State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences , Wuhan, China.
College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences , Beijing, China.
mSphere. 2023 Oct 24;8(5):e0034623. doi: 10.1128/msphere.00346-23. Epub 2023 Aug 29.
Type III secretion system (T3SS) facilitates survival and replication of . Identifying novel T3SS effectors and elucidating their functions are critical in understanding the pathogenesis of T3SS effector EseG and EseJ was highly secreted when T3SS gatekeeper-containing protein complex EsaB-EsaL-EsaM was disrupted by EsaB deficiency. Based on this observation, concentrated secretomes of Δ strain and ΔΔ strain were purified by loading them into SDS-PAGE gel for a short electrophoresis to remove impurities prior to the in-the gel digestion and mass spectrometry. Four reported T3SS effectors and two novel T3SS effector candidates EseQ (ETAE_2009) and Trx2 (ETAE_0559) were unraveled by quantitative comparison of the identified peptides. EseQ and Trx2 were revealed to be secreted and translocated in a T3SS-dependent manner through CyaA-based translocation assay and immunofluorescent staining, demonstrating that EseQ and Trx2 are the novel T3SS effectors of . Trx2 was found to suppress macrophage apoptosis as revealed by TUNEL staining and cleaved caspase-3 of infected J774A.1 monolayers. Moreover, Trx2 has been shown to inhibit the p65 phosphorylation and p65 translocation into the nucleus, thus blocking the NF-κB pathway. Furthermore, depletion of Trx2 slightly but significantly attenuates virulence in a fish infection model. Taken together, an efficient method was established in unraveling T3SS effectors in , and Trx2, one of the novel T3SS effectors identified in this study, was demonstrated to suppress apoptosis and block NF- κB pathway during infection. IMPORTANCE is an intracellular bacterial pathogen that causes intestinal inflammation and hemorrhagic sepsis in fish and human. Virulence depends on the type III secretion system (T3SS). Identifying the bacterial effector proteins secreted by T3SS and defining their role is key to understanding pathogenesis. EsaB depletion disrupts the T3SS gatekeeper-containing protein complex, resulting in increased secretion of T3SS effectors EseG and EseJ. EseQ and Trx2 were shown to be the novel T3SS effectors of by a secretome comparison between ∆ strain and ∆∆ strain (T3SS mutant), together with CyaA-based translocation assay. In addition, Trx2 has been shown to suppress macrophage apoptosis and block the NF-κB pathway. Together, this work expands the known repertoire of T3SS effectors and sheds light on the pathogenic mechanism of .
III 型分泌系统（T3SS）有助于 的存活和复制。鉴定新的 T3SS 效应子并阐明其功能对于理解 T3SS 效应子在 的发病机制中至关重要。当 T3SS 门卫蛋白复合物 EsaB-EsaL-EsaM 被 EsaB 缺乏破坏时，T3SS 效应子 EseG 和 EseJ 被高度分泌。基于这一观察结果，通过将含有 T3SS 的Δ 株和 ΔΔ 株的浓缩分泌物加载到 SDS-PAGE 凝胶中进行短电泳，在凝胶内消化和质谱之前去除杂质来纯化它们。通过定量比较鉴定的肽，揭示了四个报道的 T3SS 效应子和两个新的 T3SS 效应子候选物 EseQ（ETAE_2009）和 Trx2（ETAE_0559）。通过 CyaA 基转位测定和免疫荧光染色，揭示了 EseQ 和 Trx2 以 T3SS 依赖的方式分泌和转位，表明 EseQ 和 Trx2 是 的新的 T3SS 效应子。TUNEL 染色和感染 J774A.1 单层细胞的裂解 caspase-3 显示，Trx2 抑制巨噬细胞凋亡。此外，Trx2 已被证明抑制 p65 磷酸化和 p65 向核内易位，从而阻断 NF-κB 途径。此外，在鱼类感染模型中，Trx2 的耗竭略但显著减弱了 的毒力。总之，建立了一种有效的方法来揭示 的 T3SS 效应子，本研究鉴定的新 T3SS 效应子之一 Trx2 被证明在 感染期间抑制细胞凋亡并阻断 NF-κB 途径。
重要性： 是一种胞内细菌病原体，可引起鱼类和人类的肠道炎症和出血性败血症。毒力取决于 III 型分泌系统（T3SS）。鉴定由 T3SS 分泌的细菌效应蛋白并定义其作用是理解 发病机制的关键。EsaB 耗竭破坏了含有 T3SS 门卫蛋白复合物的蛋白质，导致 T3SS 效应子 EseG 和 EseJ 的分泌增加。通过比较 ∆ 株和 ∆∆ 株（T3SS 突变体）之间的分泌组，以及基于 CyaA 的转位测定，表明 EseQ 和 Trx2 是 的新的 T3SS 效应子。此外，Trx2 已被证明抑制巨噬细胞凋亡并阻断 NF-κB 途径。总之，这项工作扩展了已知的 T3SS 效应子谱，并揭示了 的发病机制。
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