The small regulatory RNA FasX enhances group A Streptococcus virulence and inhibits pilus expression via serotype‐specific targets

Abstract

Summary Bacterial pathogens commonly show intra-species variation in virulence factor expression and often this correlates with pathogenic potential. The group A Streptococcus (GAS) produces a small regulatory RNA (sRNA), FasX, which regulates the expression of pili and the thrombolytic agent streptokinase. As GAS serotypes are polymorphic regarding (a) FasX abundance, (b) the fibronectin, collagen, T-antigen (FCT) region of the genome, which contains the pilus genes (nine different FCT-types), and (c) the streptokinase-encoding gene (ska) sequence (two different alleles), we sought to test whether FasX regulates pilus and streptokinase expression in a serotype-specific manner. Parental, fasX mutant, and complemented derivatives of serotype M1 (ska-2, FCT-2), M2 (ska-1, FCT-6), M6 (ska-2, FCT-1), and M28 (ska-1, FCT-4) isolates were compared. While FasX reduced pilus expression in each serotype, the molecular basis differed, as FasX bound, and inhibited the translation of, different FCT-region mRNAs. FasX enhanced streptokinase expression in each serotype, although the degree of regulation varied. Finally, we established that the regulation afforded by FasX enhances GAS virulence, assessed by a model of bacteremia using human plasminogen-expressing mice. Our data are the first to identify and characterize serotype-specific regulation by an sRNA in GAS, and to show an sRNA directly contributes to GAS virulence.

The FasX Small Regulatory RNA Negatively Regulates the Expression of Two Fibronectin-Binding Proteins in Group A Streptococcus

“…2). The small but reproducible change in prtF1 and prtF2 mRNA abundance by FasX mirrors that observed for the pilus mRNAs (21,23); this points to the primary mode of regulation of PrtF1 and PrtF2 expression by FasX as potentially also being at the level of mRNA translation.…”

“…FasX downregulates the expression of other FCT region (pilus) genes via direct base pairing to target gene transcripts (21,23). Hence, we hypothesized that a similar mechanism underlies the FasX-mediated negative regulation of PrtF1 and PrtF2.…”

“…For this analysis we used the parental serotype M28 GAS isolate MGAS6180 containing empty vector (termed M28 ϩ vector), its fasX mutant derivative M28⌬fasX containing empty vector (M28⌬fasX ϩ vector), and the complemented mutant derivative containing a plasmid-carried wild-type fasX allele (M28⌬fasX ϩ pFasX). We investigated mRNA levels as, for other FasX-regulated targets, changes in mRNA abundance have been observed regardless of whether the main mechanism of regulation is the modulation of mRNA stability (i.e., regulation of streptokinase) (22) or of mRNA translation (i.e., regulation of pilus) (21,23). Relative to the parental strain, the fasX mutant had a small but statistically significant increase in the abundance of prtF2, but not prtF1, mRNA (Fig.…”

“…FasX positively regulates the expression of the thrombolytic agent streptokinase (SKA) by binding to the extreme 5= end of ska mRNA, forming a secondary structure that leads to an enhancement in ska mRNA stability and, subsequently, SKA abundance (22). FasX negatively regulates the expression of GAS pili by, in a serotype-specific manner, binding to the extreme 5= end of mRNAs encoding either a minor or the major pilus proteins and inhibiting their translation by blocking access to the ribosome binding site (21,23).…”

“…To date only one bona fide sRNA has been described in GAS, the 205-nucleotide (nt) FasX sRNA (20). FasX has both positive and negative regulatory targets, and the regulation afforded by FasX contributes to the virulence of this pathogen in a mouse soft-tissue infec-tion model (21). FasX positively regulates the expression of the thrombolytic agent streptokinase (SKA) by binding to the extreme 5= end of ska mRNA, forming a secondary structure that leads to an enhancement in ska mRNA stability and, subsequently, SKA abundance (22).…”