Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, USA.
Department of Molecular Biology and Microbiology, Graduate School of Biomedical Sciences, Tufts University School of Medicine, Boston, Massachusetts, USA.
J Bacteriol. 2021 Jun 8;203(13):e0014121. doi: 10.1128/JB.00141-21.
ICP2 is a virulent bacteriophage (phage) that preys on Vibrio cholerae. ICP2 was first isolated from cholera patient stool samples. Some of these stools also contained ICP2-resistant isogenic V. cholerae strains harboring missense mutations in the trimeric outer membrane porin protein OmpU, identifying it as the ICP2 receptor. In this study, we identify the ICP2 proteins that mediate interactions with OmpU by selecting for ICP2 host range mutants within infant rabbits infected with a mixture of wild-type and OmpU mutant strains. ICP2 host range mutants that can now infect OmpU mutant strains have missense mutations in the putative tail fiber gene and the putative adhesin gene . Using site-specific mutagenesis, we show that single or double mutations in are sufficient to generate the host range mutant phenotype. However, at least one additional mutation in is required for robust plaque formation on specific OmpU mutants. Mutations in alone were insufficient to produce a host range mutant phenotype. All ICP2 host range mutants retained the ability to form plaques on wild-type V. cholerae cells. The strength of binding of host range mutants to V. cholerae correlated with plaque morphology, indicating that the selected mutations in and restore molecular interactions with the receptor. We propose that ICP2 host range mutants evolve by a two-step process. First, mutations are selected for their broad host range, albeit accompanied by low-level phage adsorption. Subsequent selection occurs for mutations that further increase productive binding to specific OmpU alleles, allowing for near-wild-type efficiencies of adsorption and subsequent phage multiplication. Concern over multidrug-resistant bacterial pathogens, including Vibrio cholerae, has led to renewed interest in phage biology and the potential for phage therapy. ICP2 is a genetically unique virulent phage isolated from cholera patient stool samples. It is also one of three phages in a prophylactic cocktail that have been shown to be effective in animal models of infection and the only one of the three that requires a protein receptor (OmpU). This study identifies an ICP2 tail fiber and a receptor binding protein and examines how ICP2 responds to the selective pressures of phage-resistant OmpU mutants. We found that this particular coevolutionary arms race presents fitness costs to both ICP2 and V. cholerae.
ICP2 是一种烈性噬菌体（噬菌体），以霍乱弧菌为食。ICP2 最初是从霍乱患者的粪便样本中分离出来的。这些粪便中有些还含有 ICP2 抗性的同基因霍乱弧菌菌株，这些菌株的三聚体外膜孔蛋白 OmpU 中存在错义突变，将其鉴定为 ICP2 受体。在这项研究中，我们通过在感染野生型和 OmpU 突变株混合物的幼兔中选择 ICP2 宿主范围突变体，鉴定出介导与 OmpU 相互作用的 ICP2 蛋白。现在可以感染 OmpU 突变株的 ICP2 宿主范围突变体在假定的尾纤维基因和假定的粘附基因中存在错义突变。使用定点诱变，我们表明 中的单个或双突变足以产生宿主范围突变体表型。然而，在特定的 OmpU 突变体上形成强菌斑还需要 在 中至少存在一个额外的突变。 中的突变本身不足以产生宿主范围突变体表型。所有 ICP2 宿主范围突变体仍保留在野生型霍乱弧菌细胞上形成菌斑的能力。宿主范围突变体与霍乱弧菌的结合强度与菌斑形态相关，表明 中和 中的选定突变恢复了与受体的分子相互作用。我们提出，ICP2 宿主范围突变体通过两步进化过程产生。首先，选择具有广泛宿主范围的 突变体，尽管伴随着低水平的噬菌体吸附。随后选择进一步增加与特定 OmpU 等位基因有效结合的 突变体，从而允许接近野生型的吸附效率和随后的噬菌体增殖。对包括霍乱弧菌在内的多药耐药细菌病原体的担忧，导致人们重新关注噬菌体生物学和噬菌体治疗的潜力。ICP2 是一种从霍乱患者粪便样本中分离出来的具有独特遗传特性的烈性噬菌体。它也是预防鸡尾酒中的三种噬菌体之一，已在感染动物模型中证明有效，并且是三种噬菌体中唯一需要蛋白质受体（OmpU）的一种。本研究鉴定了一种 ICP2 尾纤维和一种受体结合蛋白，并研究了 ICP2 如何应对噬菌体抗性 OmpU 突变体的选择压力。我们发现，这种特殊的共同进化军备竞赛对 ICP2 和霍乱弧菌都带来了适应度代价。
