Department of Food Science, The Pennsylvania State University, University Park, Pennsylvania, USA.
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA.
Infect Immun. 2022 Feb 17;90(2):e0058721. doi: 10.1128/IAI.00587-21. Epub 2021 Dec 6.
Enterohemorrhagic Escherichia coli (EHEC) strains, including the foodborne pathogen E. coli O157:H7, are responsible for thousands of hospitalizations each year. Various environmental triggers can modulate pathogenicity in EHEC by inducing the expression of Shiga toxin (Stx), which is encoded on a lambdoid prophage and transcribed together with phage late genes. Cell-free supernatants of the sequence type 73 (ST73) E. coli strain 0.1229 are potent inducers of Stx2a production in EHEC, suggesting that 0.1229 secretes a factor that activates the SOS response and leads to phage lysis. We previously demonstrated that this factor, designated microcin 1229 (Mcc1229), was proteinaceous and plasmid-encoded. To further characterize Mcc1229 and support its classification as a microcin, we investigated its regulation, determined its receptor, and identified loci providing immunity. The production of Mcc1229 was increased upon iron limitation, as determined by an enzyme-linked immunosorbent assay (ELISA), fusions, and quantitative real-time PCR (qRT-PCR). Spontaneous Mcc1229-resistant mutants and targeted gene deletion revealed that CirA was the Mcc1229 receptor. TonB, which interacts with CirA in the periplasm, was also essential for Mcc1229 import. Subcloning of the Mcc1229 plasmid indicated that Mcc activity was neutralized by two open reading frames (ORFs), each predicted to encode a domain of unknown function (DUF)-containing protein. In a germfree mouse model of infection, colonization with 0.1229 suppressed subsequent colonization by EHEC. Although Mcc1229 was produced , it was dispensable for colonization suppression. The regulation, import, and immunity determinants identified here are consistent with features of other Mccs, suggesting that Mcc1229 should be included in this class of small molecules.
肠出血性大肠杆菌（EHEC）菌株，包括食源性病原体 E. coli O157:H7，每年导致数千人住院。各种环境触发因素可以通过诱导编码在λ噬菌体样噬菌体上的志贺毒素（Stx）的表达来调节 EHEC 的致病性，Stx 与噬菌体晚期基因一起转录。 0.1229 型大肠杆菌菌株 0.1229 的细胞外上清液是 EHEC 产生 Stx2a 的有效诱导剂，这表明 0.1229 分泌了一种因子，激活 SOS 反应并导致噬菌体裂解。我们之前证明该因子，命名为微菌素 1229（Mcc1229），是蛋白质和质粒编码的。为了进一步表征 Mcc1229 并支持将其分类为微菌素，我们研究了其调节，确定了其受体，并鉴定了提供免疫的基因座。通过酶联免疫吸附测定（ELISA）、融合和定量实时 PCR（qRT-PCR）确定，在缺铁条件下，Mcc1229 的产生增加。自发的 Mcc1229 抗性突变体和靶向基因缺失表明 CirA 是 Mcc1229 的受体。在周质中与 CirA 相互作用的 TonB 对于 Mcc1229 的导入也是必不可少的。Mcc1229 质粒的亚克隆表明，Mcc 活性被两个开放阅读框（ORF）中和，每个 ORF 都预测编码一个含有未知功能（DUF）的域的蛋白质。在无菌小鼠感染模型中，0.1229 的定植抑制了随后 EHEC 的定植。尽管产生了 Mcc1229，但它对定植抑制是可有可无的。这里确定的调节、导入和免疫决定因素与其他 Mcc 的特征一致，表明 Mcc1229 应归入此类小分子。
