Staphyloxanthin Biosynthesis Research Articles

Sub-inhibitory concentrations of tetrabromobisphenol A induce the biofilm formation of methicillin-resistant Staphylococcus aureus.

Biofilm formation by methicillin-resistant Staphylococcus aureus (MRSA) on indwelling medical devices complicates the treatment of infection. Tetrabromobisphenol A (TBBPA), a synthetic, lipophilic, halogenated aromatic compound widely used as an additive in plastics and electronic products, has raised environmental concerns due to its potential for bioaccumulation. This study investigated the impact of sub-inhibitory concentrations of TBBPA on MRSA biofilm formation. Crystal violet staining and confocal laser scanning microscopy analysis demonstrated that 1/8 MIC (0.5µg/mL) of TBBPA significantly stimulated MRSA biofilm formation (P < 0.0001). MTT assays indicated that the metabolic activity within the biofilms increased by 15.60-40.85% compared to untreated controls. Dot blot immunoassay, autolysis assay, and extracellular DNA (eDNA) quantification further revealed TBBPA enhanced the production of polysaccharide intercellular adhesin (PIA) and eDNA, which are key biofilm components. Additionally, TBBPA was found to enhance the production of staphyloxanthin, facilitating MRSA survival under oxidative conditions and in human whole blood. RT-qPCR analysis showed that TBBPA significantly upregulated genes associated with biofilm formation (icaA, atlA, sarA), staphyloxanthin biosynthesis (crtM and sigB), and oxidative stress responses (sodA and katA). These findings suggest that TBBPA promotes MRSA biofilm development and enhances bacterial resistance to adverse conditions, thereby potentially exacerbating risks to human health.

Berberine hydrochloride reduces staphyloxanthin synthesis by inhibiting fni genes in methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) poses a great health threat to humans. Looking for compounds that could reduce the resistance of S. aureus towards methicillin is an effective way to alleviate the antimicrobial resistance crisis. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), Time-killing growth curve, staphyloxanthin and penicillin-binding protein 2a (PBP2a) were detected. A quantitative polymerase chain reaction was used to measure the effect of BBH on the gene transcription profiles of MRSA. The MIC of MRSA-ST59-t437 towards oxacillin was 8µg/ml, and MBC was 128µg/ml. After adding a sub-inhibitory concentration of BBH, the MIC and MBC of MRSA-ST59-t478 towards oxacillin went down to 0.125 and 32µg/ml respectively. The amount of PBP2a and staphyloxanthin were reduced after treatment with BBH. Moreover, the transcription levels of sarA, mecA and fni genes were downregulated. It is for the first time reported that BBH could inhibit staphyloxanthin synthesis by inhibiting fni gene. Moreover, fni might be the target gene of sarA, and there might be another regulatory pathway to inhibit staphyloxanthin biosynthesis. BBH could effectively reduce the methicillin resistance of MRSA-ST59-t437 by downregulating fni, sarA and mecA genes.

Eugenol targeting CrtM inhibits the biosynthesis of staphyloxanthin in Staphylococcus aureus

Staphylococcus aureus is a serious foodborne pathogen threatening food safety and public health. Especially the emergence of methicillin-resistant Staphylococcus aureus (MRSA) increased the difficulty of S. aureus treatment. Staphyloxanthin is a crucial virulence factor of S. aureus. Blocking staphyloxanthin production could help the host immune system counteract the invading S. aureus cells. In this study, we first screened for staphyloxanthin inhibitors using a virtual screening method. The outcome of the virtual screening method resulted in the identification of eugenol (300 μg/mL), which significantly inhibits the staphyloxanthin production in S. aureus ATCC 29213, S. aureus Newman, MRSA ATCC 43300 and MRSA ATCC BAA1717 by 84.2 %, 63.5 %, 68.1 %, and 79.5 %, respectively. The outcome of the growth curve assay, field-emission scanning electron, and confocal laser scanning microscopy analyses confirmed that eugenol at the test concentration did not affect the morphology and growth of S. aureus. Moreover, the survival rate of S. aureus ATCC 29213 and MRSA ATCC 43300 under H2O2 pressure decreased to 51.9 % and 45.5 % in the presence of eugenol, respectively. The quantitative RT-PCR and molecular simulation studies revealed that eugenol targets staphyloxanthin biosynthesis by downregulating the transcription of the crtM gene and inhibiting the activity of the CrtM enzyme. Taken together, we first determined that eugenol was a prominent compound for staphyloxanthin inhibitor to combat S. aureus especially MRSA infections.

Multimodal nanoimmunotherapy engages neutrophils to eliminate Staphylococcus aureus infections.

The increasing prevalence of antimicrobial resistance in Staphylococcus aureus necessitates alternative therapeutic approaches. Neutrophils play a crucial role in the fight against S. aureus but suffer from deficiencies in function leading to increased infection. Here we report a nanoparticle-mediated immunotherapy aimed at potentiating neutrophils to eliminate S. aureus. The nanoparticles consist of naftifine, haemoglobin (Hb) and a red blood cell membrane coating. Naftifine disrupts staphyloxanthin biosynthesis, Hb reduces bacterial hydrogen sulfide levels and the red blood cell membrane modifies bacterial lipid composition. Collectively, the nanoparticles can sensitize S. aureus to host oxidant killing. Furthermore, in the infectious microenvironment, Hb triggers lipid peroxidation in S. aureus, promoting neutrophil chemotaxis. Oxygen supplied by Hb can also significantly enhance the bactericidal capability of the recruited neutrophils by restoring neutrophil respiratory burst via hypoxia relief. This multimodal nanoimmunotherapy demonstrates excellent therapeutic efficacy in treating antimicrobial-resistant S. aureus persisters, biofilms and S. aureus-induced infection in mice.

A natural inhibitor of diapophytoene desaturase attenuates methicillin-resistant Staphylococcus aureus (MRSA) pathogenicity and overcomes drug-resistance.

At present, the inhibition of staphyloxanthin biosynthesis has emerged as a prominent strategy in combating methicillin-resistant Staphylococcus aureus (MRSA) infection. Nonetheless, there remains a limited understanding regarding the bio-structural characteristics of staphyloxanthin biosynthetic enzymes, as well as the molecular mechanisms underlying the interaction between inhibitors and proteins. Furthermore, the functional scope of these inhibitors is relatively narrow. In this study, we address these limitations by harnessing the power of deep learning techniques to construct the 3D structure of diapophytoene desaturase (CrtN). We perform efficient virtual screening and unveil alnustone as a potent inhibitor of CrtN. Further investigations employing molecular modelling, site-directed mutagenesis and biolayer interferometry (BLI) confirmed that alnustone binds to the catalytic active site of CrtN. Transcriptomic analysis reveals that alnustone significantly down-regulates genes associated with staphyloxanthin, histidine and peptidoglycan biosynthesis. Under the effects of alnustone, MRSA strains exhibit enhanced sensitivity to various antibiotics and the host immune system, accompanied by increased cell membrane permeability. In a mouse model of systemic MRSA infection, the combination of alnustone and antibiotics exhibited a significant therapeutic effect, leading to reduced bacterial colony counts and attenuated pathological damage. Alnustone, as a natural inhibitor targeting CrtN, exhibits outstanding antibacterial properties that are single-targeted yet multifunctional. This finding provides a novel strategy and theoretical basis for the development of drugs targeting staphyloxanthin producing bacteria.

Investigating the bispecific lead compounds against methicillin-resistant Staphylococcus aureus SarA and CrtM using machine learning and molecular dynamics approach

Methicillin-resistant Staphylococcus aureus (MRSA) is a notorious pathogen that has emerged as a serious global health concern over the past few decades. Staphylococcal accessory regulator A (SarA) and 4,4′-diapophytoene synthase (CrtM) play a crucial role in biofilm formation and staphyloxanthin biosynthesis. Thus, the present study used a machine learning-based QSAR model to screen 1261 plant-derived natural organic compounds in order to identify a medication candidate with both biofilm and virulence inhibitory potential. Additionally, the in-silico molecular docking analysis has demonstrated significant binding efficacy of the identified hit compound, that is 85137543, with SarA and CrtM when compared to the control compound, hesperidin. Post-MD simulation analysis of the complexes depicted strong binding of 85137543 to both SarA and CrtM. Moreover, 85137543 showed hydrogen bonding with the key residues of both proteins during docking (ALA138 of SarA and ALA134 of CrtM) and post-MD simulation (LYS273 of CrtM and ASN212 of SarA). The RMSD of 85137543 was stable and consistent when bound to both CrtM and SarA with RMSDs of 1.3 and 1 nm, respectively. In addition, principal component analysis and the free energy landscape showed stable complex formation with both proteins. Low binding free energy (ΔGTotal) was observed by 85137543 for SarA (−47.92 kcal/mol) and CrtM (−36.43 kcal/mol), which showed strong binding. Overall, this study identified 85137543 as a potential inhibitor of both SarA and CrtM in MRSA. Communicated by Ramaswamy H. Sarma

Spinach protein-capped silver nanoparticles with low hemolytic activity inhibit methicillin-resistant Staphylococcus aureus biofilm formation as a new therapeutic approach

BackgroundThe methicillin-resistant Staphylococcus aureus (MRSA) was designated as a high-priority pathogen by the World Health Organization. Cardiovascular and thoracic surgery complications frequently involve MRSA infection. Treatment of newly emerging resistant S. aureus strains is clinically challenging due to the natural tendency of MRSA survival through biofilm formation. The failure of traditional antibiotic therapy necessitates development of next-generation therapeutic molecules. ObjectivesSilver nanoparticles were investigated to treat MRSA in order to overcome the challenges of antibiotic therapy. MethodsSpinach protein functionalized silver nanoparticles (SPAgNPs) were synthesized by reducing with sodium borohydride. The resazurin microtiter assay was used to determine the minimum inhibitory concentration (MIC) of SPAgNPs against MRSA. The antibiofilm activity was investigated using a crystal violet assay at sub-MIC levels and validated using scanning electron microscopy. To demonstrate the mechanism of action of SPAgNPs, the virulence factors involved in MRSA biofilm formation were examined. SEM was used to evaluate the hemocompatibility of human red blood cells. Results and conclusionThe synthesized SPAgNPs showed characteristic yellow colour with surface Plasmon resonance maximum at 418 nm. Powder X-ray diffraction and transmission electron microscopy analysis confirmed that the particles are crystalline with a size range from 14 to 40 nm. SPAgNPs have antibacterial activity at a minimum inhibitory concentration (MIC) of 1.25 mM, killing MRSA via disrupting membrane integrity and producing excess reactive oxygen species. Strikingly, SPAgNPs inhibit MRSA biofilm formation at sub-MIC (0.625 mM). Scanning electron microscopy study reveals that SPAgNPs trigger biofilm inhibition without causing cell damage. Hence the NPs are very effective in preventing pathogens from triggering stress responses. SPAgNPs suppress exopolysaccharide production, cell surface hydrophobicity, and staphyloxanthin biosynthesis. Testing SPAgNPs against human red blood cells reveals that NPs do not harm human cells, suggesting that the SPAgNPs are biocompatible. The findings show that SPAgNPs could be explored as next-generation materials for treating MDR pathogenic infection linked with biofilms.

Activity of Lipid Loaded Lectin against co-infection of Candida albicans and Staphylococcus aureus using the Zebrafish model.

Nosocomial infection caused by mixed species of methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans (CA) is difficult to manage with existing antimicrobials, particularly in the presence of mixed-species biofilm. This study evaluates the activity of cationic lipid, specifically functionalized with lectin, against mixed biofilms of MRSA and CA and their effectiveness in vivo using the zebrafish model. The present study demonstrates for the first time the antimicrobial activity of 2-((N-[2-hydroxyethyl]palmitamido)methyl)-1-methylpyridin-1-ium iodide (cN16E) against MRSA and mixed species of MRSA + CA. The cN16E functionalized with Butea monosperma seed lectin (BMSL) showed a lower minimum inhibitory concentration (MIC) as compared with cN16E. BMSL-cN16E (BcN16E) exhibited strong membrane-damaging activity at a lower concentration than cN16E. Crystal violet assay showed that BcN16E inhibits mixed-species biofilm at the concentration of 15.63 µM, which is four-fold lower than the MIC. Especially, BcN16E was found to be effective in disturbing mature mixed biofilm at 31.25 µM, which is two-fold lower than the MIC, suggesting true antibiofilm activity without pressurizing the microorganisms. The treatment with BcN16E significantly reduced the exopolysaccharide synthesis (> 78%), cell surface hydrophobicity (> 70%), hyphae formation, staphyloxanthin biosynthesis (> 41%), and antioxidant enzyme and hemolysin activity (> 70%). Notably, BcN16E was efficient in reducing the in vivo colonization of bacterial and fungal burden in the blood and muscle tissues of zebrafish. Antimicrobial and antibiofilm efficacy of BcN16E against MRSA, and mixed species of MRSA + CA were demonstrated. Importantly, BcN16E treatment rescued Zebrafish coinfected with mixed species of MRSA + CA.

Antibiofilm and staphyloxanthin inhibitory potential of terbinafine against Staphylococcus aureus: in vitro and in vivo studies

BackgroundAntimicrobial 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.MethodsTerbinafine 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.ResultsTerbinafine 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.ConclusionsPresent findings strongly suggest that terbinafine could be used safely and efficiently as an anti-virulent agent to combat S. aureus infections.

Alternative sigma factor B reduces biofilm formation and stress response in milk-derived Staphylococcus aureus

Staphylococcus aureus is one of the most important food-borne pathogens and shows strong tolerance to environmental stress. The functions of alternative sigma factor B (SigB) have been studied in S. aureus Newman and SH1000 (a derivative strain of S. aureus NCTC8325) isolated from human samples. However, the role of SigB in the stress response of food-borne S. aureus has not been reported. In this study, the functions of SigB involved in food-related stress responses and biofilm formation in S. aureus RMSA24 isolated from raw milk were investigated. The sigB mutant synthesized less biofilm than the wild-type strain and resulted in a nonpigmented phenotype. Our results indicated that SigB was essential for biofilm formation and staphyloxanthin biosynthesis. Furthermore, mutation of sigB provoked a decrease in resistance to heat, drying, and hydrogen peroxide. This study further confirmed that sigB is a transcription factor responding to biofilm formation and stress tolerance in S. aureus, and the findings of this study provide an important clue for further studying the control of biofilm formation and the tolerance to high temperature, drying, oxidation of S. aureus in the food processing environment.

Celastrol mitigates staphyloxanthin biosynthesis and biofilm formation in Staphylococcus aureus via targeting key regulators of virulence; in vitro and in vivo approach

BackgroundStaphylococcus 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.ResultsThe 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.ConclusionsCelastrol is a novel anti-virulent agent against S. aureus suggesting, a prospective therapeutic role for celastrol as a multi-targeted anti-pathogenic agent.

Carotenogenesis of Staphylococcus aureus: New insights and impact on membrane biophysical properties

Staphyloxanthin (STX) is a saccharolipid derived from a carotenoid in Staphylococcus aureus involved in oxidative-stress tolerance and antimicrobial peptide resistance. STX influences the biophysical properties of the bacterial membrane and has been associated to the formation of lipid domains in the regulation of methicillin-resistance. In this work, a targeted metabolomics and biophysical characterization study was carried out to investigate the biosynthetic pathways of carotenoids, and their impact on the membrane biophysical properties. Five different S. aureus strains were investigated, including three wild-type strains containing the crtM gene related to STX biosynthesis, a crtM-deletion mutant, and a crtMN plasmid-complemented variant. LC-DAD-MS/MS analysis of extracts allowed the identification of 34 metabolites related to carotenogenesis in S. aureus at different growth phases (8, 24 and 48 h), showing the progression of these metabolites as the bacteria advances into the stationary phase. For the first time, 22 members of a large family of carotenoids were identified, including STX and STX-homologues, as well as Dehydro-STX and Dehydro-STX-homologues. Moreover, thermotropic behavior of the CH2 stretch of lipid acyl chains in live cells by FTIR, show that the presence of STX increases acyl chain order at the bacterial growth temperature. Indeed, the cooperative melting event of the bacterial membrane, which occurs around 15 °C in the native strains, shifts with increased carotenoid content. These results show the diversity biosynthetic of carotenoids in S. aureus, and their influence on membrane biophysical properties.

Naftifine enhances photodynamic therapy against Staphylococcus aureus by inhibiting staphyloxanthin expression

Staphyloxanthin (STX) is a virulence factor produced by Staphylococcus aureus (S. aureus) including methicillin-resistant S. aureus (MRSA), a widely distributed Gram-positive bacterium related to several clinical human infections. We herein reported that Naftifine, a broad-spectrum antifungal agent, plays an important role on photodynamic antimicrobial therapy (PACT) for S. aureus. STX attenuated the PACT effect on S. aureus in a dose-dependent manner, which probably due to its biological function of scavenging reactive oxygen species (ROS). On the contrary, the sensitivity of S. aureus to PACT was significantly improved after an incubation with Naftifine (10 μM) compared with that of S. aureus untreated with Naftifine. The reason for this phenomenon is that Naftifine inhibits STX biosynthesis and improves membrane permeability. Taken together, we identified STX as a limiting factor of PACT in S. aureus. Meanwhile, we found that the combination of PACT and Naftifine, an inhibitor of STX biosynthesis, has synergistic antibacterial effect on S. aureus, and provide a new way for the rapid and efficient treatment of S. aureus infection without drug resistance.

Structure-activity relationships and mechanism of action of tetragomycin derivatives as inhibitors of Staphylococcus aureus staphyloxanthin biosynthesis

Despite the main strategy to overcome bacterial resistance has focused on the development of more potent antimicrobial agents, the evolutionary pressure caused by such drugs makes this strategy limited. Molecules that interfere with virulence factors appear as a promising alternative though, as they cause reduced selective pressure. As a matter of fact, staphyloxanthin biosynthesis inhibition (STXBI) has been pursued as promising strategy to reduce S. aureus virulence. Herein, we report the inhibitory profile of 27 tetrangomycin derivatives over staphyloxanthin production. The experimental result showed that naphthoquinone dehydro-α-lapachone (25 - EC50 = 57.29 ± 1.15 μM) and 2-Isopropylnaphtho[2,3-b]furan-4,9-dione (26 EC50 = 82.10 ± 1.09 μM) are the most potent compounds and suggest that hydrogen acceptor groups and lipophilic moieties decorating the naphthoquinone ring are crucial for STXBI. In addition, we present an in situ analysis, through RAMAN spectroscopy, that is inexpensive and might be employed to probe the mechanism of action of staphyloxanthin biosynthesis inhibitors. Therefore, our molecular simplification strategies afforded promising lead compounds for the development of drugs that modulate S. aureus staphyloxanthin biosynthesis.

Callistemon citrinus bioactive metabolites as new inhibitors of methicillin-resistant Staphylococcus aureus biofilm formation

Ethnopharmacological relevanceThe development of new inhibitors of bacterial virulence factors from natural origin has recently received significant attention. Callistemon citrinus Skeels is an important plant of great medicinal value. Its antimicrobial activity is well documented. Although several compounds were isolated from this plant, the actual bioactive compounds responsible for its antimicrobial activity are still unrevealed. Aim of the studyTo evaluate the effect of C. citrinus crude extract and isolated compounds on methicillin-resistant and sensitive Staphylococcus aureus. Materials and methodsThe methylene chloride-methanol extract (MME) of C. citrinus leaves was prepared by Soxhlet apparatus. Biologically guided fractionation of MME was accomplished using several normal and reversed phase silica gel columns. The potency of MME and its isolated compounds against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) was evaluated. In addition, the mechanism of resistance was studied using three virulence factors; antibiofilm activity, inhibition of staphyloxanthin biosynthesis and effect on acid tolerance. Ultrastructural changes in MRSA and MSSA were observed by TEM to understand mode of action of these compounds. ResultsPulverulentone A (C1), 8- desmethyl eucalyptin (C2) and eucalyptin (C3) were isolated from the most bioactive fraction of MME. Confocal scanning laser microscopy images revealed that C. citrinus isolated compounds destroyed the intact architecture of biofilm, thickness and reduced its biomass. Pulverulentone A (C1) showed the most potent anti-biofilm activity up to 71% and 62.3% against MRSA and MSSA, respectively. It also exhibited the highest inhibition of staphyloxanthin biosynthesis of MRSA and MSSA by 55.6% and 54.5%, respectively. The bacterial cell membrane was compromised, losing its integrity and releasing important cellular constituents when exposed to C1-C3 ConclusionsC. citrinus phenolics and acylphloroglucinols may serve as potential source of plant-based antibacterials and thus could be implicated to control MRSA biofilm formation.

5-Dodecanolide interferes with biofilm formation and reduces the virulence of Methicillin-resistant Staphylococcus aureus (MRSA) through up regulation of agr system

Methicillin resistant Staphylococcus aureus (MRSA) is a predominant human pathogen with high morbidity that is listed in the WHO high priority pathogen list. Being a primary cause of persistent human infections, biofilm forming ability of S. aureus plays a pivotal role in the development of antibiotic resistance. Hence, targeting biofilm is an alternative strategy to fight bacterial infections. The present study for the first time demonstrates the non-antibacterial biofilm inhibitory efficacy of 5-Dodecanolide (DD) against ATCC strain and clinical isolates of S. aureus. In addition, DD is able to inhibit adherence of MRSA on human plasma coated Titanium surface. Further, treatment with DD significantly reduced the eDNA synthesis, autoaggregation, staphyloxanthin biosynthesis and ring biofilm formation. Reduction in staphyloxanthin in turn increased the susceptibility of MRSA to healthy human blood and H2O2 exposure. Quantitative PCR analysis revealed the induced expression of agrA and agrC upon DD treatment. This resulted down regulation of genes involved in biofilm formation such as fnbA and fnbB and up regulation of RNAIII, hld, psmα and genes involved in biofilm matrix degradation such as aur and nuc. Inefficacy of DD on the biofilm formation of agr mutant further validated the agr mediated antibiofilm potential of DD. Notably, DD was efficient in reducing the in vivo colonization of MRSA in Caenorhabditis elegans. Results of gene expression studies and physiological assays unveiled the agr mediated antibiofilm efficacy of DD.

Lactobacillus plantarum USM8613 Aids in Wound Healing and Suppresses Staphylococcus aureus Infection at Wound Sites.

This study aimed to elucidate the targets and mechanisms of anti-staphylococcal effects from bioactive metabolites produced by lactic acid bacteria. We aimed to better understand the safety and efficacy of these bioactive metabolites in in vivo systems, typically at topical sites. The cell-free supernatant and protein-rich fraction from Lactobacillus plantarum USM8613 inhibited staphyloxanthin biosynthesis, reduced (p < 0.05) the cell number of Staphylococcus aureus by 106CFU/mL and reduced biofilm thickness by 55% in S. aureus-infected porcine skins. Genome-wide analysis and gene expression analysis illustrated the production of several plantaricins, especially the plantaricins EF and JK that enhanced the anti-staphylococcal effects of L. plantarum USM8613. In vivo data using rats showed that the protein-rich fraction from L. plantarum USM8613 exerted wound healing properties via direct inhibition of S. aureus and promoted innate immunity, in which the expression of β-defensin was significantly (p < 0.05) upregulated by 3.8-fold. The protein fraction from L. plantarum USM8613 also significantly enhanced (p < 0.05) the production of cytokines and chemokines through various stages of wound recovery. Using ∆atl S. aureus, the protein-rich fraction from L. plantarum USM8613 exerted inhibitory activity via targeting the atl gene in S. aureus. Taken altogether, our present study illustrates the potential of L. plantarum USM8613 in aiding wound healing, suppressing of S. aureus infection at wound sites and promoting host innate immunity.

A computational knowledge-base elucidates the response of Staphylococcus aureus to different media types.

S. aureus is classified as a serious threat pathogen and is a priority that guides the discovery and development of new antibiotics. Despite growing knowledge of S. aureus metabolic capabilities, our understanding of its systems-level responses to different media types remains incomplete. Here, we develop a manually reconstructed genome-scale model (GEM-PRO) of metabolism with 3D protein structures for S. aureus USA300 str. JE2 containing 854 genes, 1,440 reactions, 1,327 metabolites and 673 3-dimensional protein structures. Computations were in 85% agreement with gene essentiality data from random barcode transposon site sequencing (RB-TnSeq) and 68% agreement with experimental physiological data. Comparisons of computational predictions with experimental observations highlight: 1) cases of non-essential biomass precursors; 2) metabolic genes subject to transcriptional regulation involved in Staphyloxanthin biosynthesis; 3) the essentiality of purine and amino acid biosynthesis in synthetic physiological media; and 4) a switch to aerobic fermentation upon exposure to extracellular glucose elucidated as a result of integrating time-course of quantitative exo-metabolomics data. An up-to-date GEM-PRO thus serves as a knowledge-based platform to elucidate S. aureus’ metabolic response to its environment.

Candida albicans quorum-sensing molecule farnesol modulates staphyloxanthin production and activates the thiol-based oxidative-stress response in Staphylococcus aureus

ABSTRACTMicrobial species utilize secreted-signaling molecules to coordinate their behavior. Our previous investigations demonstrated a key role for the Candida albicans-secreted quorum-sensing molecule farnesol in modulating Staphylococcus aureus response to antimicrobials in mixed biofilms. In this study, we aimed to provide mechanistic insights into the impact of farnesol on S. aureus within the context of inter-species interactions. To mimic biofilm dynamics, farnesol-sensitized S. aureus cells were generated via sequential farnesol exposure. The sensitized phenotype exhibited dramatic loss of the typical pigment, which we identified as staphyloxanthin, an important virulence factor synthesized by the Crt operon in S. aureus. Additionally, farnesol exposure exerted oxidative-stress as indicated by transcriptional analysis demonstrating alterations in redox-sensors and major virulence regulators. Paradoxically, the activated stress-response conferred S. aureus with enhanced tolerance to H2O2 and phagocytic killing. Since expression of enzymes in the staphyloxanthin biosynthesis pathway was not impacted by farnesol, we generated a theoretical-binding model which indicated that farnesol may block staphyloxanthin biosynthesis via competitive-binding to the CrtM enzyme crucial for staphyloxanthin synthesis, due to high structural similarity to the CrtM substrate. Finally, mixed growth with C. albicans was found to similarly induce S. aureus depigmentation, but not during growth with a farnesol-deficient C. albicans strain. Collectively, the findings demonstrate that a fungal molecule acts as a redox-cycler eliciting a bacterial stress response via activation of the thiol-based redox system under the control of global regulators. Therefore, farnesol-induced transcriptional modulations of key regulatory networks in S. aureus may modulate the pathogenesis of C. albicans-S. aureus co-infections.

Network analytics approach towards identifying potential antivirulence drug targets within the Staphylococcus aureus staphyloxanthin biosynthetic network

Staphylococcus aureus is associated with several clinically significant infections among humans and infections associated with antibiotic-resistant strains are growing in frequency. Antivirulence strategies shift the target of drugs from bacterial growth to the infection process resulting to milder evolutionary pressure for the development of bacterial resistant strains. Staphyloxanthin (STX) is a yellowish-orange carotenoid pigment synthesized by S. aureus and this carotenoid functions as an important virulence factor for the bacteria. In this study, we elucidated whether network analytics can be used as a viable tool to identify significant components in the STX biosynthetic network which in-turn could serve as possible antivirulence drug targets. For confirmation, we correlated our results to known drugs that were able to inhibit STX biosynthesis. Throughout this study, we established that crtN(1) activity and 4,4′-diaponeurosporene amounts are significant components in the STX biosynthetic network and, moreover, network analytics can aid in identifying antivirulence drug targets within the STX biosynthetic network. Similarly, we found that network analytics is capable of identifying multiple potential targets simultaneously. Taken together, we propose that an effective antivirulence drug against S. aureus STX biosynthesis would involve targeting crtN(1) activity, 4,4′-diaponeurosporene levels, or both components.