Large-Scale Identification of Genes Required for Full Virulence ofStaphylococcus aureus

Abstract

Gene products required for in vivo growth and survival of microbial pathogens comprise a unique functional class and may represent new targets for antimicrobial chemotherapy, vaccine construction, or diagnostics. Although some factors governing Staphylococcus aureus pathogenicity have been identified and studied, a comprehensive genomic analysis of virulence functions will be a prerequisite for developing a global understanding of interactions between this pathogen and its human host. In this study, we describe a genetic screening strategy and demonstrate its use in screening a collection of 6,300 S. aureus insertion mutants for virulence attenuation in a murine model of systemic infection. Ninety-five attenuated mutants were identified, reassembled into new pools, and rescreened using the same murine model. This effort identified 24 highly attenuated mutants, each of which was further characterized for virulence attenuation in vivo and for growth phenotypes in vitro. Mutants were recovered in numbers up to 1,200-fold less than wild type in the spleens of systemically infected animals and up to 4,000-fold less than wild type in localized abscess infections. Genetic analysis of the mutants identified insertions in 23 unique genes. The largest gene classes represented by these mutants encoded enzymes involved in small-molecule biosynthesis and cell surface transmembrane proteins involved in small-molecule binding and transport. Additionally, three insertions defined two histidine kinase sensor-response regulator gene pairs important for S. aureus in vivo survival. Our findings extend the understanding of pathogenic mechanisms employed by S. aureus to ensure its successful growth and survival in vivo. Many of the gene products we have identified represent attractive new targets for antibacterial chemotherapy.

Simultaneous lack of catalase and beta-toxin in Staphylococcus aureus leads to increased intracellular survival in macrophages and epithelial cells and to attenuated virulence in murine and ovine models

“…CI is a sensitive measure of the relative degree of virulence attenuation of a particular mutant in mixed infections with the wild-type strain (Benton et al, 2004). To provide more data about the virulence of the mutants, CI was determined in vivo, using an established mouse subcutaneous model of infection (Horsburgh et al, 2001).…”

“…CI was measured as described previously (Benton et al, 2004) in an established murine abscess model of infection (Horsburgh et al, 2001), with the following modifications. Mutant and wild-type strains were separately grown to stationary phase in BHI broth, harvested, washed and resuspended in sterile PBS.…”

New Insights into the WalK/WalR (YycG/YycF) Essential Signal Transduction Pathway Reveal a Major Role in Controlling Cell Wall Metabolism and Biofilm Formation inStaphylococcus aureus

“…Genome scanning has predicted 17 potential TCSs in S. aureus, some of which have been reported to be regulators of bacterial virulence. The AgrCA TCS is a well-known global regulatory system controlling numerous genes involved in S. aureus virulence, such as cytotoxin-and hemolysin-encoding genes, but other TCSs, such as ArlSR, SaeSR, VraSR, and SsrAB, have also been reported to play roles in S. aureus virulence (2,32,41).…”

The Virulence Regulator Sae ofStaphylococcus aureus:Promoter Activities and Response to Phagocytosis-Related Signals

“…Microarray analysis and proteomics have revealed that most of the genes activated by sae are involved in bacterial adhesion, immune modulation, or toxicity (25,29,38). In addition, the importance of gene regulation by sae in vivo was shown in several animal models (3,15,16,29,43).The sae locus consists of four open reading frames, two of which (saeR and saeS) show strong sequence homology to response regulators and to histidine kinases (HKs) of bacterial two-component regulators (10). Two additional open reading frames, saeP and saeQ, both located upstream of saeRS, are likely to be important for the functionality of the sae operon (33, 39).…”

“…Microarray analysis and proteomics have revealed that most of the genes activated by sae are involved in bacterial adhesion, immune modulation, or toxicity (25,29,38). In addition, the importance of gene regulation by sae in vivo was shown in several animal models (3,15,16,29,43).…”