Prophage induction, but not production of phage particles, is required for lethal disease in a microbiome-replete murine model of enterohemorrhagic E. coli infection
Prophage induction, but not production of phage particles, is required for lethal disease in a microbiome-replete murine model of enterohemorrhagic E. coli infection
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DOI：
10.1371/journal.ppat.1007494
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发表时间：
2019-01-01
期刊：
影响因子：
6.7
通讯作者：
Leong, John M.
中科院分区：
文献类型：
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作者：
Balasubramanian, Sowmya;Osburne, Marcia S.;Leong, John M.
肠出血性大肠杆菌（EHEC）通过产生特征性的附着和消退（AE）病变来定植肠上皮。它们被编码志贺毒素2 （Stx2）的噬菌体溶原，导致严重的临床表现。作为一种溶原，导致裂解生长和stx2表达的原噬菌体基因受到抑制，而在体外和无菌或链霉素处理的小鼠中，诱导细菌对DNA损伤的SOS反应可导致裂解噬菌体生长和stx2产生。在体外，一些共生菌减少了噬菌体诱导和伴随的Stx2产生，而有人提出，噬菌体敏感的共生菌可能通过促进体内感染的连续周期来增加Stx2的产生。我们测试了噬菌体诱导在微生物组丰富的小鼠中Stx产生和致死性疾病中的作用，使用我们的小鼠模型，该模型包含了编码stx2的噬菌体Stx溶原的小鼠病原体鼠柠檬酸杆菌（Citrobacter rodentium）。该菌株在小鼠肠道上产生ehec样AE病变，并引起致命的stx介导疾病。我们发现致死性小鼠感染不需要phi stx（2dact）感染或溶原共生菌。此外，我们在感染小鼠的粪便中检测到环状噬菌体基因组，可能在复制的早期阶段，证实在微生物群丰富的小鼠感染期间发生了噬菌体诱导。此外，不响应DNA损伤或不表达stx的C. rodentium （phi stx(2dact)）突变体在体外既不产生高水平的Stx2，也不产生体内致死性感染，这证实了SOS诱导和噬菌体编码的stx基因的伴随表达是疾病发生所必需的。相比之下，不能切除原噬菌体基因组或包装噬菌体基因组的C. rodentium （phi stx(2dact)）突变体保留了在体外产生stx的能力，并在小鼠中引起致命疾病。因此，在充满微生物组的肠出血性大肠杆菌感染模型中，裂解诱导编码stx的噬菌体对致死性疾病至关重要，但实际的噬菌体生产并非如此。肠出血性大肠杆菌（EHEC）是一种产生志贺毒素的食源性病原体，与世界范围内严重的疾病爆发有关，其中包括在过去二十年中在美国爆发的390多起食物中毒事件。人类通过摄入受污染的食物或水，或通过与动物或其环境接触获得肠出血性大肠杆菌。感染和毒素的产生可能导致局部出血性结肠炎，但也可能发展为危及生命的全身性溶血性尿毒症综合征（HUS），这是儿童肾衰竭的主要原因。肠出血性大肠杆菌或溶血性尿毒综合征的治疗仍然难以捉摸，因为抗生素已被证明会加剧疾病。志贺毒素基因驻留在肠出血性大肠杆菌基因组中存在的休眠细菌病毒上，但在病毒被诱导离开休眠状态并开始复制时表达。广泛的病毒复制被认为是产生足以致病的毒素所必需的。在我们的肠出血性大肠杆菌疾病小鼠模型中使用病毒和细菌突变体，我们发现，尽管开始病毒复制所需的诱导信号是致死性疾病所必需的，但病毒的产生却不是：即使没有病毒复制，也会产生足够的志贺毒素来引起致死性小鼠疾病。未来对肠出血性大肠杆菌感染的人类样本的分析将确定这种现象是否适用，从而可能指导干预策略。
Enterohemorrhagic Escherichia coli (EHEC) colonize intestinal epithelium by generating characteristic attaching and effacing (AE) lesions. They are lysogenized by prophage that encode Shiga toxin 2 (Stx2), which is responsible for severe clinical manifestations. As a lysogen, prophage genes leading to lytic growth and stx2 expression are repressed, whereas induction of the bacterial SOS response in response to DNA damage leads to lytic phage growth and Stx2 production both in vitro and in germ-free or streptomycin-treated mice. Some commensal bacteria diminish prophage induction and concomitant Stx2 production in vitro, whereas it has been proposed that phage-susceptible commensals may amplify Stx2 production by facilitating successive cycles of infection in vivo. We tested the role of phage induction in both Stx production and lethal disease in microbiome-replete mice, using our mouse model encompassing the murine pathogen Citrobacter rodentium lysogenized with the Stx2-encoding phage stx(2dact). This strain generates EHEC-like AE lesions on the murine intestine and causes lethal Stx-mediated disease. We found that lethal mouse infection did not require that phi stx(2dact) infect or lysogenize commensal bacteria. In addition, we detected circularized phage genomes, potentially in the early stage of replication, in feces of infected mice, confirming that prophage induction occurs during infection of microbiota-replete mice. Further, C. rodentium (phi stx(2dact)) mutants that do not respond to DNA damage or express stx produced neither high levels of Stx2 in vitro or lethal infection in vivo, confirming that SOS induction and concomitant expression of phage-encoded stx genes are required for disease. In contrast, C. rodentium (phi stx(2dact)) mutants incapable of prophage genome excision or of packaging phage genomes retained the ability to produce Stx in vitro, as well as to cause lethal disease in mice. Thus, in a microbiome-replete EHEC infection model, lytic induction of Stx-encoding prophage is essential for lethal disease, but actual phage production is not.Author summary Enterohemorrhagic Escherichia coli (EHEC), a food-borne pathogen that produces Shiga toxin, is associated with serious disease outbreaks worldwide, including over 390 food poisoning outbreaks in the U.S. in the last two decades. Humans acquire EHEC by ingesting contaminated food or water, or through contact with animals or their environment. Infection and toxin production may result in localized hemorrhagic colitis, but may progress to life-threatening systemic hemolytic uremic syndrome (HUS), the leading cause of kidney failure in children. Treatment for EHEC or HUS remains elusive, as antibiotics have been shown to exacerbate disease. Shiga toxin genes reside on a dormant bacterial virus present in the EHEC genome, but are expressed when the virus is induced to leave its dormant state and begin to replicate. Extensive virus replication has been thought necessary to produce sufficient toxin to cause disease. Using viral and bacterial mutants in our EHEC disease mouse model, we showed that whereas an inducing signal needed to begin viral replication was essential for lethal disease, virus production was not: sufficient Shiga toxin was produced to cause lethal mouse disease, even without viral replication. Future analyses of EHEC-infected human samples will determine whether this same phenomenon applies, potentially directing intervention strategies.