Restoration of the wild-type phenotypes in Streptococcus pyogenes rgg mutant strains depends on the expression level of Rgg transcriptional regulator
Abstract number: P1839
Dmitriev A.V., McDowell E.J., Kappeler K.V., Anbalagan S., Chaussee M.S.
Objectives: The clinical outcomes of Streptococcus pyogenes infections range from mild pharyngitis to severe toxic shock syndrome and necrotising fasciitis. The expression of S. pyogenes virulence factors is coordinately regulated transcriptionally, translationally and post-translationally. Rgg is a global transcriptional regulatory protein necessary for SpeB expression. In a strain-specific manner, Rgg additionally affects numerous phenotypes such as virulence, tolerance to thermal and oxidative stress, fermentation of amino acids during the exponential phase of growth, and the ability to grow in enriched and chemically defined media, among others. Given the potential differences in expression level of Rgg in S. pyogenes strains, it was of interest to determine if a correlation existed between the Rgg-regulated phenotypes and the level of Rgg expression.
Methods: The wild-type S. pyogenes strains NZ131, SF370, and their rgg isogenic mutants were used. Rgg expressing plasmids were constructed by methods of genetic engineering. Complementation of the rgg mutants was done episomally and chromosomally under inducible promoter and chromosomally under native rgg promoter. Chromosomal complementation was done by the insertional mutagenesis. Phenotypes of all the strains were analyzed by the methods of biochemistry and microbiology. Gene expression levels were assessed by quantitative RT-PCR.
Results: The rgg gene was cloned into pMSP3535 vector under control of a nisin-inducible promoter and introduced into the rgg mutant strains resulting in episomal complementation of the rgg gene. Alternatively, the rgg gene was cloned into a suicide vector, which was unable to replicate in streptococci. Subsequently, an intact rgg gene was chromosomally restored either under inducible promoter or native rgg promoter. Restoration of rgg in the chromosome under the control of native promoter restored wild-type phenotypes, as determined by qRT-PCR and biochemical assays. In contrast, both episomal complementation and chromosomal complementation of rgg under control of an inducible promoter, resulting in over-expression of rgg, did not restore any of metabolic properties associated with the wild-type strain.
Conclusion: Rgg transcriptional activity depends on the rgg expression level. Given the importance of the rgg level in controlling virulence factor expression in S. pyogenes, further characterisation of the mechanism of Rgg-mediated gene transcription is necessary.
|Session name:||19th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Helsinki, Finland, 16 - 19 May 2009|
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