staphyloxanthin and Staphylococcal-Infections

The majority of antibiotics currently used to treat methicillin-resistant Staphylococus aureus (MRSA) infections target bacterial cell wall synthesis or protein synthesis. Only daptomycin has a novel mode of action. Reliance on limited targets for MRSA chemotherapy, has contributed to antimicrobial resistance. Two alternative approaches to the treatment of S. aureus infection, particularly those caused by MRSA, that have alternative mechanisms of action and that address the challenge of antimicrobial resistance are cationic host defence peptides and agents that target S. aureus virulence. Cationic host defence peptides have multiple mechanisms of action and are less likely than conventional agents to select resistant mutants. They are amenable to modifications that improve their stability, effectiveness and selectivity. Some cationic defence peptides such as bactenecin, mucroporin and imcroporin have potent in vitro bactericidal activity against MRSA. Antipathogenic agents also have potential to limit the pathogenesis of S aureus. These are generally small molecules that inhibit virulence targets in S. aureus without killing the bacterium and therefore have limited capacity to promote resistance development. Potential antipathogenic targets include the sortase enzyme system, the accessory gene regulator (agr) and the carotenoid biosynthetic pathway. Inhibitors of these targets have been identified and these may have potential for further development.

Topics: Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Bacterial Proteins; Humans; Metabolic Networks and Pathways; Methicillin-Resistant Staphylococcus aureus; Molecular Sequence Data; Molecular Targeted Therapy; Peptides, Cyclic; Porins; Staphylococcal Infections; Trans-Activators; Virulence; Xanthophylls

The rising prevalence of antibiotic resistance in Staphylococcus aureus calls for the development of innovative antimicrobial agents targeting novel pathways. S. aureus generates various virulence factors that compromise host defense mechanisms. Flavone, a core structure of flavonoids, has been shown to diminish the production of staphyloxanthin and alpha-hemolysin. Nonetheless, the influence of flavone on the majority of other virulence factors in S. aureus and its underlying molecular mechanism remain elusive. In this study, we examined the impact of flavone on the transcriptional profile of S. aureus using transcriptome sequencing. Our findings revealed that flavone substantially downregulated the expression of over 30 virulence factors implicated in immune evasion by the pathogen. Gene set enrichment analysis of the fold change-ranked gene list in relation to the Sae regulon indicated a robust association between flavone-induced downregulation and membership in the Sae regulon. Through the analysis of Sae target promoter-gfp fusion expression patterns, we observed a dose-dependent inhibition of Sae target promoter activity by flavone. Moreover, we discovered that flavone protected human neutrophils from S. aureus-mediated killing. Flavone also decreased the expression of alpha-hemolysin and other hemolytic toxins, resulting in a reduction in S. aureus' hemolytic capacity. Additionally, our data suggested that the inhibitory effect of flavone on the Sae system operates independently of its capacity to lower staphyloxanthin levels. In conclusion, our study proposes that flavone exhibits a broad inhibitory action on multiple virulence factors of S. aureus by targeting the Sae system, consequently diminishing the bacterium's pathogenicity.

Topics: Bacterial Proteins; Flavones; Hemolysin Proteins; Humans; Staphylococcal Infections; Staphylococcus aureus; Virulence; Virulence Factors

Topics: Carotenoids; Humans; Oxygen; Phospholipids; Staphylococcal Infections; Staphylococcus aureus

Strain-level variation in Staphylococcus aureus is a factor that contributes to disease burden and clinical outcomes in skin disorders and chronic wounds. However, the microbial mechanisms that drive these variable host responses are poorly understood. To identify mechanisms underlying strain-specific outcomes, we perform high-throughput phenotyping screens on S. aureus isolates cultured from diabetic foot ulcers. Isolates from non-healing wounds produce more staphyloxanthin, a cell membrane pigment. In murine diabetic wounds, staphyloxanthin-producing isolates delay wound closure significantly compared with staphyloxanthin-deficient isolates. Staphyloxanthin promotes resistance to oxidative stress and enhances bacterial survival in neutrophils. Comparative genomic and transcriptomic analysis of genetically similar clinical isolates with disparate staphyloxanthin phenotypes reveals a mutation in the sigma B operon, resulting in marked differences in stress response gene expression. Our work illustrates a framework to identify traits that underlie strain-level variation in disease burden and suggests more precise targets for therapeutic intervention in S. aureus-positive wounds.

Topics: Animals; Diabetes Mellitus; Mice; Staphylococcal Infections; Staphylococcus aureus; Wound Healing

Methicillin resistant Staphylococcus aureus is considered multidrug resistant bacterium due to developing biofilm formation associated with antimicrobial resistance mechanisms. Therefore, inhibition of biofilm formation is an alternative therapeutic action to control MRSA infections. The present study revealed the non-antibacterial biofilm inhibitory potential of hesperidin against ATCC strain and clinical isolates of S. aureus. Hesperidin is a flavanone glycoside commonly found in citrus fruit. Hesperidin has been shown to exhibits numerous pharmacological activities. The present study aimed to evaluate the antibiofilm and antivirulence potential of hesperidin against MRSA. Results showed that hesperidin treatment significantly impedes lipase, hemolysin, autolysin, autoaggregation and staphyloxanthin production. Reductions of staphyloxanthin production possibly increase the MRSA susceptibility rate to H

Topics: Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Farnesyl-Diphosphate Farnesyltransferase; Gene Expression Regulation, Bacterial; Hesperidin; Humans; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Molecular Docking Simulation; Staphylococcal Infections; Trans-Activators; Virulence; Virulence Factors; Xanthophylls

Staphylococcus aureus is a leading cause of human infections. The spread of antibiotic-resistant staphylococci has driven the search for novel strategies to supersede antibiotics use. Thus, targeting bacterial virulence rather than viability could be a possible alternative.. The influence of celastrol on staphyloxanthin (STX) biosynthesis, biofilm formation, antibiotic susceptibility and host pathogenesis in S. aureus has been investigated. Celastrol efficiently reduced STX biosynthesis in S. aureus. Liquid chromatography-mass spectrometry (LC-MS) and molecular docking revealed that celastrol inhibits STX biosynthesis through its effect on CrtM. Quantitative measurement of STX intermediates showed a significant pigment inhibition via interference of celastrol with CrtM and accumulation of its substrate, farnesyl diphosphate. Importantly, celastrol-treated S. aureus was more sensitive to environmental stresses and human blood killing than untreated bacteria. Similarly, inhibition of STX upon celastrol treatment rendered S. aureus more susceptible to membrane targeting antibiotics. In addition to its anti-pigment capability, celastrol exhibits significant anti-biofilm activity against S. aureus as indicated by crystal violet assay and microscopy. Celastrol-treated cells showed deficient exopolysaccharide production and cell hydrophobicity. Moreover, celastrol markedly synergized the action of conventional antibiotics against S. aureus and reduced bacterial pathogenesis in vivo using mice infection model. These findings were further validated using qRT-PCR, demonstrating that celastrol could alter the expression of STX biosynthesis genes as well as biofilm formation related genes and bacterial virulence.. Celastrol is a novel anti-virulent agent against S. aureus suggesting, a prospective therapeutic role for celastrol as a multi-targeted anti-pathogenic agent.

Topics: Animals; Anti-Bacterial Agents; Biofilms; Mice; Molecular Docking Simulation; Pentacyclic Triterpenes; Staphylococcal Infections; Staphylococcus aureus; Virulence; Xanthophylls

Antimicrobial resistance is growing substantially, which necessitates the search for novel therapeutic options. Terbinafine, an allylamine antifungal agent that exhibits a broad spectrum of activity and is used in the treatment of dermatophytosis, could be a possible option to disarm S. aureus virulence.. Terbinafine inhibitory effect on staphyloxanthin was characterized by quantitative measurement of staphyloxanthin intermediates and molecular docking. The effect of terbinafine on S. aureus stress survival was characterized by viable counting. The anti-biofilm activity of terbinafine on S. aureus was assessed by the crystal violet assay and microscopy. Changes in S. aureus membrane following treatment with terbinafine were determined using Fourier transform infrared (FTIR) analysis. The synergistic action of terbinafine in combination with conventional antibiotics was characterized using the checkerboard assay. qRT-PCR was used to evaluate the impact of terbinafine on S. aureus gene expression. The influence of terbinafine on S. aureus pathogenesis was investigated in mice infection model.. Terbinafine inhibits staphyloxanthin biosynthesis through targeting dehydrosqualene desaturase (CrtN). Docking analysis of terbinafine against the predicted active site of CrtN reveals a binding energy of - 9.579 kcal/mol exemplified by the formation of H-bonds, H-arene bonds, and hydrophobic/hydrophilic interactions with the conserved amino acids of the receptor pocket. Terbinafine treated S. aureus was more susceptible to both oxidative and acid stress as well as human blood killing as compared to untreated cells. Targeting staphyloxanthin by terbinafine rendered S. aureus more sensitive to membrane acting antibiotics. Terbinafine interfered with S. aureus biofilm formation through targeting cell autoaggregation, hydrophobicity, and exopolysaccharide production. Moreover, terbinafine demonstrated a synergistic interaction against S. aureus when combined with conventional antibiotics. Importantly, terbinafine attenuated S. aureus pathogenesis using mice infection model. qRT-PCR revealed that terbinafine repressed expression of the transcriptional regulators sigB, sarA, and msaB, as well as icaA in S. aureus.. Present findings strongly suggest that terbinafine could be used safely and efficiently as an anti-virulent agent to combat S. aureus infections.

Topics: Animals; Anti-Bacterial Agents; Biofilms; Humans; Mice; Molecular Docking Simulation; Staphylococcal Infections; Staphylococcus aureus; Terbinafine; Xanthophylls

The study aimed to analyze morphological and functional changes of

Topics: Adult; Allylbenzene Derivatives; Anisoles; Anti-Bacterial Agents; Antioxidants; Bacteremia; Blood Cell Count; Female; Humans; Immunity, Innate; Interleukin-8; Male; Nitroblue Tetrazolium; Phagocytes; Phagocytosis; Spectroscopy, Fourier Transform Infrared; Staphylococcal Infections; Staphylococcus aureus; Xanthophylls

Staphylococcus aureus is an opportunistic pathogen that has the ability to cause a wide range of diseases including superficial infection and severe invasive life threatening infections. The pathogenicity of S. aureus is mediated by a group of virulence factors that mediate the colonization and penetration. The antibiotic resistance of S. aureus has evolved due to the abuse of antibiotics rendering the cure of infection very difficult especially with the shortage in new antibiotic production. To combat this shortage, repurposing of FDA-approved drugs against the virulence factors is a new strategy. The analgesic drug Diclofenac was found to have anti-virulence activity against Pseudomonas aeruginosa and Proteus mirabilis. This study aimed to demonstrate the anti-virulence effect of diclofenac against clinical MRSA isolates phenotypically and genotypically using qRT-PCR. In this study, diclofenac showed significant reduction in biofilm formation when compared to controls, the inhibition ranged between 22.67% and 70%. Also, remarkable inhibition of hemolysin activity was found (5.4-66.34%). Additionally, diclofenac has inhibitory activity against the staphyloxanthin production (8-57.2%). The results were confirmed by qRT-PCR that showed significant down-regulation of tested virulence genes. The down-regulation ranged from 43 to 64.05% for SarA, 36.85-64.75% for AgrA, 50-63.2% for hla, 38.55-60.35% for FnbA, 46.75-61.05% for IcaA, 27.55-64% for SigB and 51.05-72.8% for CrtM. In conclusion, diclofenac can be used in combination with antibiotics as anti-virulence agent against MDR-MRSA which will enhance the ability of immune system to eradicate infection.

Topics: Anti-Bacterial Agents; Biofilms; Diclofenac; Drug Resistance, Multiple, Bacterial; Genotype; Hemolysin Proteins; Humans; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Proteus mirabilis; Pseudomonas aeruginosa; Staphylococcal Infections; Virulence; Virulence Factors; Xanthophylls

A polyherbal formulation (Herboheal) traditionally indicated for woundcare was investigated for its anti-virulence potential against the notorious pathogen Staphylococcus aureus.. This study aimed at evaluating anti-virulence potential of Herboheal formulation against S. aureus in vitro as well as in vivo, followed by studying its effect on target bacterium's gene expression at the whole transcriptome level.. In vitro efficacy of the test formulation was evaluated using broth dilution assay, whereas in vivo efficacy was assayed employing the nematode Caenorhabditis elegans as the model host. Molecular targets of the test formulation in S. aureus were elucidated through whole transcriptome analysis.. This formulation could exert inhibitory effect on bacterial growth and quorum sensingregulated pigment (staphyloxanthin) production at ≥ 0.025% v/v. It not only could inhibit S. aureus biofilm formation, but also eradicated pre-formed biofilm effectively. This formulation could modulate antibiotic susceptibility of S. aureus, enhanced its susceptibility to human serum heavily, while compromising its haemolytic potential. Herboheal-treated bacteria expressed notably lesser virulence towards the nematode worm Caenorhabditis elegans. Even repeated exposure of S. aureus to this polyherbal formulation did not give rise to resistant phenotype. Whole transcriptome analysis revealed genes associated with hemolysis, virulence, enzyme activity, transport, basic cellular processes, quorum sensing, and transcriptional regulators as the major targets of Herboheal in S. aureus.. This study validates the traditional use of Herboheal formulation in wound-care by demonstrating its efficacy against one of the pathogenic bacteria most commonly involved in wound infections.

Topics: Animals; Anti-Bacterial Agents; Bacterial Proteins; Biofilms; Caenorhabditis elegans; Gene Expression Profiling; Humans; Phytotherapy; Plant Preparations; Quorum Sensing; Staphylococcal Infections; Staphylococcus aureus; Virulence; Whole Genome Sequencing; Wounds and Injuries; Xanthophylls

Topics: Animals; Cell Membrane; Cold Temperature; Fatty Acids; Female; Membrane Fluidity; Mice; Mice, Inbred ICR; Staphylococcal Infections; Staphylococcus aureus; Virulence; Xanthophylls

Investigation of the ability of glyceryl trinitrate (GTN) to inhibit staphyloxanthin, biofilm and tolerance to oxidative stress.. The disk sensitivity method was used to detect the methicillin resistance of. GTN showed a significant inhibition of biofilm, staphyloxanthin and tolerance to oxidative stress. In the molecular docking study, it was found that GTN could bind to dehydrosqualene synthase enzyme by hydrogen bonding, electrostatic interaction and pi-cation interaction.. The present study revealed the ability of GTN to serve as a potential anti-virulence candidate for attenuation of

Topics: Biofilms; Drug Resistance, Microbial; Humans; Molecular Docking Simulation; Nitroglycerin; Oxidative Stress; Staphylococcal Infections; Staphylococcus aureus; Virulence; Virulence Factors; Xanthophylls

Microbial species utilize secreted-signaling molecules to coordinate their behavior. Our previous investigations demonstrated a key role for the

Topics: Biofilms; Candida albicans; Farnesol; Microbial Interactions; Oxidative Stress; Quorum Sensing; Staphylococcal Infections; Staphylococcus aureus; Virulence; Virulence Factors; Xanthophylls

Staphylococcus aureus is an opportunistic pathogen related to a variety of life-threatening infections but for which antimicrobial resistance is liming the treatment options. We report here that myricetin, but not its glycosylated form, can remarkably decrease the production of several S. aureus virulence factors, including adhesion, biofilm formation, hemolysis and staphyloxanthin production, without interfering with growth. Myricetin affects both surface proteins and secreted proteins which indicate that its action is unrelated to inhibition of the agr quorum sensing system. Analysis of virulence related gene expression and computational simulations of pivotal proteins involved in pathogenesis demonstrate that myricetin downregulates the saeR global regulator and interacts with sortase A and α-hemolysin. Furthermore, Myr confers a significant degree of protection against staphylococcal infection in the Galleria mellonella model. The present findings reveal the potential of Myr as an alternative multi-target antivirulence candidate to control S. aureus pathogenicity.

Topics: Animals; Bacterial Adhesion; Biofilms; Erythrocytes; Extracellular Matrix Proteins; Flavonoids; Gene Expression Profiling; Hemolysis; Models, Molecular; Moths; Protective Agents; Protein Binding; Protein Conformation; Staphylococcal Infections; Staphylococcus aureus; Structure-Activity Relationship; Virulence Factors; Xanthophylls

Topics: Animals; Anti-Bacterial Agents; Bacterial Proteins; Enzyme Inhibitors; Humans; Methicillin-Resistant Staphylococcus aureus; Mice; Mutation; Neutrophils; Oxidative Stress; Oxidoreductases; Pyrrolidines; Staphylococcal Infections; Staphylococcus aureus; Sulfur Compounds; Virulence; Virulence Factors; Xanthophylls

Our previous work ( Wang et al. J. Med. Chem. 2016 , 59 , 4831 - 4848 ) revealed that effective benzocycloalkane-derived staphyloxanthin inhibitors against methicillin-resistant Staphylococcus aureus (S. aureus) infections were accompanied by poor water solubility and high hERG inhibition and dosages (preadministration). In this study, 92 chroman and coumaran derivatives as novel inhibitors have been addressed for overcoming deficiencies above. Derivatives 69 and 105 displayed excellent pigment inhibitory activities and low hERG inhibition, along with improvement of solubility by salt type selection. The broad and significantly potent antibacterial spectra of 69 and 105 were displayed first with normal administration in the livers and hearts in mice against pigmented S. aureus Newman, Mu50 (vancomycin-intermediate S. aureus), and NRS271 (linezolid-resistant S. aureus), compared with linezolid and vancomycin. In summary, both 69 and 105 have the potential to be developed as good antibacterial candidates targeting virulence factors.

Topics: Animals; Anti-Bacterial Agents; Antifungal Agents; Drug Resistance, Bacterial; Ether-A-Go-Go Potassium Channels; Linezolid; Male; Methicillin-Resistant Staphylococcus aureus; Mice; Mice, Inbred BALB C; Microbial Sensitivity Tests; Potassium Channel Blockers; Rats; Rats, Sprague-Dawley; Staphylococcal Infections; Structure-Activity Relationship; Vancomycin; Vancomycin Resistance; Xanthophylls

A central question to biology is how pathogenic bacteria initiate acute or chronic infections. Here we describe a genetic program for cell-fate decision in the opportunistic human pathogen

Topics: Animals; Bacillus subtilis; Bacterial Proteins; Base Sequence; Biofilms; Cell Differentiation; Cell Wall; Disease Models, Animal; Escherichia coli; Female; Flow Cytometry; Gene Expression Regulation, Bacterial; Genes, Bacterial; Heart; Kidney; Magnesium; Mice; Mice, Inbred BALB C; Models, Theoretical; Peptidoglycan; RNA, Bacterial; Staphylococcal Infections; Staphylococcus aureus; Teichoic Acids; Trans-Activators; Xanthophylls

A number of bacterial cell processes are confined functional membrane microdomains (FMMs), structurally and functionally similar to lipid rafts of eukaryotic cells. How bacteria organize these intricate platforms and what their biological significance is remain important questions. Using the pathogen methicillin-resistant Staphylococcus aureus (MRSA), we show here that membrane-carotenoid interaction with the scaffold protein flotillin leads to FMM formation, which can be visualized using super-resolution array tomography. These membrane platforms accumulate multimeric protein complexes, for which flotillin facilitates efficient oligomerization. One of these proteins is PBP2a, responsible for penicillin resistance in MRSA. Flotillin mutants are defective in PBP2a oligomerization. Perturbation of FMM assembly using available drugs interferes with PBP2a oligomerization and disables MRSA penicillin resistance in vitro and in vivo, resulting in MRSA infections that are susceptible to penicillin treatment. Our study demonstrates that bacteria possess sophisticated cell organization programs and defines alternative therapies to fight multidrug-resistant pathogens using conventional antibiotics.

Topics: Animals; Bacterial Proteins; Carotenoids; Cell Membrane; Female; Membrane Microdomains; Membrane Proteins; Methicillin-Resistant Staphylococcus aureus; Mice; Mice, Inbred BALB C; Penicillin-Binding Proteins; Staphylococcal Infections; Xanthophylls

Staphylococcus aureus is an important opportunistic pathogen and is the etiological agent of many hospital- and community-acquired infections. The golden pigment, staphyloxanthin, of S. aureus colonies distinguishes it from other staphylococci and related Gram-positive cocci. Staphyloxanthin is the product of a series of biosynthetic steps that produce a unique membrane-embedded C

Topics: Bacterial Proteins; Gene Expression Regulation, Bacterial; Humans; Hydrogen Peroxide; Microbial Viability; Mutation; Pigments, Biological; Promoter Regions, Genetic; Protein Binding; Reactive Oxygen Species; Sigma Factor; Staphylococcal Infections; Staphylococcus aureus; Transcription, Genetic; Xanthophylls

The surge of antibiotic resistance in Staphylococcus aureus has created a dire need for innovative anti-infective agents that attack new targets, to overcome resistance. In S. aureus, carotenoid pigment is an important virulence factor because it shields the bacterium from host oxidant killing. Here we show that naftifine, a US Food and Drug Administration (FDA)-approved antifungal drug, blocks biosynthesis of carotenoid pigment at nanomolar concentrations. This effect is mediated by competitive inhibition of S. aureus diapophytoene desaturase (CrtN), an essential enzyme for carotenoid pigment synthesis. We found that naftifine attenuated the virulence of a variety of clinical S. aureus isolates, including methicillin-resistant S. aureus (MRSA) strains, in mouse infection models. Specifically, we determined that naftifine is a lead compound for potent CrtN inhibitors. In sum, these findings reveal that naftifine could serve as a chemical probe to manipulate CrtN activity, providing proof of concept that CrtN is a druggable target against S. aureus infections.

Topics: Allylamine; Animals; Anti-Bacterial Agents; Bacterial Proteins; Binding, Competitive; Carotenoids; Drug Design; Drug Resistance, Bacterial; Methicillin-Resistant Staphylococcus aureus; Mice; Microbial Sensitivity Tests; Oxidoreductases; Staphylococcal Infections; Staphylococcus aureus; Virulence Factors; Xanthophylls

Staphylococcus aureus is a major human pathogen and emergence of antibiotic resistance in clinical staphylococcal isolates raises concerns about our ability to control these infections. Cell wall-active antibiotics cause elevated synthesis of methionine sulfoxide reductases (Msrs: MsrA1 and MsrB) in S. aureus. MsrA and MsrB enzymes reduce S-epimers and R-epimers of methionine sulfoxide, respectively, that are generated under oxidative stress. In the S. aureus chromosome, there are three msrA genes (msrA1, msrA2 and msrA3) and one msrB gene. To understand the precise physiological roles of Msr proteins in S. aureus, mutations in msrA1, msrA2 and msrA3 and msrB genes were created by site-directed mutagenesis. These mutants were combined to create a triple msrA (msrA1, msrA2 and msrA3) and a quadruple msrAB (msrA1, msrA2, msrA3, msrB) mutant. These mutants were used to determine the roles of Msr proteins in staphylococcal growth, antibiotic resistance, adherence to human lung epithelial cells, pigment production, and survival in mice relative to the wild-type strains. MsrA1-deficient strains were sensitive to oxidative stress conditions, less pigmented and less adherent to human lung epithelial cells, and showed reduced survival in mouse tissues. In contrast, MsrB-deficient strains were resistant to oxidants and were highly pigmented. Lack of MsrA2 and MsrA3 caused no apparent growth defect in S. aureus. In complementation experiments with the triple and quadruple mutants, it was MsrA1 and not MsrB that was determined to be critical for adherence and phagocytic resistance of S. aureus. Overall, the data suggests that MsrA1 may be an important virulence factor and MsrB probably plays a balancing act to counter the effect of MsrA1 in S. aureus.

Topics: Animals; Anti-Bacterial Agents; Bacterial Adhesion; Cell Wall; Disease Models, Animal; Enzyme Activation; Hemolysis; Humans; Methionine Sulfoxide Reductases; Mice; Microbial Sensitivity Tests; Mutation; Oxidants; Oxidative Stress; Phagocytosis; Protein Transport; Staphylococcal Infections; Staphylococcal Protein A; Staphylococcus aureus; Xanthophylls

The defining hallmark of the newly described species, Staphylococcus argenteus, in comparison to its sister species, S. aureus and S. schweitzeri, is the absence of production of the carotenoid pigment, staphyloxanthin. Staphylococcus argenteus lacks the responsible genetic locus crtOPQMN. We examined the impact of carotenoid synthesis in two non-pigmented S. argenteus strains, MSHR1132 and SCC1165. Following complementation with a plasmid containing the carotenoid operon (pTX-crtOPQMN), compared to wild type, both complemented strains showed substantial carotenoid production, with a resultant increase in cell membrane rigidity. Surprisingly, both crtOPQMN-complemented strains exhibited increased susceptibility to the host defense peptides, LL-37 and hNP-1 in vitro, and reduced virulence in an experimental rabbit endocarditis model.

Topics: alpha-Defensins; Animals; Antimicrobial Cationic Peptides; Biosynthetic Pathways; Cathelicidins; Disease Models, Animal; Endocarditis; Genetic Complementation Test; Host-Pathogen Interactions; Operon; Plasmids; Rabbits; Staphylococcal Infections; Staphylococcus aureus; Virulence; Xanthophylls

Topics: Anti-Bacterial Agents; Atorvastatin; Coronary Artery Bypass; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Postoperative Complications; Staphylococcal Infections; Staphylococcus aureus; Xanthophylls

One of the most common pathogens causing musculoskeletal infections remains Staphylococcus aureus. The aim of this multicentre study was to perform a phenotypic and genotypic characterisation of clinical S. aureus isolates recovered from musculoskeletal infections and to investigate differences between isolates cultured from Orthopaedic Implant Related Infections (OIRI) and those from Non-Implant Related Infections (NIRI). OIRI were further differentiated in two groups: Fracture Fixation-Device Infections (FFI) and Prosthetic Joint Infections (PJI). Three-hundred and five S. aureus strains were collected from 4 different Swiss and 2 French hospitals (FFI, n=112; PJI, n=105; NIRI, n=88). NIRI cases were composed of 27 Osteomyelitis (OM), 23 Diabetic Foot Infections (DFI), 27 Soft Tissue Infections (STI) and 11 postoperative Spinal Infections (SI). All isolates were tested for their ability to form biofilm, to produce staphyloxanthin and their haemolytic activity. They were typed by agr (accessory gene regulator) group, spa type and screened by PCR for the presence of genes of the most relevant virulence factors such as MSCRAMMs, Panton Valentine Leukotoxin (PVL), enterotoxins, exotoxins and toxic shock syndrome toxin. Overall, methicillin susceptible S. aureus (MSSA) was more prevalent than methicillin resistant S. aureus (MRSA) in this collection. The OIRI group trended towards a higher incidence of MRSA, gentamicin resistance and haemolysis activity than the NIRI group. Within the OIRI group, PJI isolates were more frequently strong biofilm formers than isolates from the FFI group. A statistically significant difference was observed between OIRI and NIRI isolates for the sdrE gene, the cna gene, the clfA gene and the bbp gene. Certain spa types (t230 and t041) with a specific genetic virulence pattern were only found in isolates cultured from OIRI. In conclusion, our study highlights significant trends regarding the virulence requirements displayed by S. aureus isolates associated with implant related infections in comparison to non-implant related infections. However, future studies including whole genome sequencing will be required to further examine genomic differences among the different infection cases.

Topics: Biofilms; Diabetic Foot; France; Genes, Bacterial; Genotype; Hemolysis; Hospitals; Humans; Musculoskeletal Diseases; Osteomyelitis; Phenotype; Polymerase Chain Reaction; Prosthesis-Related Infections; Retrospective Studies; Soft Tissue Infections; Staphylococcal Infections; Staphylococcus aureus; Switzerland; Virulence Factors; Xanthophylls

Staphylococcus aureus clonal complex 75 (herein referred to as S. argenteus) lacks the carotenoid pigment operon, crtOPQMN, responsible for production of the putative virulence factor, staphyloxanthin. Although a common cause of community-onset skin infections among Indigenous populations in northern Australia, this clone is infrequently isolated from hospital-based patients with either bacteremic or nonbacteremic infections. We hypothesized that S. argenteus would have attenuated virulence compared to other S. aureus strains due to its staphyloxanthin "deficiency." Compared to prototypical S. aureus strains, S. argenteus was more susceptible to oxidative stress and neutrophil killing in vitro and had reduced virulence in murine sepsis and skin infection models. Transformation with pTX-crtOPQMN resulted in staphyloxanthin expression and increased resistance to oxidative stress in vitro. However, neither resistance to neutrophil killing nor in vivo virulence was increased. Thus, reduced virulence of S. argenteus in these models is due to mechanisms unrelated to lack of staphyloxanthin production.

Topics: Animals; Australia; Child; Disease Models, Animal; Genetic Complementation Test; Humans; Male; Mice; Mice, Inbred BALB C; Operon; Sepsis; Staphylococcal Infections; Staphylococcal Skin Infections; Staphylococcus aureus; Virulence; Virulence Factors; Xanthophylls

Staphyloxanthin, a yellow pigment produced by methicillin-resistant Staphylococcus aureus (MRSA), is a virulent factor escaping from the host immune system. A new screening method for inhibitors of staphyloxanthin production by MRSA was established using paper disks. By this screening method, inhibitors of staphyloxanthin production were selected from the natural product library (ca. 300) and from actinomycete culture broths (ca. 1000). From the natural product library, four known inhibitors of lipid metabolism, cerulenin, dihydrobisvertinol, xanthohumol and zaragozic acid, were found to inhibit staphyloxanthin production; however, typical antibiotics used clinically, including vancomycin, had no effect on staphyloxanthin production. From actinomycete culture broths, two known anthraquinones, 6-deoxy-8-O-methylrabelomycin and tetrangomycin, were found to inhibit staphyloxanthin production by MRSA in the paper disk assay. These results suggested that this screening method is useful and effective to find compounds targeting staphyloxanthin production, leading to a new type of chemotherapeutics against MRSA infection.

Topics: Actinobacteria; Alkenes; Anthraquinones; Anti-Bacterial Agents; Benz(a)Anthracenes; Biological Products; Cerulenin; Flavonoids; Heterocyclic Compounds, 3-Ring; Lipid Metabolism; Methicillin Resistance; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Propiophenones; Staphylococcal Infections; Vancomycin; Xanthophylls

The isoprenoid biosynthesis pathways produce the largest class of small molecules in Nature: isoprenoids (also called terpenoids). Not surprisingly then, isoprenoid biosynthesis is a target for drug discovery, and many drugs--such as Lipitor (used to lower cholesterol), Fosamax (used to treat osteoporosis), and many anti-infectives--target isoprenoid biosynthesis. However, drug resistance in malaria, tuberculosis, and staph infections is rising, cheap and effective drugs for the neglected tropical diseases are lacking, and progress in the development of anticancer drugs is relatively slow. Isoprenoid biosynthesis is thus an attractive target, and in this Account, I describe developments in four areas, using in each case knowledge derived from one area of chemistry to guide the development of inhibitors (or drug leads) in another, seemingly unrelated, area. First, I describe mechanistic studies of the enzyme IspH, which is present in malaria parasites and most pathogenic bacteria, but not in humans. IspH is a 4Fe-4S protein and produces the five-carbon (C5) isoprenoids IPP (isopentenyl diphosphate) and DMAPP (dimethylallyl diphosphate) from HMBPP (E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate) via a 2H(+)/2e(-) reduction (of an allyl alcohol to an alkene). The mechanism is unusual in that it involves organometallic species: "metallacycles" (η(2)-alkenes) and η(1)/η(3)-allyls. These observations lead to novel alkyne inhibitors, which also form metallacycles. Second, I describe structure-function-inhibition studies of FPP synthase, the macromolecule that condenses IPP and DMAPP to the sesquiterpene farnesyl diphosphate (FPP) in a "head-to-tail" manner. This enzyme uses a carbocation mechanism and is potently inhibited by bone resorption drugs (bisphosphonates), which I show are also antiparasitic agents that block sterol biosynthesis in protozoa. Moreover, "lipophilic" bisphosphonates inhibit protein prenylation and invasiveness in tumor cells, in addition to activating γδ T-cells to kill tumor cells, and are important new leads in oncology. Third, I describe structural and inhibition studies of a "head-to-head" triterpene synthase, dehydrosqualene synthase (CrtM), from Staphylococcus aureus. CrtM catalyzes the first committed step in biosynthesis of the carotenoid virulence factor staphyloxanthin: the condensation of two FPP molecules to produce a cyclopropane (presqualene diphosphate). The structure of CrtM is similar to that of human squalene synthase (SQ

Topics: Animals; Anti-Infective Agents; Bacterial Proteins; Diphosphonates; Drug Evaluation, Preclinical; Farnesyl-Diphosphate Farnesyltransferase; Humans; Leishmaniasis; Mice; Pamidronate; Staphylococcal Infections; Staphylococcus aureus; Terpenes; Xanthophylls

Topics: Animals; Anticholesteremic Agents; Cholesterol; Enzyme Inhibitors; Farnesyl-Diphosphate Farnesyltransferase; Humans; Methicillin Resistance; Mice; Staphylococcal Infections; Staphylococcus aureus; Virulence; Xanthophylls

Topics: Cholesterol; Color; Drug Resistance, Bacterial; Drug Resistance, Multiple, Bacterial; Humans; Immune System; Staphylococcal Infections; Staphylococcus aureus; Virulence Factors; Xanthophylls

Staphylococcus aureus produces hospital- and community-acquired infections, with methicillin-resistant S. aureus posing a serious public health threat. The golden carotenoid pigment of S. aureus, staphyloxanthin, promotes resistance to reactive oxygen species and host neutrophil-based killing, and early enzymatic steps in staphyloxanthin production resemble those for cholesterol biosynthesis. We determined the crystal structures of S. aureus dehydrosqualene synthase (CrtM) at 1.58 angstrom resolution, finding structural similarity to human squalene synthase (SQS). We screened nine SQS inhibitors and determined the structures of three, bound to CrtM. One, previously tested for cholesterol-lowering activity in humans, blocked staphyloxanthin biosynthesis in vitro (median inhibitory concentration approximately 100 nM), resulting in colorless bacteria with increased susceptibility to killing by human blood and to innate immune clearance in a mouse infection model. This finding represents proof of principle for a virulence factor-based therapy against S. aureus.

Topics: Amino Acid Sequence; Animals; Anti-Bacterial Agents; Bacterial Proteins; Cell Line; Cell Proliferation; Cholesterol; Crystallography, X-Ray; Enzyme Inhibitors; Farnesyl-Diphosphate Farnesyltransferase; Humans; Mice; Molecular Sequence Data; Organothiophosphorus Compounds; Polyisoprenyl Phosphates; Protein Structure, Secondary; Sesquiterpenes; Staphylococcal Infections; Staphylococcus aureus; Virulence; Xanthophylls

Topics: Abscess; Animals; Anticholesteremic Agents; Bacterial Proteins; Chelating Agents; Humans; Leukocyte L1 Antigen Complex; Manganese; Neutrophils; Reactive Oxygen Species; Repressor Proteins; Staphylococcal Infections; Staphylococcus aureus; Transcription Factors; Xanthophylls