Project description:Staphylococcus aureus is a Gram-positive human pathogen causing a variety of human diseases in both hospital and community settings. This bacterium is so closely associated with prophages that it is rare to find S. aureus isolates without prophages. Two phages are known to be important for staphylococcal virulence: the beta-hemolysin (hlb) converting phage and the Panton-Valentine Leukocidin (PVL) converting phage. The hlb-converting phage is found in more than 90% of clinical isolates of S. aureus. This phage produces exotoxins and immune modulatory molecules, which inhibit human innate immune responses. The PVL-converting phage produces the two-component exotoxin PVL, which can kill human leucocytes. This phage is wide-spread among community-associated methicillin resistant S. aureus (CA-MRSA). It also shows strong association with soft tissue infections and necrotizing pneumonia. Several lines of evidence suggest that staphylococcal prophages increase bacterial virulence not only by providing virulence factors but also by altering bacterial gene expression: 1) Transposon insertion into prophage regulatory genes, but not into the genes of virulence factors, reduced S. aureus killing of Caenorhabditis elegans.; 2) Although the toxins and immune modulatory molecules encoded by the hlb- converting phages do not function in the murine system, deletion of ϕNM3, the hlb-converting phage in S. aureus Newman, reduced staphylococcal virulence in the murine abscess formation model. 3) In a preliminary microarray experiment, prophages in S. aureus Newman altered the expression of more than 300 genes. In this research proposal, using microarray and high-throughput quantitative RT-PCR (qRT-PCR) technologies, we will identify the effects of the two important staphylococcal phages on the gene expression of S. aureus in both in vitro and in vivo conditions. This project is intended to be completed within one year. All the data – microarray, qRT-PCR and all the primer sequences- will be made available to public 6 month after completion. Data from this project will help us to understand the role of prophages in the S. aureus pathogenesis and can lead to development of a strategy to interfere with the pathogenesis process. Following strains were grown in TSA broth: Staphylococcus aureus USA300 (reference) Staphylococcus aureus USA300 with deletion of ϕSa2usa (Query) Staphylococcus aureus USA300 with deletion of ϕSa3usa (Query) Staphylococcus aureus USA300 Prophage-free mutant (Query) Staphylococcus aureus USA300 Prophage-free mutant lysogenized with ϕSa2mw (Query) Staphylococcus aureus USA300 Prophage-free mutant lysogenized with ϕSa3usa (Query) strain: Staphylococcus aureus USA300 Prophage-free mutant lysogenized with both ϕSa2mw and ϕSa3usa (Query) RNA samples were harvested at early log, midlog and stationary phase.Samples were hybridized on aminosilane coated slides with 70-mer oligos.

Project description:Staphylococcus aureus causes a spectrum of human infection. Diagnostic delays and uncertainty lead to treatment delays and inappropriate antibiotic use. A growing literature suggests the hostM-bM-^@M-^Ys inflammatory response to the pathogen represents a potential tool to improve upon current diagnostics. The hypothesis of this study is that the host responds differently to S. aureus than to E. coli infection in a quantifiable way, providing a new diagnostic avenue. This study uses Bayesian sparse factor modeling and penalized binary regression to define peripheral blood gene-expression classifiers of murine and human S. aureus infection. The murine-derived classifier distinguished S. aureus infection from healthy controls and Escherichia coli-infected mice across a range of conditions (mouse and bacterial strain, time post infection) and was validated in outbred mice (AUC>0.97). A S. aureus classifier derived from a cohort of 95 human subjects distinguished S. aureus blood stream infection (BSI) from healthy subjects (AUC 0.99) and E. coli BSI (AUC 0.82). Murine and human responses to S. aureus infection share common biological pathways, allowing the murine model to classify S. aureus BSI in humans (AUC 0.84). Both murine and human S. aureus classifiers were validated in an independent human cohort (AUC 0.95 and 0.94, respectively). The approach described here lends insight into the conserved and disparate pathways utilized by mice and humans in response to these infections. Furthermore, this study advances our understanding of S. aureus infection; the host response to it; and identifies new diagnostic and therapeutic avenues. To create a host gene expression-based classifier for S. aureus infection, mice from a variety of experimental conditions were utilized. Seven different strains of inbred mice (n=187 total) were challenged with four different S. aureus strains via intraperitoneal inoculation and sacrificed at various time points as described in Methods. The comparator group for model derivation included 50 A/J or C57BL/6J mice inoculated with E. coli (O18:K1:H7) as well as 54 uninfected mice. Next, the murine S. aureus classifier was externally validated in outbred CD-1 mice with S. aureus infection (Sanger 476 or USA300), E. coli infection (O18:K1:H7), or uninfected controls (10 animals per condition). Method: All experiments were performed on mice 6-8 weeks old. For the murine S. aureus predictor, seven inbred mouse strains (3 mice/strain: 129S1/SvImJ, A/J, AKR/J, BALB/cByJ, C57BL/6J, C3H/HeJ, and NOD/LtJ) were IP inoculated with 107 CFU/g of S. aureus Sanger476, euthanized at 2h after injection, and bled. This was repeated using four different S. aureus strains (USA100, USA300, MW2, and Sanger476) in A/J mice (n=3 per S. aureus strain). For time series experiments, both A/J and C57BL/6J mouse strains were IP inoculated with S. aureus Sanger476 as above, and sacrificed at 2, 4, 6, and 12h after injection (n=5 per time point).

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Staphylococcus aureus

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