tRNA Modification by GidA/MnmE is Necessary for Streptococcus pyogenes Virulence: a New Strategy to Make Live Attenuated Strains

Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, MO 63110-1093

^* To whom correspondence should be addressed. Email: caparon{at}borcim.wustl.edu.

	Abstract

Studies directed at vaccine development and mucosal immunity against Streptococcus pyogenes would benefit from the availability of live attenuated strains. Our approach for production of candidate live attenuated strains was to identify mutations that did not alter growth in vitro, did not alter the overall complement of virulence factors produced, but did result in reduced levels of expression of multiple secreted virulence factors. A global reduction but not elimination of expression would likely lead to attenuation while maximizing the number of antigenic targets available for stimulation of immunity. Adaptation of Tn5-based transposome mutagenesis to S. pyogenes with initial screening for reduced expression of the SpeB protease, resulted in the identification of mutations in gidA, which encodes an enzyme involved in tRNA modification. Reduced SpeB expression was due to delayed onset of speB transcription resulting from reduced translation efficiency of the message for RopB, a transcriptional activator. Overall, GidA^- mutants had a near-normal global transcription profile, but expressed significantly reduced levels of multiple virulence factors due to impaired translation efficiencies. A translation defect was supported by the observation that mutants lacking MnmE, which functions in the same tRNA modification pathway as GidA, phenocopied GidA deficiency. Mutants stimulated an identical cytokine response in cultured macrophages, with the exception of reduced levels of TNF-alpha and IL-23. Significantly, GidA^- mutants were highly attenuated in the murine ulcer model of soft tissue infection. These characteristics suggest that GidA pathway tRNA modification mutants are attractive candidates for further evaluation as live attenuated strains.