Lara-Ochoa Cristina, Huerta-Saquero Alejandro, Medrano-López Abraham, Deng Wanyin, Finlay B Brett, Martínez-Laguna Ygnacio, Puente José L
Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mexico.
Centro de Detección Biomolecular, Benemérita Universidad Autónoma de Puebla, Puebla, Mexico.
Front Microbiol. 2023 Feb 16;14:1063368. doi: 10.3389/fmicb.2023.1063368. eCollection 2023.
Enteropathogenic (EPEC), enterohemorrhagic (EHEC) and (CR) belong to a group of pathogens that share the ability to form "attaching and effacing" (A/E) lesions on the intestinal epithelia. A pathogenicity island known as the locus of enterocyte effacement (LEE) contains the genes required for A/E lesion formation. The specific regulation of LEE genes relies on three LEE-encoded regulators: Ler activates the expression of the LEE operons by antagonizing the silencing effect mediated by the global regulator H-NS, GrlA activates expression and GrlR represses the expression of the LEE by interacting with GrlA. However, despite the existing knowledge of LEE regulation, the interplay between GrlR and GrlA and their independent roles in gene regulation in A/E pathogens are still not fully understood.
To further explore the role that GrlR and GrlA in the regulation of the LEE, we used different EPEC regulatory mutants and transcriptional fusions, and performed protein secretion and expression assays, western blotting and native polyacrylamide gel electrophoresis.
We showed that the transcriptional activity of LEE operons increased under LEE-repressing growth conditions in the absence of GrlR. Interestingly, GrlR overexpression exerted a strong repression effect over LEE genes in wild-type EPEC and, unexpectedly, even in the absence of H-NS, suggesting that GrlR plays an alternative repressor role. Moreover, GrlR repressed the expression of LEE promoters in a non-EPEC background. Experiments with single and double mutants showed that GrlR and H-NS negatively regulate the expression of LEE operons at two cooperative yet independent levels. In addition to the notion that GrlR acts as a repressor by inactivating GrlA through protein-protein interactions, here we showed that a DNA-binding defective GrlA mutant that still interacts with GrlR prevented GrlR-mediated repression, suggesting that GrlA has a dual role as a positive regulator by antagonizing GrlR's alternative repressor role. In line with the importance of the GrlR-GrlA complex in modulating LEE gene expression, we showed that GrlR and GrlA are expressed and interact under both inducing and repressing conditions. Further studies will be required to determine whether the GrlR alternative repressor function depends on its interaction with DNA, RNA, or another protein. These findings provide insight into an alternative regulatory pathway that GrlR employs to function as a negative regulator of LEE genes.
肠致病性大肠杆菌（EPEC）、肠出血性大肠杆菌（EHEC）和聚集性黏附性大肠杆菌（CR）属于一组病原体，它们都具有在肠道上皮细胞上形成“紧密黏附并抹平”（A/E）损伤的能力。一个被称为肠上皮细胞抹平位点（LEE）的致病岛包含了形成A/E损伤所需的基因。LEE基因的特异性调控依赖于三种由LEE编码的调控因子：Ler通过拮抗全局调控因子H-NS介导的沉默效应来激活LEE操纵子的表达，GrlA激活表达，而GrlR通过与GrlA相互作用来抑制LEE的表达。然而，尽管已有关于LEE调控的知识，但GrlR与GrlA之间的相互作用及其在A/E病原体基因调控中的独立作用仍未完全清楚。
为了进一步探究GrlR和GrlA在LEE调控中的作用，我们使用了不同的EPEC调控突变体和转录融合体，并进行了蛋白质分泌和表达分析、蛋白质印迹法以及非变性聚丙烯酰胺凝胶电泳。
我们发现，在没有GrlR的情况下，LEE操纵子的转录活性在LEE抑制生长条件下增加。有趣的是，GrlR的过表达对野生型EPEC中的LEE基因产生了强烈的抑制作用，而且出乎意料的是，即使在没有H-NS的情况下也是如此，这表明GrlR发挥了替代抑制因子的作用。此外，GrlR在非EPEC背景下抑制LEE启动子的表达。单突变体和双突变体实验表明，GrlR和H-NS在两个协同但独立的水平上对LEE操纵子的表达进行负调控。除了GrlR通过蛋白质-蛋白质相互作用使GrlA失活从而作为抑制因子发挥作用这一观点外，我们在此还表明，一个仍与GrlR相互作用的DNA结合缺陷型GrlA突变体可阻止GrlR介导的抑制作用，这表明GrlA通过拮抗GrlR的替代抑制因子作用而具有作为正调控因子的双重作用。鉴于GrlR-GrlA复合物在调节LEE基因表达中的重要性，我们表明GrlR和GrlA在诱导和抑制条件下均有表达且相互作用。还需要进一步研究来确定GrlR的替代抑制因子功能是否依赖于其与DNA、RNA或另一种蛋白质的相互作用。这些发现为GrlR作为LEE基因负调控因子所采用的替代调控途径提供了见解。
