Reversible threonine phosphorylation of a response regulator, SptR, modulates Group A Streptococcus (GAS, S. pyogenes) virulence
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Date
2014-05
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Publisher
The Ohio State University
Abstract
GAS bacterium is a successful human pathogen that causes a variety of diseases of the skin
and pharynx and results in about 500,000 deaths worldwide. The pathogenesis of GAS
diseases is highly complex. GAS virulence is regulated by several two-component regulatory
systems (TCS) constituted by a response regulator (RR) and its cognate histidine kinase (HK).
HK autophosphorylates on its His residue and transfers the phosphate to the conserved Aspresidue
of RR, which acts as a transcription factor and regulates the expression of specific
genes. SptR/S (Spy874/875) is an important TCS that regulates the expression of many
carbohydrate-metabolism and transport (CMT)-related genes. GAS mutants lacking SptR
display down-regulated CMT-related genes and are avirulent. Previously, our lab discovered
that similar phenotypic characteristics were also observed in GAS mutants when the gene
encoding for ser/thr kinase (STK) was deleted. We therefore hypothesize that STK
phosphorylates SptR and its phosphorylated form regulates the expression of CMT-related
genes. STK phosphorylates threonine residues. Whether Thr-phosphorylation of SptR plays any
role in the regulation of gene expression is not known. In in vitro phosphorylation assays, we
found that the purified SptR serves as a substrate for STK and ser/thr phosphatase (STP)-
mediated reversible phosphorylation. To confirm this finding in vivo and appreciate its
physiological significance, we immunoprecipitated (ip) SptR from the whole cell lysate derived
from the wild-type GAS, and previously characterized GAS mutants M1ΔSTK and M1ΔSTP
using SptR specific antibodies. We confirmed isolation of the 27kDa ip-protein to be SptR by
anti-SptR antibody and its threonine phosphorylation status by Anti-ThrP antibody by Western
blot analysis. Our results showed decreased and increased anti-ThrP-specific binding with
ipSptR from GAS mutants M1ΔSTK and M1ΔSTP, respectively, confirming that SptR indeed
serves as a substrate for the STK/STP-mediated reversible Thr-phosphorylation. We thus
demonstrate a novel virulence-related gene regulatory mechanism in GAS.
Description
Awarded "Outstanding Project" at the Natural and Mathematical Sciences Undergraduate Research Forum (The Ohio State University, 2012)
Keywords
Reversible threonine phosphorylation, Response Regulator, SptR, Group A Streptococcus, Virulence