Dual-Site Phosphorylation of the Control of Virulence Regulator Impacts Group A Streptococcal Global Gene Expression and Pathogenesis

Abstract

Phosphorylation relays are a major mechanism by which bacteria alter transcription in response to environmental signals, but understanding of the functional consequences of bacterial response regulator phosphorylation is limited. We sought to characterize how phosphorylation of the control of virulence regulator (CovR) protein from the major human pathogen group A Streptococcus (GAS) influences GAS global gene expression and pathogenesis. CovR mainly serves to repress GAS virulence factor-encoding genes and has been shown to homodimerize following phosphorylation on aspartate-53 (D53) in vitro. We discovered that CovR is phosphorylated in vivo and that such phosphorylation is partially heat-stable, suggesting additional phosphorylation at non-aspartate residues. Using mass spectroscopy along with targeted mutagenesis, we identified threonine-65 (T65) as an additional CovR phosphorylation site under control of the serine/threonine kinase (Stk). Phosphorylation on T65, as mimicked by the recombinant CovR T65E variant, abolished in vitro CovR D53 phosphorylation. Similarly, isoallelic GAS strains that were either unable to be phosphorylated at D53 (CovR-D53A) or had functional constitutive phosphorylation at T65 (CovR-T65E) had essentially an identical gene repression profile to each other and to a CovR-inactivated strain. However, the CovR-D53A and CovR-T65E isoallelic strains retained the ability to positively influence gene expression that was abolished in the CovR-inactivated strain. Consistent with these observations, the CovR-D53A and CovR-T65E strains were hypervirulent compared to the CovR-inactivated strain in a mouse model of invasive GAS disease. Surprisingly, an isoalleic strain unable to be phosphorylated at CovR T65 (CovR-T65A) was hypervirulent compared to the wild-type strain, as auto-regulation of covR gene expression resulted in lower covR gene transcript and CovR protein levels in the CovR-T65A strain. Taken together, these data establish that CovR is phosphorylated in vivo and elucidate how the complex interplay between CovR D53 activating phosphorylation, T65 inhibiting phosphorylation, and auto-regulation impacts streptococcal host-pathogen interaction.

Sequence type 1 group B Streptococcus , an emerging cause of invasive disease in adults, evolves by small genetic changes

“…For RNA-Seq analysis, strains were grown in quadruplicate to midexponential phase in Todd-Hewitt broth (Difco) and RNA was isolated using a Qiagen RNeasy kit. RNA-Seq analysis was performed in quadruplicate per strain as described previously (40) and as detailed in SI Appendix, SI Materials and Methods. Transcript levels were considered significantly different if the mean transcript level difference was ≥ 2.0-fold and the final adjusted P value was less than 0.05.…”

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

“…SGAS0004 is a serotype M1 strain that caused a case of bacteremia in Houston, TX, in 2012, and Sanger sequencing was used to determine that SGAS0004 contains a wild-type covRS operon. Strains 10870⌬covS, 2221⌬covS, GAS-SC-1 (covRS wild-type progenitor of GAS-LC-1), and GAS-LC-1 were previously described (17,20,24). Derivatives of strain MGAS10870 that differed only by the presence of a single-amino-acid replacement in CovS were created by using chloramphenicol-resistant, temperature-sensitive plasmid pJL1055 (gift of D. Kasper), as described previously (31).…”

“…Recently, our laboratory used Phos-Tag technology (29) to provide the first qualitative evidence that GAS CovR is phosphorylated in vivo (20). Here, we extend our study of CovR phosphorylation by using quantitative assays to address key knowledge gaps regarding how CovS influences the CovR phosphorylation status, which in turn determines global GAS expression and infectivity.…”

“…For example, it has been established that Asp53 is a key site of CovR phosphorylation resulting in CovR-mediated repression of virulence factor-encoding genes (20,21). However, whether CovS actually influences the CovR phosphorylation status is unknown, as the phenotypes of CovS-and CovR-inactivated strains are distinct (22).…”

Two-component signal transduction systems in oral bacteria

“…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]. Additional tyrosine/serine/threonine sites for CovR Spy phosphorylation were also suggested [171] indicating complexity of mechanisms for activation of this RR.…”