Expression analysis of Staphylococcus aureus USA300 LAC in response to vehicle and benzbromarone.
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ABSTRACT: Compilation fo whole genome gene expression changes in Staphylococcus aureus USA300 LAC cultures grown in the presence of vehicle or the anti-gout drug benzbromarone. The drug was intially screened as effective against the agr quorum sensing system in Staphylococcus aureus AH1677. A microarray study using total RNA harvested from three cultures of Staphylococcus aureus USA300 LAC plus vehicle control and three cultures of Staphylococcus aureus USA300 LAC plus 12 uM benzbromarone.
Project description:Staphylococcus aureus is a leading cause of bloodstream infections worldwide. In the United States, many of these infections are caused by a strain known as USA300. Although progress has been made, our understanding of the S. aureus molecules that promote bacteremia and survival in human blood is incomplete. To that end, we analyzed the USA300 transcriptome during culture in human blood, human serum, and trypticase soy broth (TSB), a standard laboratory culture media. Notably, genes encoding several cytolytic toxins were up-regulated in human blood over time, and hlgA, hlgB, and hlgC (encoding gamma-hemolysin subunits HlgA, HlgB, and HlgC) were among the most highly up-regulated genes at all time points. Culture supernatants derived from a USA300 isogenic hlgABC-deletion strain (LAC?hlgABC) had significantly reduced capacity to form pores in human neutrophils and ultimately cause neutrophil lysis. Compared with the wild-type USA300 strain (LAC), LAC?hlgABC had modestly reduced ability to cause mortality in a mouse bacteremia model. On the other hand, wild-type and LAC?hlgABC strains caused virtually identical disease in a mouse skin infection model, and bacterial survival and neutrophil lysis after phagocytosis in vitro was similar between these strains. Comparison of the cytolytic capacity of culture supernatants from wild-type and isogenic deletion strains lacking hlgABC, lukS/F-PV (encoding PVL), and/or lukDE revealed significant functional redundancy among two-component leukotoxins in vitro. These findings may explain in part the apparent limited contribution of any single two-component leukotoxin to USA300 immune evasion and virulence. S. aureus strain USA300 transcriptome during culture in human blood, human serum, and trypticase soy broth (TSB): time course.
Project description:Compilation fo whole genome gene expression changes in Staphylococcus aureus USA300 LAC cultures grown in the presence of vehicle or the anti-gout drug benzbromarone. The drug was intially screened as effective against the agr quorum sensing system in Staphylococcus aureus AH1677.
Project description:S. aureus biofilms are associated with the organism's ability to cause disease. Biofilm associated bacteria must cope with the host's innate immune system. We used commercially available Affymetrix S. aureus GeneChips to compare the gene expression properties of 4 and 6 day established biofilms following short (1 hr)- and long (24 hr)- term exposure to macrophages and neutrophils. S. aureus strain USA300 LAC biofilms where formed for 4 or 6 days. Established biofilms were then exposed to macrophages for 1 or 24 hr. Alternatively, biofilms were exposed to neutrophils for 1 or 4 hr. Total bacterial RNA was isolated and subjected to GeneChip hybridization and analysis. We sought to determine the regulatory effects of Macrophages and Neutrophils on established S. aureus biofilms.
Project description:Staphylococcus aureus USA300 wild type strain was cultivated in RPMI medium in 3 biological replicates and harvested at an OD500 of 0.5 before (as control) and at 30 min after exposure to 1.5 mM in RPMI HOCl stress. Cells were disrupted in 3 mM EDTA/ 200 mM NaCl lysis buffer with a Precellys24 ribolyzer followed by RNA isolation using the acid phenol extraction protocol as described. The RNA quality was approved by Trinean Xpose (Gentbrugge, Belgium) and the Agilent RNA Nano 6000 kit using an Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA, USA) was used to remove the rRNA. TruSeq Stranded mRNA Library Prep Kit from Illumina (San Diego, CA, USA) was applied to prepare the cDNA libraries. The cDNAs were sequenced paired end on an Illumina HiSeq 1500 (San Diego, CA, USA) using 70 and 75 bp read length and a minimum sequencing depth of 10 million reads per library. The paired end cDNA reads were mapped to the Staphylococcus aureus USA300 genome sequence (accession number FPR3757_CP255) using bowtie2 v2.2.7 (Langmead and Salzberg, 2012) with default settings for paired-end read mapping. All mapped sequence data were converted from SAM to BAM format with SAMtools v1.3 (Li et al., 2009) and imported to the software ReadXplorer v.2.2 (Hilker et al., 2016).
Project description:The staphylococcal accessory regulator A (sarA) impacts the extracellular accumulation of Staphylococcus aureus virulence factors at the level of intracellular production and extracellular protease-mediated degradation. To assess the relative impact of these two functions, we previously used a proteomics approach that measures protein abundance as a function of all proteoforms to demonstrate that mutation of sarA results in increased levels of extracellular proteases and assess the impact of this on the accumulation of S. aureus exoproteins1. While this approach confirmed that protease-mediated degradation has a significant impact on the S. aureus exoproteome, it was potentially limited in that it did not take into account the possibility that large, stable proteolytic products from a given protein could result in false negatives when quantified by total proteoforms. Here, we present an expanded proteomics approach that utilizes a dual quantitative method for measuring abundance at both the total proteoform and full-length exoprotein levels. Specifically, proteins present in conditioned medium from overnight, stationary phase cultures of the USA300 strain LAC, an isogenic sarA mutant, and a sarA mutant unable to produce any of the known extracellular proteases (sarA/protease) were resolved using one-dimensional gel electrophoresis. Using methods that focus on total proteoforms vs. methods that focus specifically on full-length proteins, quantitative proteomic comparisons of sarA vs sarA/protease mutants identified proteins that were degraded in a protease dependent manner owing to mutation of sarA, while comparisons of a sarA/protease mutant vs the LAC parent strain identified proteins in which abundance was altered in a sarA mutant in a protease-independent manner. Furthermore, the proteins uniquely identified by the full-length data analysis approach eliminated false negatives observed in the total proteoform analysis. This approach provided for a more comprehensive and robust analysis of the impact of mutating sarA and protease-mediated degradation on the S. aureus exoproteome.
Project description:S. aureus response to fakA deletion Gene expression profiles were generated by microarray analysis of S. aureus WT (USA300) or fakAKO S. aureus was grown in LB to an OD600nm of 0.45, and RNA was extracted to look at the gobal gene expression.
Project description:Subinhibitory concentrations of the neuroleptic drug thioridazine (TDZ) are well-known to enhance the killing of methicillin-resistant S. aureus (MRSA) by β-lactam antibiotics, however, the mechanism underlying the synergy between TDZ and β-lactams is not fully understood. In the present study we have examined the effect of a subinhibitory concentration of TDZ on antimicrobial resistance, the global transcriptome, and the cell wall composition of MRSA USA300. We show that TDZ is able to sensitize the bacteria to several classes of antimicrobials targeting the late stages of peptidoglycan synthesis. Furthermore, our microarray analysis demonstrates that TDZ modulates the expression of genes encoding membrane and surface proteins, transporters, and enzymes involved in amino acid biosynthesis. Interestingly, resemblance between the transcriptional profile of TDZ treatment and the transcriptomic response of S. aureus to known inhibitors of cell wall synthesis suggests that TDZ disturbs peptidoglycan biosynthesis at a stage that precedes transpeptidation. In support of this notion, dramatic changes in the muropeptide profile of USA300 were observed following growth in the presence of TDZ, indicating that TDZ can interfere with the formation of the pentaglycine branches. Strikingly, the addition of glycine to the growth medium relieved the effect of TDZ on the muropeptide profile. Furthermore, exogenous glycine offered a modest protective effect against TDZ-induced β-lactam sensitivity. We propose that TDZ exposure leads to a shortage of intracellular amino acids, including glycine, which is required for the production of normal peptidoglycan precursors with pentaglycine branches, the correct substrate of S. aureus penicillin-binding proteins. Collectively, this work demonstrates that TDZ has a major impact on the cell wall biosynthesis pathway in S. aureus and provides new insights into how MRSA may be sensitized towards β-lactam antibiotics. Staphylococcus aureus USA300 was grown to early exponential phase and treated with TDZ (16 µg/ml) alone or in combination with DCX (0.125 µg/ml) for 30 min. Changes in global gene expression were analyzed using the untreated culture as control. Hybridizations were performed in triplicate using RNA isolated from independent cultures.
Project description:A MT02 sensitive and resistant strain of Staphylococcus LAC USA300 JE2 were exposed to MT02. From those cell RNA was extracted and anylsed by RNA-Seq
Project description:Recurrent epidemics of methicillin-resistant Staphylococcus aureus (MRSA) have illustrated that the effectiveness of antibiotics in clinical application is rapidly fading. A feasible approach is to combine natural products with existing antibiotics to achieve an antibacterial effect. In this molecular docking study, we found that theaflavin (TF) preferentially binds the allosteric site of penicillin-binding protein 2a (PBP2a), inducing the PBP2a active site to open, which is convenient for β-lactam antibiotics to treat MRSA infection, instead of directly exerting antibacterial activity at the active site. Subsequent TMT-labeled proteomics analysis showed that TF treatment did not significantly change the landscape of the Staphylococcus aureus (S. aureus) USA300 proteome.Checkerboard dilution tests and kill curve assays were performed to validate the synergistic effect of TF and ceftiofur, and the fractional inhibitory concentration index (FICI) was 0.1875.Our findings provide a potential therapeutic strategy to combine existing antibiotics with natural products to resolve the prevalent infections of multidrug-resistant pathogens.
Project description:Staphylococcus aureus USA300 and Pseudomonas aeruginosa PAO1 were cultured in microaerobiosis (sealed bottles using a 1:4 medium-to-flask volume ratio without agitation) in Triptic Soy Broth (TSB; Oxoid) supplemented with 0.5% KNO3. S. aureus and P. aeruginosa monocultures were inoculated at a DO600 of 0.05 and incubated for 2h at 37°C. After that, cultures were split in two and furtherly incubated for 2h at 37°C (control) or 39°C (heat shock). A similar protocol was employed for co-culture experiments, where S. aureus and P. aeruginosa were co-inoculated each at a DO600 of 0.05 and followed the same protocol.