Dual-Site Phosphorylation of the Control of Virulence Regulator Impacts Group A Streptococcal Global Gene Expression and Pathogenesis
Dual-Site Phosphorylation of the Control of Virulence Regulator Impacts Group A Streptococcal Global Gene Expression and Pathogenesis
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DOI：
10.1371/journal.ppat.1004088
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发表时间：
2014-05-01
期刊：
影响因子：
6.7
通讯作者：
Shelburne, Samuel A., III
中科院分区：
文献类型：
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作者：
Horstmann, Nicola;Saldana, Miguel;Shelburne, Samuel A., III
磷酸化中继是细菌响应环境信号改变转录的主要机制，但对细菌反应调节因子磷酸化的功能后果的理解有限。我们试图描述来自主要人类病原体A群链球菌（GAS）的毒力调节因子（CovR）蛋白控制的磷酸化如何影响GAS全球基因表达和发病机制。CovR主要用于抑制GAS毒力因子编码基因，并在体外被证明在天冬氨酸53 （D53）磷酸化后发生同二聚体化。我们发现CovR在体内被磷酸化，并且这种磷酸化是部分热稳定的，这表明在非天冬氨酸残基上有额外的磷酸化。利用质谱和靶向诱变，我们确定了苏氨酸-65 （T65）是丝氨酸/苏氨酸激酶（Stk）控制下的另一个CovR磷酸化位点。重组CovR T65E变体模拟了T65上的磷酸化，消除了CovR D53的体外磷酸化。同样，不能在D53位点磷酸化（CovR-D53A）或在T65位点功能性组成磷酸化（CovR-T65E）的等等位基因GAS菌株基本上具有相同的基因抑制谱，彼此之间以及与covr灭活菌株相同。然而，CovR-D53A和CovR-T65E等等位基因菌株保留了积极影响基因表达的能力，而这种能力在covr灭活菌株中被取消。与这些观察结果一致，在侵袭性GAS疾病小鼠模型中，与covr灭活菌株相比，CovR-D53A和CovR-T65E菌株具有高毒力。令人惊讶的是，与野生型菌株相比，无法磷酸化CovR T65的等等位菌株（CovR- t65a）具有高毒力，因为CovR基因表达的自动调节导致CovR- t65a菌株中CovR基因转录和CovR蛋白水平较低。综上所述，这些数据证实了CovR在体内被磷酸化，并阐明了CovR D53激活磷酸化、T65抑制磷酸化和自调节之间的复杂相互作用如何影响链球菌宿主-病原体相互作用。
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.