Lysogeny with Shiga Toxin 2-Encoding Bacteriophages Represses Type III Secretion in Enterohemorrhagic Escherichia coli

Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins.噬菌体的裂解性或溶原性感染驱动肠道细菌的演化。肠出血性大肠埃希菌（Enterohemorrhagic Escherichia coli, EHEC）近年来已成为一类重要的人类人畜共患病病原体，其主要血清型由反刍动物携带。典型EHEC菌株的定义特征包括：表达Ⅲ型分泌系统（type III secretion system, T3S）、产生志贺毒素（Shiga toxins, Stx），以及伴随特定临床症状。志贺毒素的编码基因存在于整合至大肠埃希菌基因组的λ样噬菌体（lambdoid bacteriophages）中。 噬菌体型（phage type, PT）21/28是英国与人类EHEC感染关联最普遍的菌株型别，且相较于PT32菌株，其更易与高水平排菌的牛只相关联。本研究证实，无论分离来源如何，90%的PT21/28菌株同时携带Stx2和Stx2c噬菌体；而PT32菌株中仅少数（28%）同时携带这两种噬菌体。与PT32菌株相比，PT21/28菌株的T3S中位数水平更低，因此本研究探究了Stx噬菌体溶原性与T3S之间的调控关系。 研究发现，敲除EHEC菌株中的Stx2噬菌体可提升T3S水平，而溶原状态则会降低T3S水平。这一调控现象在大肠埃希菌K12背景菌株中得到验证：该菌株经标记的Stx2噬菌体转导后，通过检测受LEE编码调节因子（LEE-encoded regulator, Ler）诱导水平调控的T3S报告基因表达量，证实了整合的Stx2噬菌体会抑制LEE1的Ler诱导表达，且该调控过程依赖噬菌体CII调节蛋白。通过异位表达同源CI调节蛋白可解除这一抑制作用。 本研究提出了一个调控模型：编码Stx2的噬菌体可调控T3S系统，以协调由Stx和分泌效应蛋白共同促进的上皮细胞定植过程。