The orphan regulator RocA plays a critical role in the colonization and pathogenesis of the obligate human pathogen group A Streptococcus Despite multiple lines of evidence supporting a role for RocA as an auxiliary regulator of the control of virulence two-component regulatory system CsrRS (or CovRS), the mechanism of action of RocA remains unknown. Using a combination of in vitro and in vivo techniques, we now find that RocA interacts with CsrS in the streptococcal membrane via its N-terminal region, which contains seven transmembrane domains. This interaction is essential for RocA-mediated regulation of CsrRS function. Furthermore, we demonstrate that RocA forms homodimers via its cytoplasmic domain. The serotype-specific RocA truncation in M3 isolates alters this homotypic interaction, resulting in protein aggregation and impairment of RocA-mediated regulation. Taken together, our findings provide insight into the molecular requirements for functional interaction of RocA with CsrS to modulate CsrRS-mediated gene regulation.IMPORTANCE Bacterial two-component regulatory systems, comprising a membrane-bound sensor kinase and cytosolic response regulator, are critical in coordinating the bacterial response to changing environmental conditions. More recently, auxiliary regulators which act to modulate the activity of two-component systems, allowing integration of multiple signals and fine-tuning of bacterial responses, have been identified. RocA is a regulatory protein encoded by all serotypes of the important human pathogen group A Streptococcus Although RocA is known to exert its regulatory activity via the streptococcal two-component regulatory system CsrRS, the mechanism by which it functions was unknown. Based on new experimental evidence, we propose a model whereby RocA interacts with CsrS in the streptococcal cell membrane to enhance CsrS autokinase activity and subsequent phosphotransfer to the response regulator CsrR, which mediates transcriptional repression of target genes.

中文翻译：

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RocA 结合 CsrS 来调节 A 组链球菌中 CsrRS 介导的基因调控。


孤儿调节因子 RocA 在专性人类病原体 A 组链球菌的定植和发病机制中发挥着关键作用，尽管多种证据支持 RocA 作为毒力双组分调节系统 CsrRS（或 CovRS）控制的辅助调节因子的作用，RocA 的作用机制仍不清楚。通过结合体外和体内技术，我们现在发现 RocA 通过其 N 端区域（包含七个跨膜结构域）与链球菌膜中的 CsrS 相互作用。这种相互作用对于 RocA 介导的 CsrRS 功能调节至关重要。此外，我们证明 RocA 通过其细胞质结构域形成同二聚体。 M3 分离株中血清型特异性的 RocA 截短改变了这种同型相互作用，导致蛋白质聚集和 RocA 介导的调节受损。总而言之，我们的研究结果提供了对 RocA 与 CsrS 功能相互作用以调节 CsrRS 介导的基因调控的分子要求的见解。 重要性 细菌双组分调控系统，包括膜结合传感器激酶和胞质反应调节器，对于协调细菌对不断变化的环境条件的反应。最近，已经确定了辅助调节剂，其作用是调节双组分系统的活性，从而允许整合多个信号并微调细菌反应。 RocA 是由重要的人类病原体 A 组链球菌的所有血清型编码的调节蛋白。 尽管已知 RocA 通过链球菌双组分调节系统 CsrRS 发挥其调节活性，但其发挥作用的机制尚不清楚。 基于新的实验证据，我们提出了一个模型，通过该模型，RocA 与链球菌细胞膜中的 CsrS 相互作用，增强 CsrS 自激酶活性，并随后磷酸转移至反应调节剂 CsrR，从而介导靶基因的转录抑制。