S. aureus continues to be a major human problem because of emerging antibiotic resistance. The pathogenesis of S. aureus is complex and entails coordinate expression of extracellular and cell-wall associated virulence proteins in response to host signals. Recognizing that host factors may play a major role in modulating bacterial gene expression in vivo, we proposed to construct transcriptional vector using gfp as the reporter gene, evaluate these transcriptional fusion with promoters of putative virulence determinants in the rabbit endocarditis model, apply a DPI (differential fluorescence induction) strategy to screen by FACS and then to characterize some of the novel genes selectively expressed in vivo during the previous 5-year funding period. We have achieved many of these aims by creating an assortment of gfp variant transcriptional fusion vector, Dsred variant and also vector with the luxABCDE reporter gene. We have tested five promoters (sarA, agr, fnbA, cap5 and hla) in the GFPuvr transcriptional vector that has been optimized for the 488/515 excitation and emission spectra. Our findings demonstrated the following: 1) gene expression in vitro differs significantly from those in vivo; 2) promoter activation is dependent on the type of promoters; 3) there is organ and tissue specificity in gene activation; 4) the specific locale within a lesion also affects gene expression (e.g. on the surface vs. deep down in the lesion). In screening for mutants that affect hla expression in the rabbit endocarditis model, we found that, contrary to the in vitro model, hla was still expressed in the double sarA/agr mutant, but not in the sae mutant. We thus reason that sae is an important locus that controls hla and possibly other exoproteins in S. aureus in vivo. Our hypothesis is that additional factors likely control sae and that sae may interact with regulatory factors downstream in the cascade to modulate target gene expression. Based on this hypothesis, we propose the following specific aims: I) The interaction of saeRS with rot and sarT in the control of hla expression in vitro, using Northern, RT-PCR and transcriptional fusions with GFP reporter gene; II) Identification and characterization of genes directly regulated by sae; III) Identification and characterization of novel genes that regulate sae using a Tn917 transposon library with sae promoter driving the luxABCDE reporter gene and IV) Determination of the relative virulence and in vivo gene expression of keys construct within the sae-hla regulatory network. Upon the completion of these studies, we hope to understand how sae is activated in vivo and what target genes sae impacts during infection.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Special Emphasis Panel (ZRG1-DDR (01))
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Peters, Kent
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Dartmouth College
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Carnes, Eric C; Lopez, Deanna M; Donegan, Niles P et al. (2010) Confinement-induced quorum sensing of individual Staphylococcus aureus bacteria. Nat Chem Biol 6:41-5
Tamber, Sandeep; Schwartzman, Joseph; Cheung, Ambrose L (2010) Role of PknB kinase in antibiotic resistance and virulence in community-acquired methicillin-resistant Staphylococcus aureus strain USA300. Infect Immun 78:3637-46
Cheung, Ambrose L; Yang, Soo-Jin; Bayer, Arnold S et al. (2009) Disparity in the in vitro versus in vivo regulation of fibronectin-binding proteins by 2 global regulators, saeRS and sigB, in Staphylococcus aureus. J Infect Dis 200:1371-4
Peterson, M Michal; Mack, Jessica L; Hall, Pamela R et al. (2008) Apolipoprotein B Is an innate barrier against invasive Staphylococcus aureus infection. Cell Host Microbe 4:555-66
Shanks, Robert M Q; Meehl, Michael A; Brothers, Kimberly M et al. (2008) Genetic evidence for an alternative citrate-dependent biofilm formation pathway in Staphylococcus aureus that is dependent on fibronectin binding proteins and the GraRS two-component regulatory system. Infect Immun 76:2469-77
Fu, Zhibiao; Donegan, Niles P; Memmi, Guido et al. (2007) Characterization of MazFSa, an endoribonuclease from Staphylococcus aureus. J Bacteriol 189:8871-9
Bayer, Arnold S; McNamara, Peter; Yeaman, Michael R et al. (2006) Transposon disruption of the complex I NADH oxidoreductase gene (snoD) in Staphylococcus aureus is associated with reduced susceptibility to the microbicidal activity of thrombin-induced platelet microbicidal protein 1. J Bacteriol 188:211-22
Xiong, Yan Q; Willard, Julie; Yeaman, Michael R et al. (2006) Regulation of Staphylococcus aureus alpha-toxin gene (hla) expression by agr, sarA, and sae in vitro and in experimental infective endocarditis. J Infect Dis 194:1267-75
Ingavale, Susham; van Wamel, Willem; Luong, Thanh T et al. (2005) Rat/MgrA, a regulator of autolysis, is a regulator of virulence genes in Staphylococcus aureus. Infect Immun 73:1423-31
Trotonda, Maria Pilar; Manna, Adhar C; Cheung, Ambrose L et al. (2005) SarA positively controls bap-dependent biofilm formation in Staphylococcus aureus. J Bacteriol 187:5790-8

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