Date Published: May 1, 2014
Publisher: Public Library of Science
Author(s): Nicola Horstmann, Miguel Saldaña, Pranoti Sahasrabhojane, Hui Yao, Xiaoping Su, Erika Thompson, Antonius Koller, Samuel A. Shelburne, Frank R. DeLeo.
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.
Bacteria causing infections in humans must closely modulate virulence factor production in response to different environmental challenges , , . It has long been recognized that two-component gene regulatory systems (TCS) are a major mechanism by which bacteria react to external stimuli, and thus are critical to the virulence of numerous pathogenic bacteria , , , . Although there is diversity in TCS composition , standard TCS consist of a membrane-embedded histidine kinase that can respond to environmental signals by either phosphorylating or dephosphorylating a cognate response regulator, usually on an aspartate residue in the N-terminal receiver domain . The aspartate phosphorylation status of the response regulator alters its gene regulation effect thereby allowing the organism to remodel its expression profile .
Until recently, studies of how protein phosphorylation participates in prokaryotic signal transduction have mainly focused on linear TCS pathways that involve phosphorylation of aspartate in response regulators by their cognate histidine kinase . Increasingly, however, it is being recognized that the phosphorylation status of response regulators can be influenced by a multitude of factors, including phosphorylation on non-aspartate residues, clearly placing response regulators into the wider gene regulation network , , . Understanding the mechanisms influencing the phosphorylation status of bacterial regulator proteins and determining how such phosphorylation ultimately influences bacterial gene expression is critical to a more complete understanding of bacterial pathogenesis. Herein we demonstrate that the key GAS response regulator CovR is phosphorylated in vivo and that CovR phosphorylation at both aspartate and threonine residues profoundly influences GAS global gene expression and virulence.