Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mor., Mexico.
Programa de Medicina Humana, Universidad Privada Antenor Orrego, Trujillo, Peru.
J Med Microbiol. 2024 Oct;73(10). doi: 10.1099/jmm.0.001907.
Enteropathogenic (EPEC) strains pose a significant threat as a leading cause of severe childhood diarrhoea in developing nations. EPEC pathogenicity relies on the type III secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE), facilitating the secretion and translocation of bacterial effector proteins. While the regulatory roles of PerC (plasmid-encoded regulator) and GrlA (global regulator of LEE-activator) in expression and LEE gene activation are well-documented in the EPEC prototype strain E2348/69, understanding the variability in LEE gene expression control mechanisms among clinical EPEC isolates remains an area requiring further investigation. This study aims to explore the diversity in LEE gene expression control mechanisms among clinical EPEC isolates through a comparative analysis of secretion profiles under defined growth conditions favouring either PerC- or GrlA-mediated activation of LEE expression. We compared T3SS-dependent secretion patterns and promoter expression in both typical EPEC (tEPEC) and atypical EPEC (aEPEC) clinical isolates under growth conditions favouring either PerC- or GrlA-mediated activation of LEE expression. Additionally, we conducted promoter reporter activity assays, quantitative real-time PCR and Western blot experiments to assess gene expression activity. Significant differences in T3SS-dependent secretion were observed among tEPEC and aEPEC strains, independent of LEE sequence variations or T3SS gene functionality. Notably, a clinical tEPEC isolate exhibited increased secretion levels under repressive growth conditions and in the absence of both PerC and GrlA, implicating an alternative mechanism in the activation of Ler (LEE-encoded regulator) expression. Our findings indicate that uncharacterized LEE regulatory mechanisms contribute to phenotypic diversity among clinical EPEC isolates, though their impact on clinical outcomes remains unknown. This challenges the conventional understanding based on reference strains and highlights the need to investigate beyond established models to comprehensively elucidate EPEC pathogenesis.
肠致病性大肠杆菌（EPEC）菌株是发展中国家严重儿童腹泻的主要致病因素，构成重大威胁。EPEC 的致病性依赖于由肠上皮细胞消失（LEE）位点编码的 III 型分泌系统（T3SS），促进细菌效应蛋白的分泌和易位。虽然质粒编码调控因子（PerC）和全局调控因子 LEE 激活（GrlA）在 EPEC 原型株 E2348/69 中对 表达和 LEE 基因激活的调控作用已有详细记载，但在临床 EPEC 分离株中 LEE 基因表达控制机制的变异性仍然是一个需要进一步研究的领域。本研究旨在通过比较在有利于 PerC 或 GrlA 介导的 LEE 表达激活的特定生长条件下的分泌谱，探讨临床 EPEC 分离株中 LEE 基因表达控制机制的多样性。我们比较了典型 EPEC（tEPEC）和非典型 EPEC（aEPEC）临床分离株在有利于 PerC 或 GrlA 介导的 LEE 表达激活的生长条件下的 T3SS 依赖性分泌模式和启动子表达。此外，我们进行了启动子报告基因活性测定、定量实时 PCR 和 Western blot 实验，以评估基因表达活性。tEPEC 和 aEPEC 菌株之间观察到 T3SS 依赖性分泌的显著差异，与 LEE 序列变异或 T3SS 基因功能无关。值得注意的是，临床 tEPEC 分离株在抑制生长条件下和缺乏 PerC 和 GrlA 的情况下表现出增加的分泌水平，暗示 Ler（LEE 编码调节剂）表达的激活存在替代机制。我们的研究结果表明，未被表征的 LEE 调控机制有助于临床 EPEC 分离株的表型多样性，尽管它们对临床结果的影响尚不清楚。这对基于参考菌株的传统理解提出了挑战，并强调需要超越既定模型进行研究，以全面阐明 EPEC 发病机制。
