The human bacterial pathogen group A Streptococcus (GAS) causes a broad spectrum of diseases, including pharyngitis, impetigo, and necrotizing fasciitis. The ability of GAS to cause such disease diversity is in part due to the coordinated expression of specific subsets of virulence factors. Small regulatory RNAs (sRNAs) represent a poorly understood area of regulation in GAS and related pathogens. The goal of the proposed research is to characterize the mechanism of action of the virulence-regulating GAS sRNA FASX as a means to identify new targets for manipulation by novel antimicrobial agents. This research is of interest to the infectious diseases community as a consequence of the following observations. First, preliminary data indicates that FASX regulates GAS virulence. Second, the growth-phase-dependent transcription of FASX is consistent with a role in the transition of GAS between phases of infection. Third, the mechanism by which FASX, or indeed any GAS sRNA, regulates expression is unknown. Fourth, while sRNA-mediated regulation has been well-studied in pathogens that encode a homologue of the RNA-binding protein Hfq, little is known in those that lack an Hfq homologue (e.g. pathogens of the genera Streptococcus, Enterococcus, and Mycobacterium). We will achieve our goal by testing the following hypotheses: (i) FASX is a major regulator of GAS virulence factors. To identify the breath of FASX-mediated regulation in GAS we will use two-dimensional liquid chromatography mass spectrometry analysis to compare the proteomes of a clinical GAS isolate with its isogenic fasX mutant derivative. Proteomes will be compared in vitro and ex vivo, with ex vivo conditions being growth in human plasma (an invasive infection model) and human saliva (a pharyngeal infection model). (ii) FASX binds mRNAs and/or proteins to regulate virulence factor production. We have fused a streptomycin-binding RNA aptamer to FASX that enables the hybrid RNA to be retained within a streptomycin affinity matrix. We will isolate GAS mRNAs and/or proteins that interact with our FASX hybrid by performing pull-down assays. The identity of FASX-binding mRNAs and/or proteins will be determined through use of a custom microarray or by mass spec analysis, respectively, and confirmed using in vitro binding assays. (iii) Specific nucleotides within FASX are required for activity. To facilitate investigation of FASX regulatory targets and mechanism/s of action we will perform site-directed mutagenesis on fasX. FASX nucleotides will be scored as having no role, a moderate role, or a major role in activity based upon the ability of mutant fasX alleles to restore streptokinase activity to fasX mutant strain 2221FASX. Candidate FASX:mRNA interactions will be analyzed bioinformatically to highlight regions of complementary base-pairing. Putative base-pairing will be tested in vivo by fusing those mRNA regions predicted to hybridize with FASX to a lacZ reporter gene, and measuring 2-gal activity in the presence and absence of FASX.
Each year in the U.S. there are ~30 million cases of GAS pharyngitis. The proposed research would provide molecular insight into an understudied field of virulence regulation in GAS and related pathogens. Public health may be enhanced through the long-term goal of translating knowledge of FASX regulatory pathways into new treatment and/or preventative regimes based upon the inhibition of these pathways by novel antimicrobial agents.
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