Abstract
ABSTRACTSpxA is a unique transcriptional regulator highly conserved among members of the phylum Firmicutes that binds RNA polymerase and can act as an antiactivator. Why some Firmicutes members have two highly similar SpxA paralogs is not understood. Here, we show that the SpxA paralogs of the pathogen Streptococcus pyogenes, SpxA1 and SpxA2, act coordinately to regulate virulence by fine-tuning toxin expression and stress resistance. Construction and analysis of mutants revealed that SpxA1− mutants were defective for growth under aerobic conditions, while SpxA2− mutants had severely attenuated responses to multiple stresses, including thermal and oxidative stresses. SpxA1− mutants had enhanced resistance to the cationic antimicrobial molecule polymyxin B, while SpxA2− mutants were more sensitive. In a murine model of soft tissue infection, a SpxA1− mutant was highly attenuated. In contrast, the highly stress-sensitive SpxA2− mutant was hypervirulent, exhibiting more extensive tissue damage and a greater bacterial burden than the wild-type strain. SpxA1− attenuation was associated with reduced expression of several toxins, including the SpeB cysteine protease. In contrast, SpxA2− hypervirulence correlated with toxin overexpression and could be suppressed to wild-type levels by deletion of speB. These data show that SpxA1 and SpxA2 have opposing roles in virulence and stress resistance, suggesting that they act coordinately to fine-tune toxin expression in response to stress. SpxA2− hypervirulence also shows that stress resistance is not always essential for S. pyogenes pathogenesis in soft tissue.
Highlights
IMPORTANCE For many pathogens, it is generally assumed that stress resistance is essential for pathogenesis
To determine if additional Clp proteins participate in SpeB regulation and whether ClpX acts in a ClpP-dependent or -independent manner, mutants were generated by a gene insertion strategy in clpP, the genes for the three additional members of the Clp chaperone family (ClpC, ClpE, and ClpL), and a regulator of the transcription of these genes (CtsR) that are found in serotype M14 S. pyogenes HSC5
Of the Clp chaperones, only the loss of ClpX was associated with a complete loss of proteolytic activity when examined on protease indicator plates and a 90% reduction relative to the wild type (WT) as measured by a protease assay (Fig. 1A and B)
Summary
IMPORTANCE For many pathogens, it is generally assumed that stress resistance is essential for pathogenesis. To protect themselves against these stresses, bacteria employ numerous countermeasures via both general and stress-specific mechanisms Examples of the former include alteration of membrane fluidity and degradation of damaged or misfolded proteins. ® mbio.asm.org 1 defensive responses can act as markers of specific temporal stages of disease progression or host compartments, pathogens often coordinately regulate stress response programs with virulence factor expression [2]. Because of the diversity of host tissues that it can infect and the types of diseases that it can cause, host defensive responses challenge Streptococcus pyogenes with numerous environmental stresses This Gram-positive bacterium primarily infects soft tissues of the pharynx and skin to cause diseases that include pharyngitis (strep throat) and impetigo to more severe diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome [3]. The ClpP proteolytic subunit is essential for growth under stress conditions in a variety of Gram-positive bacteria [12,13,14], including various streptococcal species [11, 15, 16]
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