霍乱弧菌毒力调控蛋白AphB抗ROS和RNS的巯基调控机制及其对致病过程的影响

Bacterial pathogens have abilities to retool their genetic programs to adapt different host niches along infections. However, most bacterial sensors of the host environment are unknown, including those for Vibrio cholerae, the cause of the widespread diarrheal disease cholera. In addition to establishing colonization, V. cholerae must cope with changes in the intestinal environment caused by the response of the host innate immune system. Several studies suggest that reactive nitrogen species (RNS), such as nitric oxide (NO), and oxidative stress from reactive oxygen species (ROS) are challenges for V. cholerae during infection. Previously we made the discovery that the transcription factor AphB, a LysR-family protein, possesses a cysteine residue that undergoes modification in response to the microoxic conditions of the intestines, leading to activation of virulence. We now have further evidence that diverse cysteine modifications in AphB lead to diverse effects on cell physiology through changes in gene transcription. We have found that V. cholerae harboring the unreactive cysteine→serine mutation at residue 235 (C235S) is sensitive to NO and organic hydroperoxide, and is defective in colonization of an adult mouse model. We hypothesize that AphB is the central processor of environmental information relevant to infection for V. cholerae. Specifically, AphB serves as thiol-based switches to integrate the presence of reductive, oxidative and nitrosative reactants into the gene expression decisions necessary to adapt host environments. We will use a mix of biochemical and genetic techniques to define mechanistically how AphB orchestrates the pathogenic life cycle of V. cholerae. All sequenced Vibrio species contain AphB homologs with conserved cysteine residues. We believe that by understanding the way AphB monitors the microenvironment for V. cholerae, we then may be able to extend the paradigm of cysteine-based environmental sensing to other V. cholerae proteins and other pathogens.

病原细菌在感染宿主过程中，常会根据宿主环境调整自身基因表达，但包括霍乱弧菌在内，大多数细菌感受宿主环境的具体过程还是未知的。就霍乱弧菌而言，从水体到宿主肠道的感染过程中，环境发生了巨大变化，包括面对RNS和ROS造成的压力，能否成功处理宿主免疫系统给肠道环境带来的变化，是成功感染的关键。AphB是霍乱弧菌中非常重要的毒力调控蛋白，前期我们发现AphB会通过修饰半胱氨酸残基来响应肠道的微氧环境，并激活毒力因子的表达；而将其中关键性的第235位半胱氨酸突变成丝氨酸后，该突变株对NO和H2O2敏感，并在成年鼠中具有定殖缺陷。本项目将以AphB为对象，研究AphB如何感知宿主体内的ROS和RNS，并通过巯基修饰的方式进行自身氧化还原和亚硝基化修饰，从而调控抗ROS和RNS基因的表达，适应宿主环境。这也将为其它病原细菌的巯基修饰相关的调控提供相关理论依据。

病原细菌在感染宿主过程中，常会根据宿主环境调整自身基因表达。就霍乱弧菌而言，从水体到宿主肠道的感染过程中，环境发生了巨大变化，能否成功处理包括宿主内氧化环境在内的压力，都是成功感染的关键。AphB是霍乱弧菌中非常重要的毒力调控蛋白，前期我们发现霍乱弧菌的重要毒力激活因子AphB 会通过修饰半胱氨酸残基来响应肠道的微氧环境，并激活毒力因子的表达。本项目以 AphB 为主要对象，发现霍乱弧菌中有两套“双保险”调控抗氧化和毒力表达的机制。一套由AphB和OhrR同时抑制ROS抗性基因ohrA，还原态的AphB和OhrR的直接结合到ohrA启动子上并抑制其表达，而有机氢过氧化物（如氢过氧化枯烯CHP）则会使AphB和OhrR接触对ohrA的抑制。第二套系统中，像AphB一样，还原态OhrR会与tcpP启动子结合加以调控，面对氧化还原压力时，OhrR比AphB还原速度更快，对OhrR的巯基氧化还原状态的切换是这种快速激活毒力表达和成功定殖过程所必须的。此外，OxyR调控还会调控dps来抗有机和无机过氧化物，及多种环境压力。在H2O2低浓度时，则由OxyR2激活AhpC去修饰OxyR1活性，从而实现分级调控抗氧化。以上系统揭示了病原细菌通过对多个转录因子进行翻译后调控来感知和适应环境变化的机制，对其它病原细菌的研究也有借鉴意义。