Use of a Phosphorylation Site Mutant To Identify Distinct Modes of Gene Repression by the Control of Virulence Regulator (CovR) in Streptococcus pyogenes

Abstract

Control of the virulence regulator/sensor kinase (CovRS) two-component system (TCS) serves as a model for investigating the impact of signaling pathways on the pathogenesis of Gram-positive bacteria. However, the molecular mechanisms by which CovR, an OmpR/PhoB family response regulator, controls virulence gene expression are poorly defined, partly due to the labile nature of its aspartate phosphorylation site. To better understand the regulatory effect of phosphorylated CovR, we generated the phosphorylation site mutant strain 10870-CovR-D53E, which we predicted to have a constitutive CovR phosphorylation phenotype. Interestingly, this strain showed CovR activity only for a subset of the CovR regulon, which allowed for classification of CovR-influenced genes into D53E-regulated and D53E-nonregulated groups. Inspection of the promoter sequences of genes belonging to each group revealed distinct promoter architectures with respect to the location and number of putative CovR-binding sites. Electrophoretic mobility shift analysis demonstrated that recombinant CovR-D53E protein retains its ability to bind promoter DNA from both CovR-D53E-regulated and -nonregulated groups, implying that factors other than mere DNA binding are crucial for gene regulation. In fact, we found that CovR-D53E is incapable of dimerization, a process thought to be critical to OmpR/PhoB family regulator function. Thus, our global analysis of CovR-D53E indicates dimerization-dependent and dimerization-independent modes of CovR-mediated repression, thereby establishing distinct mechanisms by which this critical regulator coordinates virulence gene expression.IMPORTANCE Streptococcus pyogenes causes a wide variety of diseases, ranging from superficial skin and throat infections to life-threatening invasive infections. To establish these various disease manifestations, Streptococcus pyogenes requires tightly coordinated production of its virulence factor repertoire. Here, the response regulator CovR plays a crucial role. As an OmpR/PhoB family member, CovR is activated by phosphorylation on a conserved aspartate residue, leading to protein dimerization and subsequent binding to operator sites. Our transcriptome analysis using the monomeric phosphorylation mimic mutant CovR-D53E broadens this general notion by revealing dimerization-independent repression of a subset of CovR-regulated genes. Combined with promoter analyses, these data suggest distinct mechanisms of CovR transcriptional control, which allow for differential expression of virulence genes in response to environmental cues.KEYWORDS Streptococcus pyogenes, dimerization, pathogenesis, regulation of gene expression, transcriptional regulation, two-component regulatory systems, virulence regulation

Phosphatase activity of the control of virulence sensor kinase CovS is critical for the pathogenesis of group A streptococcus

“…In accordance with the CovR~P levels, strains CovR-A81T and CovS-P285S had reduced SpeB activity on milk plates, while SpeB activity was not affected in CovR-R66H or CovR-L155I ( S1 Fig ). Next, we performed TaqMan qRT-PCR of various known CovR-regulated genes that have previously shown to be regulated by CovR via different mechanisms [ 44 ] to evaluate the effect of the mutations on CovR-mediated transcription regulation ( Fig 4B–4D ). Consistent with the CovR~P level analysis, in strains CovR-A81T and CovS-P285S transcript levels of spyM3_0105 , prtS , sagB , and cbp (which encode a cell surface protein, an IL-8 degrading protease, a pore-forming toxin, and a pilus protein, respectively) resembled that of a covS deletion strain.…”

“…In addition, we have previously described a group of CovR-regulated genes (e.g. sagB , cbp , covR ), whose transcription regulation is independent of CovS [ 14 , 44 ]. Hence, for this group, which we designate as class 0 in this context, CovR~P of only 20% is sufficient to repress gene expression.…”

The group A Streptococcus accessory protein RocA: regulatory activity, interacting partners and influence on disease potential

“…the hemolysin streptolysin O, the thrombolytic agent streptokinase, the chemokine protease SpyCEP, the protease SpeB and the anti‐phagocytic hyaluronic acid capsule) (Graham et al , ; Sumby et al , ; Gryllos et al , ). CovS functions as both a kinase and a phosphatase, with these opposing activities altering the ratio of phosphorylated to non‐phosphorylated CovR, which impacts regulation as most CovR/S‐regulated promoters, are repressed by the phosphorylated form of CovR (Gusa et al , ; Churchward, ; Horstmann et al , ).…”

Tools to map target genes of bacterial two‐component system response regulators

“…However, this approach is not universally applicable because there are examples of RRs where the Asp‐Glu substitution results in an inactive RR (Pazour et al ., ; Webber and Kadner, ). There are also instances where the Asp‐Glu mutant retains a fraction of the activity of the WT, and such mutants can be useful in generating insights on the relationship between phosphorylation and regulation of different targets (Horstmann et al ., ). The in vitro assays may be performed with and without activation to determine the effect of phosphorylation on DNA binding.…”