antimicrobial peptides (AMPs). Lysyl-PG synthesis and translocation to the outer leaflet of the bacterial membrane are achieved by the membrane protein MprF. Consequently, mutants lacking a functional mprF gene are in particular vulnerable to the action of AMPs. Hence, we aim at elucidating whether and to which extent lysyl-PG modulates membrane binding, insertion, and permeabilization by various AMPs. Lysyl-PG was incorporated into artificial lipid bilayers, mimicking the cytoplasmic membrane of S. aureus. Moreover, we determined the activity of the peptides against a clinical isolate of S. aureus strain SA113 and two mutants lacking a functional mprF gene and visualized peptide-induced ultrastructural changes of bacteria by transmission electron microscopy. The studied peptides were: (i) NK-2, an ?-helical fragment of mammalian NK-lysin, (ii) arenicin-1, a lugworm ?-sheet peptide, and (iii) bee venom melittin. Biophysical data obtained by FRET spectroscopy, Fourier transform infrared spectroscopy, and electrical measurements with planar lipid bilayers were correlated with the biological activities of the peptides. They strongly support the hypothesis that peptide-membrane interactions are a prerequisite for eradication of S. aureus. However, degree and mode of modulation of membrane properties such as fluidity, capacitance, and conductivity were unique for each of the peptides. Altogether, our data support and underline the significance of lysyl-PG for S. aureus resistance to AMPs.
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Broad-spectrum antimicrobial peptide resistance by MprF-mediated aminoacylation and flipping of phospholipids.
Mol. Microbiol.
PUBLISHED: 03-01-2011
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Bacteria are frequently exposed to cationic antimicrobial peptides (CAMPs) from eukaryotic hosts (host defence peptides) or from prokaryotic competitors (bacteriocins). However, many bacteria, among them most of the major human pathogens, achieve CAMP resistance by MprF, a unique enzyme that modifies anionic phospholipids with l-lysine or l-alanine thereby introducing positive charges into the membrane surface and reducing the affinity for CAMPs. The lysyl or alanyl groups are derived from aminoacyl tRNAs and are usually transferred to phosphatidylglycerol (PG). Recent studies with MprF from Staphylococcus aureus demonstrated that production of Lys-PG only leads to CAMP resistance when an additional flippase domain of MprF is present that translocates Lys-PG and exposes it at the outer leaflet of the membrane. Thus, MprF exerts two specific functions that have hardly been found in other bacterial proteins. MprF proteins are crucial virulence factors of many human pathogens, which recommends them as targets for new anti-virulence drugs. Intriguingly, specific point mutations in mprF cause resistance to the CAMP-like antibiotic daptomycin in a yet unclear way that may involve altered Lys-PG synthesis and/or Lys-PG flipping capacities. Thus, a thorough characterization of MprF domains and functions will help to unravel how bacteria maintain and protect their cytoplasmic membranes.
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Neutrophil responses to staphylococcal pathogens and commensals via the formyl peptide receptor 2 relates to phenol-soluble modulin release and virulence.
FASEB J.
PUBLISHED: 12-23-2010
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The mechanisms used by the immune system to discriminate between pathogenic and commensal bacteria have remained largely unclear. Recently, we have shown that virulence of Staphylococcus aureus depends on secretion of phenol-soluble modulin (PSM) peptides that disrupt neutrophils at micromolar concentrations. Moreover, all S. aureus PSMs stimulate and attract neutrophils at nanomolar concentrations via interaction with the formyl-peptide receptor 2 (FPR2). Here, we demonstrate that FPR2 allows neutrophils to adjust their responses in relation to the aggressiveness of staphylococcal species, which differ largely in their capacity to infect or colonize humans and animals. PSM-related peptides were detected in all human and animal pathogenic staphylococci, but were absent from most commensal species. Three PSM?-like peptides produced by the serious human pathogen Staphylococcus lugdunensis were identified as the previously described S. lugdunensis-synergistic hemolysins (SLUSHs). SLUSHs attracted and stimulated human leukocytes in a FPR2-dependent manner, indicating that FPR2 is a general receptor for all PSM-like peptide toxins. Remarkably, the release of PSMs correlated closely with the apparent capacity of staphylococcal species to cause invasive infections and with their ability to activate FPR2. These findings suggest that the innate immune system may be able to respond in different ways to pathogenic or innocuous staphylococci by monitoring the presence of PSMs via FPR2.
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Pyruvate formate lyase acts as a formate supplier for metabolic processes during anaerobiosis in Staphylococcus aureus.
J. Bacteriol.
PUBLISHED: 12-17-2010
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Previous studies demonstrated an upregulation of pyruvate formate lyase (Pfl) and NAD-dependent formate dehydrogenase (Fdh) in Staphylococcus aureus biofilms. To investigate their physiological role, we constructed fdh and pfl deletion mutants (?fdh and ?pfl). Although formate dehydrogenase activity in the fdh mutant was lost, it showed little phenotypic alterations under oxygen-limited conditions. In contrast, the pfl mutant displayed pleiotropic effects and revealed the importance of formate production for anabolic metabolism. In the pfl mutant, no formate was produced, glucose consumption was delayed, and ethanol production was decreased, whereas acetate and lactate production were unaffected. All metabolic alterations could be restored by addition of formate or complementation of the ?pfl mutant. In compensation reactions, serine and threonine were consumed better by the ?pfl mutant than by the wild type, suggesting that their catabolism contributes to the refilling of formyl-tetrahydrofolate, which acts as a donor of formyl groups in, e.g., purine and protein biosynthesis. This notion was supported by reduced production of formylated peptides by the ?pfl mutant compared to that of the parental strain, as demonstrated by weaker formyl-peptide receptor 1 (FPR1)-mediated activation of leukocytes with the mutant. FPR1 stimulation could also be restored either by addition of formate or by complementation of the mutation. Furthermore, arginine consumption and arc operon transcription were increased in the ?pfl mutant. Unlike what occurred with the investigated anaerobic conditions, a biofilm is distinguished by nutrient, oxygen, and pH gradients, and we thus assume that Pfl plays a significant role in the anaerobic layer of a biofilm. Fdh might be critical in (micro)aerobic layers, as formate oxidation is correlated with the generation of NADH/H(+), whose regeneration requires respiration.
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Skin commensals amplify the innate immune response to pathogens by activation of distinct signaling pathways.
J. Invest. Dermatol.
PUBLISHED: 11-04-2010
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Little is known about the impact of different microbial signals on skin barrier organ function and the interdependency between resident microflora and pathogenic microorganisms. This study shows that commensal and pathogenic staphylococci differ in their ability to induce expression of antimicrobial peptides/proteins (AMPs) and activate different signaling pathways in human primary keratinocytes. Whereas secreted factors of skin commensals induce expression of the AMPs HBD-3 and RNase7 in primary human keratinocytes via Toll-like receptor (TLR)-2, EGFR, and NF-?B activation, those of pathogenic staphylococci activate the mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT signaling pathways and suppress NF-?B activation. Interestingly, commensal bacteria are able to amplify the innate immune response of human keratinocytes to pathogens by increased induction of AMP expression and abrogation of NF-?B suppression, suggesting that the two activation pathways can act in a synergistic way. These data indicate that commensal and pathogenic microorganisms evolved specific mechanisms to modulate innate immunity of the skin.
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Natural Staphylococcus aureus-derived peptidoglycan fragments activate NOD2 and act as potent costimulators of the innate immune system exclusively in the presence of TLR signals.
FASEB J.
PUBLISHED: 06-03-2010
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Innate immune sensing of Staphylococcus aureus unravels basic mechanisms leading to either effective antibacterial immune responses or harmful inflammation. The nature and properties of S. aureus-derived pathogen-associated molecular pattern (PAMPs) are still not completely understood. We investigated the innate immune sensing of peptidoglycan (PGN) structures and subsequent immune consequences. Macromolecular PGN (PGN(polymer)) preparations activated NF-?B through human Toll-like receptors 2 (TLR2), as shown by luciferase reporter assays, and induced murine dendritic cell (DC) maturation and cytokine production. In contrast, PGN(polymer) from lgt-mutant S. aureus failed to stimulate human TLR2, demonstrating that lipoproteins within the macromolecular structures of PGN(polymer), but not PGN itself, activate TLR2. Thus, HPLC-purified monomeric PGN (PGN(monomer)) structures were investigated. Strikingly, PGN(monomer) completely lacked NF-?B activation, lacked TLR2 activity, and failed to functionally activate murine DCs. However, PGN(monomer) in concert with various TLR ligands most effectively stimulated DCs to up-regulate IL-12p70 and IL-23 by ?3- to 5-fold. Consequently, DCs coactivated by PGN(monomer) markedly up-regulated Th1 and Th17 while suppressing Th2 cell priming. Notably, PGN(monomer) failed to coactivate NOD2(-/-) DCs. This demonstrates that PGN(monomer) is a natural ligand of NOD2, which was previously only demonstrated for synthetic compounds like muramyl dipeptide. Interestingly, murine DCs lacking TLR2 remained mute in response to the combinative immune sensing of S. aureus-derived PAMPs, including PGN(monomer), providing for the first time an explanation of why S. aureus can colonize the nasal mucosa in the absence of inflammation. This is very likely based on the lack of TLR2 expression in mucosal epithelial cells under normal conditions, which determines the unresponsiveness to S. aureus PAMPs.
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Human formyl peptide receptor 2 senses highly pathogenic Staphylococcus aureus.
Cell Host Microbe
PUBLISHED: 04-09-2010
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Virulence of emerging community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) and other highly pathogenic S. aureus strains depends on their production of phenol-soluble modulin (PSM) peptide toxins, which combine the capacities to attract and lyse neutrophils. The molecular basis of PSM-stimulated neutrophil recruitment has remained unclear. Here, we demonstrate that the human formyl peptide receptor 2 (FPR2/ALX), which has previously been implicated in control of endogenous inflammatory processes, senses PSMs at nanomolar concentrations and initiates proinflammatory neutrophil responses to CA-MRSA. Specific blocking of FPR2/ALX or deletion of PSM genes in CA-MRSA severely diminished neutrophil detection of CA-MRSA. Furthermore, a specific inhibitor of FPR2/ALX and of its functional mouse counterpart blocked PSM-mediated leukocyte infiltration in vivo in a mouse model. Thus, the innate immune system uses a distinct FPR2/ALX-dependent mechanism to specifically sense bacterial peptide toxins and detect highly virulent bacterial pathogens. FPR2/ALX represents an attractive target for new anti-infective or anti-inflammatory strategies.
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Temporal expression of adhesion factors and activity of global regulators during establishment of Staphylococcus aureus nasal colonization.
J. Infect. Dis.
PUBLISHED: 03-24-2010
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The human pathogen Staphylococcus aureus successfully colonizes its primary reservoir, the nasal cavity, most likely by regulatory adaptation to the nose environment. Cotton rats represent an excellent model for the study of bacterial gene expression in the initial phases of colonization. To gain insight into the expression profile necessary for the establishment of colonization, we performed direct transcript analysis by quantitative real-time reverse-transcription polymerase chain reaction on cotton rat noses removed from euthanized animals on days 1, 4, or 10 after instillation of 2 human S. aureus nose isolates. Global virulence regulators (agr, sae) were not active in this early phase, but the essential 2-component regulatory system WalKR seems to play an important role. Accordingly, an elevated expression of walKR target genes (sak, sceD) could be detected. In agreement with previous studies that demonstrated the essential role played by wall teichoic acid (WTA) polymers in nasal colonization, we detected a strongly increased expression of WTA-biosynthetic genes. The expression profile switched to production of the adhesive proteins ClfB and IsdA at later stages of the colonization process. These data underscore the temporal differences in the roles of WTA and surface proteins in nasal colonization, and they provide the first evidence for a regulation of WTA biosynthesis in vivo.
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Activation of hypoxia inducible factor 1 is a general phenomenon in infections with human pathogens.
PLoS ONE
PUBLISHED: 03-11-2010
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Hypoxia inducible factor (HIF)-1 is the key transcriptional factor involved in the adaptation process of cells and organisms to hypoxia. Recent findings suggest that HIF-1 plays also a crucial role in inflammatory and infectious diseases.
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Glycosylation of wall teichoic acid in Staphylococcus aureus by TarM.
J. Biol. Chem.
PUBLISHED: 02-25-2010
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Wall teichoic acid (WTA) glycopolymers are major constituents of cell envelopes in Staphylococcus aureus and related gram-positive bacteria with important roles in cell wall maintenance, susceptibility to antimicrobial molecules, biofilm formation, and host interaction. Most S. aureus strains express polyribitol phosphate WTA substituted with D-alanine and N-acetylglucosamine (GlcNAc). WTA sugar modifications are highly variable and have been implicated in bacteriophage susceptibility and immunogenicity, but the pathway and enzymes of staphylococcal WTA glycosylation have remained unknown. Revisiting the structure of S. aureus RN4220 WTA by NMR analysis revealed the presence of canonical polyribitol phosphate WTA bearing only alpha-linked GlcNAc substituents. A RN4220 transposon mutant resistant to WTA-dependent phages was identified and shown to produce altered WTA, which exhibited faster electrophoretic migration and lacked completely the WTA alpha-GlcNAc residues. Disruption of a gene of unknown function, renamed tarM, was responsible for this phenotype. Recombinant TarM was capable of glycosylating WTA in vitro in a UDP-GlcNAc-dependent manner, thereby confirming its WTA GlcNAc-transferase activity. Deletion of the last seven amino acids from the C terminus abolished the activity of TarM. tarM-related genes were found in the genomes of several WTA-producing bacteria, suggesting that TarM-mediated WTA glycosylation is a general pathway in gram-positive bacteria. Our study represents a basis for dissecting the biosynthesis and function of glycosylated WTA in S. aureus and other bacteria.
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Changing the phospholipid composition of Staphylococcus aureus causes distinct changes in membrane proteome and membrane-sensory regulators.
Proteomics
PUBLISHED: 02-18-2010
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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.
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Vibrational and electronic characterisation of Staphylococcus aureus wall teichoic acids and relevant components in thin films.
Anal Bioanal Chem
PUBLISHED: 01-28-2010
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This work reports an investigation of S. aureus wall teichoic acid (WTA) and compares this biopolymer with its major occurring components, D: -alanine and glycerol phosphate. Detailed insight into molecular structures and electronic properties is obtained by vibrational and photoemission spectroscopy. Calculations are performed to support the analysis of our experimental vibrational spectra. It is shown that there are contributions of positive and negative charges in WTAs, but the number of negative charges is expected to be higher. The presence of both positive and negative charges on WTA may offer a route for modification of surfaces with the objective of avoiding the formation of biofilms.
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Formyl peptide receptor-mediated proinflammatory consequences of peptide deformylase inhibition in Staphylococcus aureus.
Microbes Infect.
PUBLISHED: 01-28-2010
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The biosynthesis of proteins with N-terminal formylated methionine residues and subsequent protein deformylation are unique and invariant bacterial processes. They are exploited by the capacity of the human innate immune system to sense formylated peptides (FPs) and targeted by the deformylation-blocking antibiotic actinonin. We show that human polymorphonuclear leukocytes respond via the formyl peptide receptor (FPR) with increased calcium ion fluxes, chemotactic migration, IL-8 release, and CD11b upregulation to the human pathogen Staphylococcus aureus upon actinonin treatment. These data underscore the crucial role of bacterial FPs in innate immunity and indicate that deformylase inhibition may have considerable proinflammatory consequences.
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Role of staphylococcal wall teichoic acid in targeting the major autolysin Atl.
Mol. Microbiol.
PUBLISHED: 01-25-2010
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Staphylococcal cell separation depends largely on the bifunctional autolysin Atl that is processed to amidase-R(1,2) and R(3)-glucosaminidase. These murein hydrolases are targeted via repeat domains (R) to the septal region of the cell surface, thereby allowing localized peptidoglycan hydrolysis and separation of the dividing cells. Here we show that targeting of the amidase repeats is based on an exclusion strategy mediated by wall teichoic acid (WTA). In Staphylococcus aureus wild-type, externally applied repeats (R(1,2)) or endogenously expressed amidase were localized exclusively at the cross-wall region, while in Delta tagO mutant that lacks WTA binding was evenly distributed on the cell surface, which explains the increased fragility and autolysis susceptibility of the mutant. WTA prevented binding of Atl to the old cell wall but not to the cross-wall region suggesting a lower WTA content. In binding studies with ConcanavalinA-fluorescein (ConA-FITC) conjugate that binds preferentially to teichoic acids, ConA-FITC was bound throughout the cell surface with the exception of the cross wall. ConA binding suggest that either content or polymerization of WTA gradually increases with distance from the cross-wall. By preventing binding of Atl, WTA directs Atl to the cross-wall to perform the last step of cell division, namely separation of the daughter cells.
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Penetration of the blood-brain barrier by Staphylococcus aureus: contribution of membrane-anchored lipoteichoic acid.
J. Mol. Med.
PUBLISHED: 01-05-2010
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Staphylococcus aureus is one of the most prevalent organisms responsible for nosocomial infections, and cases of community-acquired S. aureus infection have continued to increase despite widespread preventative measures. Pathologies attributed to S. aureus infection are diverse; ranging from dermal lesions to bacteremia, abscesses, and endocarditis. Reported cases of S. aureus-associated meningitis and brain abscesses have also increased in recent years, however, the precise mechanism whereby S. aureus leave the bloodstream and gain access to the central nervous system (CNS) are not known. Here we demonstrate for the first time that S. aureus efficiently adheres to and invades human brain microvascular endothelial cells (hBMEC), the single-cell layer which constitutes the blood-brain barrier (BBB). The addition of cytochalasin D, an actin microfilament aggregation inhibitor, strongly reduced bacterial invasion, suggesting an active hBMEC process is required for efficient staphylococcal uptake. Furthermore, mice injected with S. aureus exhibited significant levels of brain bacterial counts and histopathologic evidence of meningeal inflammation and brain abscess formation, indicating that S. aureus was able to breech the BBB in an experimental model of hematogenous meningitis. We found that a YpfP-deficient mutant, defective in lipoteichoic acid (LTA) membrane anchoring, exhibited a decreased ability to invade hBMEC and correlated to a reduced risk for the development of meningitis in vivo. Our results demonstrate that LTA-mediated penetration of the BBB may be a primary step in the pathogenesis of staphylococcal CNS disease.
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The wall teichoic acid and lipoteichoic acid polymers of Staphylococcus aureus.
Int. J. Med. Microbiol.
PUBLISHED: 11-06-2009
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Staphylococci and most other Gram-positive bacteria incorporate complex teichoic acid (TA) polymers into their cell envelopes. Several crucial roles in Staphylococcus aureus fitness and cell wall maintenance have been assigned to these polymers, which are either covalently linked to peptidoglycan (wall teichoic acid, WTA) or to the cytoplasmic membrane (lipoteichoic acid, LTA). However, the exact TA structures, functions, and biosynthetic pathways are only superficially understood. Recently, most of the enzymes mediating TA biosynthesis have been identified and mutants lacking or with defined changes in WTA or LTA have become available. Their characterization has revealed crucial roles of TAs in protection against harmful molecules and environmental stresses; in control of enzymes directing cell division or morphogenesis and of cation homeostasis; and in interaction with host or bacteriophage receptors and biomaterials. Accordingly, several in vivo studies have demonstrated the importance of WTA and LTA in S. aureus colonization, infection, and immune evasion. TAs and enzymes required for TA biosynthesis represent attractive candidates for novel vaccines and antibiotics and are targeted by recently developed antibacterial therapeutics.
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Manipulation of charged residues within the two-peptide lantibiotic lacticin 3147.
Microb Biotechnol
PUBLISHED: 08-23-2009
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Lantibiotics are antimicrobial peptides which contain a high percentage of post-translationally modified residues. While most attention has been paid to the role of these critical structural features, evidence continues to emerge that charged amino acids also play a key role in these peptides. Here 16 charge mutants of the two-peptide lantibiotic lacticin 3147 [composed of Ltn? (2+, 2-) and Ltn? (2+)] were constructed which, when supplemented with previously generated peptides, results in a total bank of 23 derivatives altered in one or more charged residues. When examined individually, in combination with a wild-type partner or, in some instances, in combination with one another, these mutants reveal the importance of charge at specific locations within Ltn? and Ltn?, confirm the critical role of the negatively charged glutamate residue in Ltn? and facilitate an investigation of the contribution of positively charged residues to the cationic Ltn?. From these investigations it is also apparent that the relative importance of the overall charge of lacticin 3147 varies depending on the target bacteria and is most evident when strains with more negatively charged cell envelopes are targeted. These studies also result in, for the first time, the creation of a derivative of a lacticin 3147 peptide (Ltn?R27A) which displays enhanced specific activity.
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Neutrophil antimicrobial defense against Staphylococcus aureus is mediated by phagolysosomal but not extracellular trap-associated cathelicidin.
J. Leukoc. Biol.
PUBLISHED: 07-28-2009
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Neutrophils kill invading pathogens by AMPs, including cathelicidins, ROS, and NETs. The human pathogen Staphylococcus aureus exhibits enhanced resistance to neutrophil AMPs, including the murine cathelicidin CRAMP, in part, as a result of alanylation of teichoic acids by the dlt operon. In this study, we took advantage of the hypersusceptible phenotype of S. aureus DeltadltA against cationic AMPs to study the impact of the murine cathelicidin CRAMP on staphylococcal killing and to identify its key site of action in murine neutrophils. We demonstrate that CRAMP remained intracellular during PMN exudation from blood and was secreted upon PMA stimulation. We show first evidence that CRAMP was recruited to phagolysosomes in infected neutrophils and exhibited intracellular activity against S. aureus. Later in infection, neutrophils produced NETs, and immunofluorescence revealed association of CRAMP with S. aureus in NETs, which similarly killed S. aureus wt and DeltadltA, indicating that CRAMP activity was reduced when associated with NETs. Indeed, the presence of DNA reduced the antimicrobial activity of CRAMP, and CRAMP localization in response to S. aureus was independent of the NADPH oxidase, whereas killing was partially dependent on a functional NADPH oxidase. Our study indicates that neutrophils use CRAMP in a timed and locally coordinated manner in defense against S. aureus.
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Staphylococcus aureus mutant screen reveals interaction of the human antimicrobial peptide dermcidin with membrane phospholipids.
Antimicrob. Agents Chemother.
PUBLISHED: 07-13-2009
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Antimicrobial peptides (AMPs) form an important part of the innate host defense. In contrast to most AMPs, human dermcidin has an anionic net charge. To investigate whether bacteria have developed specific mechanisms of resistance to dermcidin, we screened for mutants of the leading human pathogen, Staphylococcus aureus, with altered resistance to dermcidin. To that end, we constructed a plasmid for use in mariner-based transposon mutagenesis and developed a high-throughput cell viability screening method based on luminescence. In a large screen, we did not find mutants with strongly increased susceptibility to dermcidin, indicating that S. aureus has no specific mechanism of resistance to this AMP. Furthermore, we detected a mutation in a gene of unknown function that resulted in significantly increased resistance to dermcidin. The mutant strain had an altered membrane phospholipid pattern and showed decreased binding of dermcidin to the bacterial surface, indicating that dermcidin interacts with membrane phospholipids. The mode of this interaction was direct, as shown by assays of dermcidin binding to phospholipid preparations, and specific, as the resistance to other AMPs was not affected. Our findings indicate that dermcidin has an exceptional value for the human innate host defense and lend support to the idea that it evolved to evade bacterial resistance mechanisms targeted at the cationic character of most AMPs. Moreover, they suggest that the antimicrobial activity of dermcidin is dependent on the interaction with the bacterial membrane and might thus assist with the determination of the yet unknown mode of action of this important human AMP.
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Wall teichoic acid protects Staphylococcus aureus against antimicrobial fatty acids from human skin.
J. Bacteriol.
PUBLISHED: 05-08-2009
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Skin-colonizing gram-positive bacteria produce wall teichoic acids (WTAs) or related glycopolymers for unclear reasons. Using a WTA-deficient Staphylococcus aureus mutant, we demonstrated that WTA confers resistance to antimicrobial fatty acids from human sebaceous glands by preventing fatty acid binding. Thus, WTA is probably important for bacterial skin colonization.
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Dermcidin-derived peptides show a different mode of action than the cathelicidin LL-37 against Staphylococcus aureus.
Antimicrob. Agents Chemother.
PUBLISHED: 04-13-2009
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Dermcidin (DCD) is an antimicrobial peptide which is constitutively expressed in eccrine sweat glands. By postsecretory proteolytic processing in sweat, the DCD protein gives rise to anionic and cationic DCD peptides with a broad spectrum of antimicrobial activity. Many antimicrobial peptides induce membrane permeabilization as part of their killing mechanism, which is accompanied by a loss of the bacterial membrane potential. In this study we show that there is a time-dependent bactericidal activity of anionic and cationic DCD-derived peptides which is followed by bacterial membrane depolarization. However, DCD-derived peptides do not induce pore formation in the membranes of gram-negative and gram-positive bacteria. This is in contrast to the mode of action of the cathelicidin LL-37. Interestingly, LL-37 as well as DCD-derived peptides inhibit bacterial macromolecular synthesis, especially RNA and protein synthesis, without binding to microbial DNA or RNA. Binding studies with components of the cell envelope of gram-positive and gram-negative bacteria and with model membranes indicated that DCD-derived peptides bind to the bacterial envelope but show only a weak binding to lipopolysaccharide (LPS) from gram-negative bacteria or to peptidoglycan, lipoteichoic acid, and wall teichoic acid, isolated from Staphylococcus aureus. In contrast, LL-37 binds strongly in a dose-dependent fashion to these components. Altogether, these data indicate that the mode of action of DCD-derived peptides is different from that of the cathelicidin LL-37 and that components of the bacterial cell envelope play a role in the antimicrobial activity of DCD.
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Mobile genetic element-encoded cytolysin connects virulence to methicillin resistance in MRSA.
PLoS Pathog.
PUBLISHED: 04-03-2009
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Bacterial virulence and antibiotic resistance have a significant influence on disease severity and treatment options during bacterial infections. Frequently, the underlying genetic determinants are encoded on mobile genetic elements (MGEs). In the leading human pathogen Staphylococcus aureus, MGEs that contain antibiotic resistance genes commonly do not contain genes for virulence determinants. The phenol-soluble modulins (PSMs) are staphylococcal cytolytic toxins with a crucial role in immune evasion. While all known PSMs are core genome-encoded, we here describe a previously unidentified psm gene, psm-mec, within the staphylococcal methicillin resistance-encoding MGE SCCmec. PSM-mec was strongly expressed in many strains and showed the physico-chemical, pro-inflammatory, and cytolytic characteristics typical of PSMs. Notably, in an S. aureus strain with low production of core genome-encoded PSMs, expression of PSM-mec had a significant impact on immune evasion and disease. In addition to providing high-level resistance to methicillin, acquisition of SCCmec elements encoding PSM-mec by horizontal gene transfer may therefore contribute to staphylococcal virulence by substituting for the lack of expression of core genome-encoded PSMs. Thus, our study reveals a previously unknown role of methicillin resistance clusters in staphylococcal pathogenesis and shows that important virulence and antibiotic resistance determinants may be combined in staphylococcal MGEs.
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Regulation of mprF in daptomycin-nonsusceptible Staphylococcus aureus strains.
Antimicrob. Agents Chemother.
PUBLISHED: 03-16-2009
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We used a well-characterized isogenic set of clinical bloodstream Staphylococcus aureus strains to study (i) regulation of mprF-mediated phosphatidylglycerol lysinylation in the contexts of in vitro daptomycin (DAP) nonsuceptibility and (ii) the role of mprF mutation in endovascular virulence. We observed a correlation between increased expression of a mutant mprF gene and reduced in vitro DAP susceptibility. There were no detectable fitness differences between strains in experimental infective endocarditis.
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The bacterial defensin resistance protein MprF consists of separable domains for lipid lysinylation and antimicrobial peptide repulsion.
PLoS Pathog.
PUBLISHED: 03-13-2009
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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.
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Methicillin resistance in Staphylococcus aureus requires glycosylated wall teichoic acids.
Proc. Natl. Acad. Sci. U.S.A.
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Staphylococcus aureus peptidoglycan (PG) is densely functionalized with anionic polymers called wall teichoic acids (WTAs). These polymers contain three tailoring modifications: d-alanylation, ?-O-GlcNAcylation, and ?-O-GlcNAcylation. Here we describe the discovery and biochemical characterization of a unique glycosyltransferase, TarS, that attaches ?-O-GlcNAc (?-O-N-acetyl-D-glucosamine) residues to S. aureus WTAs. We report that methicillin resistant S. aureus (MRSA) is sensitized to ?-lactams upon tarS deletion. Unlike strains completely lacking WTAs, which are also sensitive to ?-lactams, ?tarS strains have no growth or cell division defects. Because neither ?-O-GlcNAc nor ?-O-Glucose modifications can confer resistance, the resistance phenotype requires a highly specific chemical modification of the WTA backbone, ?-O-GlcNAc residues. These data suggest ?-O-GlcNAcylated WTAs scaffold factors required for MRSA resistance. The ?-O-GlcNAc transferase identified here, TarS, is a unique target for antimicrobials that sensitize MRSA to ?-lactams.
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Proton-binding capacity of Staphylococcus aureus wall teichoic acid and its role in controlling autolysin activity.
PLoS ONE
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Wall teichoic acid (WTA) or related polyanionic cell wall glycopolymers are produced by most gram-positive bacterial species and have been implicated in various cellular functions. WTA and the proton gradient across bacterial membranes are known to control the activity of autolysins but the molecular details of these interactions are poorly understood. We demonstrate that WTA contributes substantially to the proton-binding capacity of Staphylococcus aureus cell walls and controls autolysis largely via the major autolysin AtlA whose activity is known to decline at acidic pH values. Compounds that increase or decrease the activity of the respiratory chain, a main source of protons in the cell wall, modulated autolysis rates in WTA-producing cells but did not affect the augmented autolytic activity observed in a WTA-deficient mutant. We propose that WTA represents a cation-exchanger like mesh in the gram-positive cell envelopes that is required for creating a locally acidified milieu to govern the pH-dependent activity of autolysins.
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Enterococcus faecium stimulates human neutrophils via the formyl-peptide receptor 2.
PLoS ONE
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The human formyl-peptide receptor 2 (FPR2/ALX) senses phenol-soluble modulin (PSM) peptide toxins produced by pathogenic staphylococcal species and plays a crucial role in directing neutrophil influx during staphylococcal infection. However, it has remained unclear if FPR2 responds also to molecules from other bacterial pathogens. Here we analyzed a variety of gram-positive and gram-negative pathogens and found that apart from staphylococci only certain enterococcal strains have the capacity to stimulate FPR2/ALX. Most of the analyzed Enterococcus faecium but only sporadic Enterococcus faecalis strains released FPR2/ALX-stimulating molecules leading to neutrophil calcium ion fluxes, chemotaxis, and complement receptor upregulation. Among ten test strains vancomycin-resistant E. faecium had a significantly higher capacity to stimulate FPR2/ALX than vancomycin-susceptible strains, suggesting an association of strong FPR2/ALX activation with health-care associated strains. The enterococcal FPR2/ALX agonists were found to be peptides or proteins, which appear, however, to be unrelated to staphylococcal PSMs in sequence and physicochemical properties. Enterococci are among the most frequent invasive bacterial pathogens but the basis of enterococcal virulence and immune activation has remained incompletely understood. Our study indicates that previously unrecognized proteinaceous agonists contribute to Enterococcus-host interaction and underscores the importance of FPR2/ALX in host defense against major endogenous bacterial pathogens.
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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.
Antimicrob. Agents Chemother.
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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.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.