HilE Regulates HilD by Blocking DNA Binding in Salmonella enterica Serovar Typhimurium
HilE Regulates HilD by Blocking DNA Binding in Salmonella enterica Serovar Typhimurium
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DOI：
10.1128/jb.00750-17
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发表时间：
2018-04-01
影响因子：
3.2
通讯作者：
Slauch, James M.
中科院分区：
文献类型：
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作者：
Grenz, Jesse R.;Chubiz, Jessica E. Cott;Slauch, James M.
沙门氏菌3型分泌系统（T3SS）编码于沙门氏菌致病性岛1 （SPI1）位点，介导宿主肠上皮的侵袭。SPI1的表达依赖于三种类似arac的调节因子：HilD、HilC和RtsA。这些调节因子在一个复杂的前馈回路中相互激活和激活hilA， hilA编码T3SS结构基因的激活子。HilD已被证明是大多数已知激活SPI1 T3SS表达的信号的主要整合点，作为控制系统诱导的开关。HilD是一个作用于HilD的负调节器。在这里，我们提供的遗传和生化数据表明，HilE特异性结合HilD，而不是HilC或RtsA。体内报告系统和体外凝胶转移试验表明，这种蛋白质-蛋白质相互作用阻断了HilD结合DNA的能力。hil不影响HilD二聚化，也不控制HilD蛋白的稳定性。我们还利用竞争实验研究了HilE在小鼠感染过程中的作用。虽然hilE的缺失不会产生表型，但hilE突变确实抑制了FliZ缺失带来的入侵缺陷，FliZ是控制HilD蛋白活性的积极信号。综上所述，这些数据表明HilE的功能限制了低水平的HilD活性，防止SPI1过早激活，直到正输入达到完全诱导系统所需的阈值。沙门氏菌是全球胃肠道和全身性疾病的主要原因。沙门氏菌诱导炎症性腹泻和进入下层组织需要SPI1 T3SS。一个复杂的调控网络控制着SPI1的表达，以响应许多生理输入。大多数这些信号主要影响HilD的翻译或活动。当HilD活性超过一个允许有效激活其自身启动子的阈值时，系统被触发。这个阈值是由HilD设定的，它与HilD绑定，以防止HilD活动不可避免的微小波动导致系统不适当地激活。电路也作为一个范例系统，必须集成众多的环境参数来控制调节输出。
The Salmonella type three secretion system (T3SS), encoded in the Salmonella pathogenicity island 1 (SPI1) locus, mediates the invasion of the host intestinal epithelium. SPI1 expression is dependent upon three AraC-like regulators: HilD, HilC, and RtsA. These regulators act in a complex feed-forward loop to activate each other and hilA, which encodes the activator of the T3SS structural genes. HilD has been shown to be the major integration point of most signals known to activate the expression of the SPI1 T3SS, acting as a switch to control induction of the system. HilE is a negative regulator that acts upon HilD. Here we provide genetic and biochemical data showing that HilE specifically binds to HilD but not to HilC or RtsA. This protein-protein interaction blocks the ability of HilD to bind DNA as shown by both an in vivo reporter system and an in vitro gel shift assay. HilE does not affect HilD dimerization, nor does it control the stability of the HilD protein. We also investigated the role of HilE during the infection of mice using competition assays. Although deletion of hilE does not confer a phenotype, the hilE mutation does suppress the invasion defect conferred by loss of FliZ, which acts as a positive signal controlling HilD protein activity. Together, these data suggest that HilE functions to restrict low-level HilD activity, preventing premature activation of SPI1 until positive inputs reach a threshold required to fully induce the system.IMPORTANCE Salmonella is a leading cause of gastrointestinal and systemic disease throughout the world. The SPI1 T3SS is required for Salmonella to induce inflammatory diarrhea and to gain access to underlying tissue. A complex regulatory network controls expression of SPI1 in response to numerous physiological inputs. Most of these signals impinge primarily on HilD translation or activity. The system is triggered when HilD activity crosses a threshold that allows efficient activation of its own promoter. This threshold is set by HilE, which binds to HilD to prevent the inevitable minor fluctuations in HilD activity from inappropriately activating the system. The circuit also serves as a paradigm for systems that must integrate numerous environmental parameters to control regulatory output.