Signal Transduction through CsrRS Confers an Invasive Phenotype in Group A Streptococcus

Abstract

The CsrRS (or CovRS) two component system controls expression of up to 15% of the genome of group A Streptococcus (GAS). While some studies have suggested that the sensor histidine kinase CsrS responds to membrane perturbations as a result of various environmental stresses, other data have implicated the human antimicrobial peptide LL-37 and extracellular Mg2+ as specific signals. We now report that Mg2+ and LL-37 have opposite effects on expression of multiple genes that are activated or repressed by the transcriptional regulator CsrR. Using a GAS isolate representative of the recently emerged and widely disseminated M1T1 clone implicated in severe invasive disease, we found marked up-regulation by CsrRS of multiple virulence factors including pyrogenic exotoxin A, DNase Sda1, streptolysin O, and the hyaluronic acid capsular polysaccharide, among others. Topology and surface protein labeling studies indicated that CsrS is associated with the bacterial cell membrane and has a surface-exposed extracellular domain accessible to environmental ligands. Replacement of a cluster of three acidic amino acids with uncharged residues in the extracellular domain of CsrS abrogated LL-37 signaling and conferred a hyporesponsive phenotype consistent with tonic activation of CsrS autokinase activity, an effect that could be overridden by mutation of the CsrS active site histidine. Both loss- and gain-of-function mutations of a conserved site in the receiver domain of CsrR established an essential role for lysine 102 in CsrS-to-CsrR signal transduction. These results provide strong evidence that Mg2+ and LL-37 are specific signals that function by altering CsrS autokinase activity and downstream phosphotransfer to CsrR to modulate its activity as a transcriptional regulator. The representation of multiple antiphagocytic and cytotoxic factors in the CsrRS regulon together with results of in vitro phagocytic killing assays support the hypothesis that CsrRS mediates conversion of GAS from a colonizing to an invasive phenotype in response to signaling by host LL-37.

Characterization of the Effect of the Histidine Kinase CovS on Response Regulator Phosphorylation in Group A Streptococcus

“…To this end, the CovRS wild-type strains MGAS10870 (serotype M3) and MGAS2221 (serotype M1) were grown in THY, high-Mg 2ϩ THY, or THY with a sublethal dose of the human antimicrobial peptide LL-37. This dose of LL-37 was previously shown to relieve CovR-mediated gene repression in a CovS-dependent manner (2). Surprisingly, CovRϳP levels as well as the impacts of distinct growth media differed between the strains of the two GAS serotypes (Fig.…”

“…The strains used in this work are presented in Table 1, and primers used for strain creation are listed in Table S3 in the supplemental material. Strains were grown in a nutrientrich standard laboratory medium (Todd-Hewitt broth with 0.2% yeast extract [THY]) at 37°C in 5% CO 2 with 15 mM MgCl 2 or 100 nM LL-37 added when noted to create high-Mg 2ϩ or LL-37-supplemented conditions, respectively (2). THY without supplemental MgCl 2 was previously determined to have an Mg 2ϩ concentration of ϳ1 mM (30).…”

“…T wo-component gene regulatory systems (TCSs) are a major mechanism by which bacteria detect and respond to external stimuli (1,2). Although there is a diverse array of TCS types, in the canonical TCS, a membrane-embedded histidine kinase reacts to an extracellular signal by altering the phosphorylation status of an aspartate (Asp) residue on a cognate response regulator (3).…”

“…Among the most extensively studied TCSs in terms of contributions to bacterial infectivity is the control of virulence regulator/sensor kinase (CovRS; also known as CsrRS) of group A Streptococcus (GAS) (2,8,9). GAS, also called Streptococcus pyogenes, is a Gram-positive bacterium that causes a diverse array of human infections ranging from uncomplicated pharyngitis (i.e., strep throat) to the highly lethal necrotizing fasciitis (i.e., flesh-eating disease) (10).…”

Two-component signal transduction systems in oral bacteria

“…Because multiple stress conditions promote CovS Spy phosphatase activity, it was proposed that CovS Spy is responsive to general stresses affecting membrane structure [164]. On the other hand, there is also evidence that CovS Spy activities are modulated by specific external ligands, including Mg 2+ , but not other divalent cations, and the human antimicrobial peptide LL-37 at subinhibitory concentrations, but not other antimicrobial peptides [165–168]. CovR Spy is also phosphorylated at the threonine residue T-65 by an STK [145,169], similar to CovR Sag of S. agalactiae [170].…”