A Quorum Sensing-Regulated Protein Binds Cell Wall Components and Enhances Lysozyme Resistance in Streptococcus pyogenes

Abstract

The Rgg2/3 quorum sensing (QS) system is conserved among all sequenced isolates of group A (GAS;). The molecular architecture of the system consists of a transcriptional activator (Rgg2) and a transcriptional repressor (Rgg3) under the control of autoinducing peptide pheromones (SHP2 and SHP3). Activation of the Rgg2/3 pathway leads to increases in biofilm formation and resistance to the bactericidal effects of the host factor lysozyme. In this work, we show that deletion of a small gene, , abolished both phenotypes in response to pheromone signaling. The gene encodes a small, positively charged, secreted protein, referred to as StcA. Analysis of recombinant StcA showed that it can directly interact with GAS cell wall preparations containing phosphodiester-linked carbohydrate polymers but not with preparations devoid of them. Immunofluorescence microscopy detected antibody against StcA bound to the surface of paraformaldehyde-fixed wild-type cells. Expression of StcA in bacterial culture induced a shift in the electrostatic potential of the bacterial cell surface, which became more positively charged. These results suggest that StcA promotes phenotypes by way of ionic interactions with the GAS cell wall, most likely with negatively charged cell wall-associated polysaccharides. This study focuses on a small protein, StcA, that is expressed and secreted under induction of Rgg2/3 QS, ionically associating with negatively charged domains on the cell surface. These data present a novel mechanism of resistance to the host factor lysozyme by GAS and have implications in the relevance of this circuit in the interaction between the bacterium and the human host that is mediated by the bacterial cell surface.

Identification of a novel cationic glycolipid inStreptococcus agalactiaethat contributes to brain entry and meningitis

“…Cytochrome C assay was performed as described previously (55,56) with minor modifications. Briefly, overnight cultures of strains were diluted to an OD 600nm of 0.05 in 20 mL prewarmed THB, with no antibiotic, and grown to mid exponential phase (OD 600nm between 0.48 – 0.6) before cells were pelleted to an OD 600nm of 7 in 1 mL and washed twice with morpholinepropanesulfonic acid (MOPS) at pH 7.…”

AStreptococcusquorum sensing system enables suppression of innate immunity

“…Our previous work illustrated that cell surface properties of GAS are altered via Rgg2/3 QS. 3,4,14 To examine if these surface changes include alterations of the repertoire or display of antigens, macrophages were infected with GAS inactivated by different methods, including heat (heat killed, HK), short wave ultraviolet light (UV), and 4% paraformaldehyde (PFA) treatment. While heat has been shown to alter cell wall peptidoglycans and denature proteins, UV treatment damages DNA and RNA and is less likely to interfere with cell surface properties.…”

“…3 We previously described that activation of Rgg2/3 QS mediates production of biofilms and resistance to the host antimicrobial factor lysozyme, two phenotypes that rely on surface modifications and were shown to require QSregulation of StcA. 3,4 We wondered whether regulation of StcA also enabled suppression of inflammatory pathways in innate immune cells. Infecting macrophages with 0414c and 0450-0460 grown in the presence of SHP (QS-ON) or inactive revSHP (QS-OFF) showed that while spy49_0414c is not required for QS-regulated attenuation of cytokine responses, spy49_0450-0460 is required (Fig 5C).…”

“…Rgg2/3-mediated regulation of stcA promotes aggregation and biofilm development (two phenotypes that require adhesive surface structures) and resistance to lysozyme (an antimicrobial host factor that targets components of the bacterial cell wall). 3,4 Because the surface of GAS interfaces with host cells, we hypothesized that Rgg2/3-mediated alterations to GAS would result in altered host immune responses.…”

24th Annual Midwest Microbial Pathogenesis Conference

“…Since the earliest reports of population-density regulation of bioluminescence in Vibrio fischeri and competence in Streptococcus pneumoniae, we now appreciate that many virulence functions are also regulated via quorum sensing signaling cascades. In this issue of JB, Gogos et al describe a quorum sensing-regulated protein that affects the cell wall and enhances lysozyme resistance of Streptococcus pyogenes (29). Hustmyer et al introduce a new method, rapid arbitrary PCR insertion libraries (RAIL), to define the promoter regions important to acyl homoserine lactone-regulated functions in Vibrio harveyi (30) (see also the accompanying commentary by Matson describing RAIL and its potential for other researchers [31]).…”