RNA-Seq of WT Staphylococcus aureus and dpurR grown to exponential and stationary phase
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ABSTRACT:
ORGANISM(S): Staphylococcus aureus
PROVIDER: GSE132179 | GEO | 2019/06/12
REPOSITORIES: GEO
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Project description:Staphylococcus aureus is a significant cause of human infection. Here, we demonstrate that mutations in the transcriptional repressor of purine biosynthesis, purR, enhance the pathogenic potential of S. aureus. Indeed, systemic infection with purR mutants causes accelerated mortality in mice, which is due to aberrant up-regulation of fibronectin binding proteins (FnBPs). Remarkably, purR mutations can arise upon exposure of S. aureus to stress, such as an intact immune system. In humans, naturally occurring anti-FnBP antibodies exist that, while not protective against recurrent S. aureus infection, ostensibly protect against hypervirulent S. aureus infections. Vaccination studies support this notion, where anti-Fnb antibodies in mice protect against purR hypervirulence. These findings provide a novel link between purine metabolism and virulence in S. aureus.
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Project description:The success of Staphylococcus aureus as a pathogen results from the production of a wealth of virulence determinants that aid in immune evasion, host cell invasion and dissemination of infection. Given the niche specific roles of these factors in infection, their production is controlled by a complex network of regulatory factors. In a continued effort to understand this network, the present study is aimed at characterizing the role of the transcriptional regulator XdrA and its effects on S. aureus gene expression. Using an unbiased global analysis, we find that XdrA has a broad impact on gene expression, influencing the transcription of several important virulence determinants, and factors involved in gene regulation. When assessing the role of XdrA in virulence, we find that an xdrA mutant has an increased ability to survive in whole human blood, mediated in part by increased survival within neutrophils, and an upregulation in expression of several factors involved in immune evasion, including sbi, fnbpA and efb. Furthermore, the increased survival within neutrophils appears to result from an upregulation in expression of sodM, recA, and sae, all of which assist bacterial cells in combating the effects of oxidative stress. In addition to these changes, we find that the xdrA mutant has a decreased abundance of cytolytic toxins, likely resulting from changes in agr and sae activity. We suggest that the broad impact of XdrA on the expression of genes involved in immune evasion, DNA damage, and oxidative stress tolerance, collectively result in a survival advantage allowing for the increased ability to causes disease in vivo, when xdrA is disrupted. In sum, our findings shed new light on the role of XdrA and its seemingly novel influence on S. aureus survival during infection. The dataset contained herein was used to elucidate the whole shotgun secreted proteome (secretome) obtained from 5 hour (mid exponential phase) and 15 hour (stationary phase) planktonic cultures of Staphylococcus aureus USA300 LAC strain, and a derivative of this strain deficient in the gene encoding XdrA.
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Project description:Staphylococcus aureus is an important human pathogen that causes life-threatening infections, and is resistant to the majority of our antibiotic arsenal. This resistance is complicated by the observation that most antibacterial agents target actively growing cells, thus, proving ineffective against slow growing populations, such as cells within a biofilm or in stationary phase. Recently, our group generated updated genome annotation files for S. aureus that not only include protein-coding genes but also regulatory and small RNAs. As such, these annotation files were used to perform a transcriptomic analysis in order to understand the metabolic and physiological changes that occur during transition from active growth to stationary phase; with a focus on sRNAs. We observed ∼24% of protein-coding and 34% of sRNA genes displaying changes in expression by ≥3-fold. Collectively, this study adds to our understanding of S. aureus adaptation to nutrient-limiting conditions, and sheds new light onto the contribution of sRNAs to this process.
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Project description:Staphylococcus aureus is an important human pathogen that causes life-threatening infections, and is resistant to the majority of our antibiotic arsenal. This resistance is complicated by the observation that most antibacterial agents target actively growing cells, thus, proving ineffective against slow growing populations, such as cells within a biofilm or in stationary phase. Recently, our group generated updated genome annotation files for S. aureus that not only include protein-coding genes but also regulatory and small RNAs. As such, these annotation files were used to perform a transcriptomic analysis in order to understand the metabolic and physiological changes that occur during transition from active growth to stationary phase; with a focus on sRNAs. We observed â¼24% of protein-coding and 34% of sRNA genes displaying changes in expression by â¥3-fold. Collectively, this study adds to our understanding of S. aureus adaptation to nutrient-limiting conditions, and sheds new light onto the contribution of sRNAs to this process. Bacterial cells were grown in TSB medium at 37°C with shaking for 3h (exponential growth phase) or 16h (stationary growth phase).
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Project description:RNA-Seq of WT Staphylococcus aureus and dpurR grown to exponential and stationary phase
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Project description:Staphylococcus aureus is a leading pathogen that is currently the most common cause of infection in hospitalized patients. An in-depth genetic analysis of S. aureus virulence genes contributing to pathogenesis is needed to develop novel antimicrobial therapies. However, tools for genetic manipulation in S. aureus are limited, particularly those for gene expression. Here, 38 highly expressed genes were identified in S. aureus USA300_FPR3757 via RNA-seq. Promoter regions from 30 of these genes were successfully cloned, of which 20 promoters exhibited a wide range of activity. By utilizing these active promoters, 20 S. aureus-E. coli shuttle vectors were constructed and evaluated by expressing an egfp reporter gene. Expression of egfp gene under the control of different promoters was confirmed and quantified by Western-blot and qPCR, which suggested that the activity of these promoters varied from 18% to 650% of the activity of PsarA, a widely used promoter for gene expression currently. In addition, our constructed vectors were verified to be of high compatibility on gene expression in different S. aureus strains. Furthermore, these vectors were evaluated and used in overexpressing two endogenous proteins in S. aureus, namely, catalase and the transcriptional repressor of purine biosynthesis (purR). Meanwhile, the physiological functions and phenotypes of overexpressed purR and catalase in S. aureus were validated. Altogether, this evidence indicates that our constructed vectors provide a wide range of promoter activity on gene expression in S. aureus. This developed system will provide a powerful tool for the direct analysis of target gene function in staphylococcal cells.
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