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

<div><p>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 <u>co</u>ntrol of <u>v</u>irulence <u>r</u>egulator (CovR) protein from the major human pathogen group A <i>Streptococcus</i> (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) <i>in vitro</i>. We discovered that CovR is phosphorylated <i>in vivo</i> 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 <i>in vitro</i> 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 <i>covR</i> gene expression resulted in lower <i>covR</i> gene transcript and CovR protein levels in the CovR-T65A strain. Taken together, these data establish that CovR is phosphorylated <i>in vivo</i> and elucidate how the complex interplay between CovR D53 activating phosphorylation, T65 inhibiting phosphorylation, and auto-regulation impacts streptococcal host-pathogen interaction.</p></div

Mass spectra showing that CovR is phosphorylated on threonine 65.

<p>Recombinant CovR was incubated with Stk_KD and then subjected to chymotrypsin digestion. (A) Mass spectra of the MS/MS fragmentation of a 2⁺ ion of the CovR-derived peptide EVTphosRRLQTEKTTY. Fragment marked with blue arrow shows the peptide that has lost phosphoric acid, thus decreasing the apparent mass from 852.92 to 804.27. The peptide marked with the red star corresponds to the y₁₀²⁺ fragment (RRLQTEKTTY) and has not lost phosphoric acid. Thus, T3 must be the position of phosphorylation. (B) Mass spectra of the MS/MS/MS fragmentation of the y₁₀²⁺ ion confirmed that the y₁₀²⁺ fragment is indeed RRLQTEKTTY. Fragments of corresponding b or y ions are indicated.</p

Strains and plasmids used in this work.

<p>Strains and plasmids used in this work.</p

Mass spectra showing that CovR is phosphorylated on threonine 65.

<p>Recombinant CovR was incubated with Stk_KD and then subjected to chymotrypsin digestion. (A) Mass spectra of the MS/MS fragmentation of a 2⁺ ion of the CovR-derived peptide EVTphosRRLQTEKTTY. Fragment marked with blue arrow shows the peptide that has lost phosphoric acid, thus decreasing the apparent mass from 852.92 to 804.27. The peptide marked with the red star corresponds to the y₁₀²⁺ fragment (RRLQTEKTTY) and has not lost phosphoric acid. Thus, T3 must be the position of phosphorylation. (B) Mass spectra of the MS/MS/MS fragmentation of the y₁₀²⁺ ion confirmed that the y₁₀²⁺ fragment is indeed RRLQTEKTTY. Fragments of corresponding b or y ions are indicated.</p

CovR-D53A and CovR-T65E bind CovR-regulated promoters with similar affinity to unphosphorylated CovR.

<p>Binding of recombinant CovR, CovR-D53A, and CovR-T65E proteins to the promoters of (<i>A</i>) <i>hasA</i> and (B) <i>prtS</i>. Increasing concentrations of unphosphorylated CovR, CovR-D53A, and CovR-T65E (monomer concentration given in µM) were used as indicated. Samples were incubated for 15 min at 37°C and electrophoresed on a 6% TBE polyacrylamide gel for 60 min at 120 V. The gels were stained with ethidium bromide. ns, non-specific DNA, f, free DNA, cI, lower molecular weight complex, cII, higher molecular weight complex. Gels shown are representatives of identical results obtained on two separate occasions.</p

CovR levels are decreased in the CovR-T65A strain.

<p>(A) Western blot analysis. Indicated strains were grown to mid-exponential phase in regular THY medium (−) and in THY medium supplemented with 15 mM MgCl₂ (+). Cell lysates containing 70 µg of total protein were loaded on a 12% SDS-gel. The gel was divided into two parts; the upper part was blotted against anti-CovR antibody while the lower part was blotted against anti-HPr antibody which served as a loading control. (B) qRT-PCR analysis of <i>covR</i> transcript levels. For “Low MgCl₂” and “High MgCl₂” samples, the indicated strains were grown as for the Western blot analysis in duplicate on two separate occasions and analyzed in duplicate. Data shown are mean ± standard deviation of 8 data points. For the “During infection” samples, four animals were infected with each strain and data were analyzed in triplicate as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004088#ppat-1004088-g008" target="_blank">Figure 8</a>. Data graphed are mean ± standard deviation of 12 data points. For all three conditions the lack of a bar for 10870Δ<i>covR</i> strain indicates no <i>covR</i> transcript level was detectable. * designates a significant difference in <i>covR</i> transcript level between strain MGAS10870 and CovR-T65A as determined by Student's t-test.</p

Strains MGAS10870 and CovR-T65A respond to changes in Mg²⁺.

<p>For all panels, data shown are gene transcript levels as measured by TaqMan quantitative real-time PCR (qRT-PCR). Strains were grown in normal THY (low Mg²⁺) or in THY with 15 mM MgCl₂ (high Mg²⁺) to mid-exponential phase in duplicate on two separate occasions and analyzed in duplicate. Data shown are mean ± standard deviation of 8 data points. (A) Log₂ ratio of transcript levels of indicated genes during growth in low vs. high Mg²⁺ medium in strain MGAS10870. <i>P</i> value refers to Student's t-test comparing gene transcript levels in low vs. high Mg²⁺ medium. (B–F) Data shown are transcript levels of indicated genes in indicated strains relative to the endogenous control gene <i>tufA</i>. * indicates significant difference in gene transcript for the indicated strain between growth in a low Mg²⁺ vs. high Mg²⁺ conditions as determined by Student's t-test.</p

CovR phosphorylation affects GAS virulence and gene expression during infection.

<p>(A) 20 CD-1 mice per strains were challenged intra-peritoneally with 1×10⁷ colony forming units of the indicated strains and followed for near-mortality. Data graphed are survival. <i>P</i> values refer to Mantel-Cox (log-rank) test adjusted for multiple comparisons. For (B–F), RNA was isolated from infected animals as described in the Material and Methods and converted to cDNA. Data graphed are gene transcript levels of the indicated genes relative to the endogenous control gene <i>tufA</i>. Four animals were infected with each strain and data were analyzed in triplicate. Data graphed are mean ± standard deviation of 12 data points. * indicates that gene transcript level for that particular strain was significantly different compared to all other strains as determined by analysis of variance (ANOVA) and Bonferroni's post-hoc test. For panel (C), ** indicates that the transcript level of <i>ska</i> was significantly lower in strains 10870Δ<i>covS</i> and CovR-T65A compared to strains 10870Δ<i>covR</i>, CovR-D53A, and CovR-T65E.</p

CovR dual-site phosphorylation events influence each other.

<p>(A) Western blot using anti-CovR antibody. Samples were separated on a 12% Phos-tag-SDS polyacrylamide gel which was loaded as follows: CovR phosphorylated with acetyl-phosphate as positive control, unphosphorylated CovR as negative control, Stk_KD alone, Stk_KD in Mg²⁺/ATP buffer or Mn²⁺/ATP buffer; CovR-T65A phosphorylated with Stk_KD in Mg²⁺/ATP or Mn²⁺/ATP buffer; CovR phosphorylated with Stk_KD in Mg²⁺/ATP or Mn²⁺/ATP buffer. Phosphorylated and unphosphorylated CovR species are labeled. (B) Recombinant wild type and variant CovR proteins were incubated with kinase buffer in the absence (<b>−</b>) and presence (<b>+</b>) of acetyl-phosphate, separated on a 12% Phos-tag SDS polyacrylamide gel and blotted with anti-CovR antibodies. Note that CovR-D53A and T65E variants cannot be phosphorylated by acetyl-phosphate. (C) Recombinant wild type and variant CovR proteins were incubated with kinase buffer in the absence (<b>−</b>) and presence (<b>+</b>) of acetyl-phosphate or Stk_KD, separated on a 12% Phos-tag SDS polyacrylamide gel and blotted with anti-CovR antibody. The retarded bands represent the phosphorylated CovR species. The * denotes unspecific background noted for lanes 4 and 5. Note that the CovR-T65A and T65E variants cannot be phosphorylated by Stk_KD.</p

Strains and plasmids used in this work.

<p>Strains and plasmids used in this work.</p