Blackburn Elizabeth A, Chintoan-Uta Cosmin, Bease Andrew G, Tuijtel Maarten W, Hateley Max, Wear Martin, Longbottom David, Cassady-Cain Robin, Stevens Mark P
The Edinburgh Protein Production Facility, University of Edinburgh, Edinburgh, UK.
The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, UK.
Virulence. 2025 Dec;16(1):2506500. doi: 10.1080/21505594.2025.2506500. Epub 2025 May 22.
Pathogens frequently produce proteins to evade or inhibit host immune responses. One such protein is lymphostatin from attaching and effacing (also known as lymphocyte inhibitory factor A; LifA), which influences intestinal colonization and inhibits mitogen- and antigen-activated proliferation of T lymphocytes and pro-inflammatory cytokine synthesis. Here, we report the cloning, purification and characterization of a LifA homologue from . The predicted 382 KDa protein (CPE2_0552) exhibited 36% identity and 55% similarity over 3171 amino acids to lymphostatin from enteropathogenic strain E2348/69. CPE2_0552 shares glycosyltransferase and cysteine protease motifs required for lymphostatin activity, including similarity in the tertiary structure of these domains predicted by AlphaFold 3. Purified CPE2_0552 did not share the L-shaped globular structure of lymphostatin when analyzed by transmission electron microscopy. CPE2_0552 inhibited concanavalin A-stimulated proliferation of bovine T cells in a concentration-dependent manner, with an inhibitory dose 50 (ID) of 812 pg/mL. This was 38-fold higher than the ID of E2348/69 lymphostatin tested in parallel on T cells from the same donors (21 pg/mL), but was similar to another LifA homologue from O157:H7 (ToxB). Moreover, CPE2_0552 inhibited the secretion of interferon gamma (IFNγ), a key cytokine that influences the outcome of infections. At the concentrations at which CPE2_0552 inhibited T lymphocyte proliferation and IFNγ secretion, negligible cytotoxicity was observed after 72 h of stimulation. Our study indicates that lymphostatin belongs to a wider family of lymphocyte-inhibitory molecules that exist in distantly related bacterial pathogens.
病原体经常产生蛋白质以逃避或抑制宿主免疫反应。其中一种蛋白质是来自黏附性和毁损性大肠杆菌（也称为淋巴细胞抑制因子A；LifA）的淋巴细胞抑制素，它影响肠道定植，并抑制丝裂原和抗原激活的T淋巴细胞增殖以及促炎细胞因子合成。在此，我们报告了来自[具体菌种]的LifA同源物的克隆、纯化和特性分析。预测的382 kDa蛋白（CPE2_0552）在3171个氨基酸上与肠致病性大肠杆菌E2348/69的淋巴细胞抑制素具有36%的同一性和55%的相似性。CPE2_0552具有淋巴细胞抑制素活性所需的糖基转移酶和半胱氨酸蛋白酶基序，包括通过AlphaFold 3预测的这些结构域三级结构的相似性。通过透射电子显微镜分析时，纯化的CPE2_0552不具有淋巴细胞抑制素的L形球状结构。CPE2_0552以浓度依赖的方式抑制伴刀豆球蛋白A刺激的牛T细胞增殖，半数抑制剂量（ID50）为812 pg/mL。这比在来自相同供体的T细胞上平行测试的E2348/69淋巴细胞抑制素的ID50（21 pg/mL）高38倍，但与来自O157:H7的另一种LifA同源物（ToxB）相似。此外，CPE2_0552抑制干扰素γ（IFNγ）的分泌，IFNγ是一种影响大肠杆菌感染结果的关键细胞因子。在CPE2_0552抑制T淋巴细胞增殖和IFNγ分泌的浓度下，刺激72小时后观察到的细胞毒性可忽略不计。我们的研究表明，大肠杆菌淋巴细胞抑制素属于存在于远缘相关细菌病原体中的更广泛的淋巴细胞抑制分子家族。
