The Streptococcus pyogenes serotype M49 Nra-Ralp3 Transcriptional Regulatory Network and its Control on Virulence Factor Expression from the Novel ERES Pathogenicity Region

Department of Medical Microbiology and Hospital Hygiene, Institute of Medical Microbiology, Virology and Hygiene, Department of Proteome Research, Institute of Immunology, Rostock University Hospital, Schillingallee 70, 18057 Rostock, Germany, Department of Oral and Molecular Microbiology, Osaka University Graduate School of Dentistry, 1-8, Yamadaoka, Suita-Osaka, 565-0871, Japan

^* To whom correspondence should be addressed. Email: bernd.kreikemeyer{at}med.uni-rostock.de.

	Abstract

Many Streptococcus pyogenes (group A streptococcus, GAS) virulence factor and transcriptional regulator encoding genes cluster together in discrete genomic regions. Nra is a central regulator of the FCT-region. Past studies exclusively described Nra as a transcriptional repressor of adhesin and toxin genes. Transcriptome and proteome analysis of a serotype M49 GAS strain and its isogenic Nra-mutant now uncovered the complete Nra regulon profile. Nra is active in all growth-phases tested, with the largest regulon in the transition phase. Almost exclusively virulence factor encoding genes are under Nra repression, including the GAS pilus operon, the capsule synthesis operon, the CMT-translocation system genes, all Mga-region core virulence genes, and other regulators like the Ihk/Irr system, Rgg, and two additional RALP regulators. Surprisingly, our experiments reveal that Nra additionally acts as positive regulator, mostly for genes encoding proteins and enzymes of metabolic function. Epidemiological investigations discovered a strong genetic linkage of one particular regulator under Nra repression, Ralp3 (Spy 0735), with a gene encoding Epf (extracellular protein factor from S. suis). In a serotype-specific fashion, this RE-(Ralp3/epf) gene block is integrated, most likely via transposition, in the ES-(eno/sagA) virulence gene block, which is present in all GAS serotypes. In GAS serotypes M1, M4, M12, M28, and M49 this novel discrete genetic region is therefore designated ERES-(eno/ralp3/epf/sagA) pathogenicicty region. Functional experiments typify Epf as novel GAS plasminogen-binding protein and revealed that Ralp3 activity counteracts Nra and MsmR regulatory activity. Together, next to Mga- and FCT-regions, the ERES-region is the third discrete chromosomal pathogenicity region. All regions are transcriptionally linked, adding another level of complexity to the already existing GAS growth phase-dependent regulatory network.