Genetic Characterization and Virulence Role of the RALP3/LSA Locus Upstream of the Streptolysin S Operon in Invasive M1T1 Group AStreptococcus

Abstract

Group A Streptococcus (GAS) is a leading human pathogen associated with a wide spectrum of mucosal and invasive infections. GAS expresses a large number of virulence determinants whose expression is under the control of several transcriptional regulatory networks. Here we performed the first mutational analysis of a genetic locus immediately upstream of the streptolysin S biosynthetic operon in several GAS genome sequences, including that of the M1T1 serotype, the leading isolates associated with serious invasive disease. The locus consists of a predicted RofA-like stand-alone transcriptional regulator (RALP3) and the largest open reading frame in the GAS genome, encoding a predicted LPXSG motif cell wall-anchored protein we have named LSA (for "large surface-anchored" protein). Comparative reverse transcription-PCR analysis of wildtype M1T1 GAS and an isogenic RALP3-deficient mutant identifies RALP3 as a global transcriptional regulator affecting expression of numerous virulence factor genes, including those for strong repression of the hyaluronic acid capsule and cysteine protease production. RALP3 contributed to GAS epithelial cell invasion and bloodstream survival. LSA was found to be under negative regulation by RALP3 and to influence GAS-epithelial cell interactions and GAS antimicrobial peptide sensitivity. Isogenic M1T1 GAS mutants lacking either RALP3 or LSA were attenuated in a murine model of systemic infection, indicating that this locus plays a role in the virulence potential of the organism.

Effects of the ERES Pathogenicity Region Regulator Ralp3 on Streptococcus pyogenes Serotype M49 Virulence Factor Expression

“…Analysis of the GAS serotype M1 genome sequence (8) led to the identification of two additional Ralp regulators, Ralp3 and Ralp4 (13). Both were partially characterized (19,22). Our analyses indicate that Ralp3 is a transcriptional regulator involved in GAS virulence, thereby confirming the general observation from Kwinn et al (22).…”

“…Capsule expression is strongly linked to survival of GAS in human blood (42). Kwinn et al described Ralp3 as a transcriptional regulator of hasA in GAS serotype M1 (22). hasA encodes a hyaluronate synthase involved in capsule formation, which has a known role in resistance to phagocytic clearance (42).…”

Innate barriers against skin infection and associated disorders

“…Through a complex interaction, secreted SpeB is trapped on the bacterial surface by host α2-macroglobulin that is bound by the cell-wall anchored GAS protein GRAB; the retained SpeB is capable of cleaving and inactivating LL-37 and protecting the bacteria against its antimicrobial action [43]. A surface-anchored protein known as LSA, representing the largest ORF in the GAS genome, also affords the pathogen a level protection from cathelicidin AMP action through an as yet undetermined mechanism [44].…”

Small RNAs in streptococci

“…CodY, Irr, SLS and SptR regulate positively the operon [56][57][58][59] whereas CcpA, CovR, FasX, LuxS, Mga and RopB and members of the Ralp regulator family including RofA, Nra and Ralp3 have repressing effects. 42,56,57,[60][61][62][63][64][65][66][67][68][69][70][71] The nutritional status of the cells also has regulatory roles on sag/pel expression. 46,72 How Pel controls the expression of virulence factors has not yet been deciphered and no primary targets are known.…”