The University of Aucklandgrid.9654.e, School of Biological Sciences, Auckland, New Zealand.
Forage Science, AgResearch, Lincoln Research Centre, Christchurch, New Zealand.
J Bacteriol. 2021 Sep 23;203(20):e0010421. doi: 10.1128/JB.00104-21. Epub 2021 Aug 9.
The antifeeding prophage (Afp) produced by the bacterium Serratia entomophila is the archetypical external contractile injection system (eCIS). Afp and its orthologues are characterized by three sheath proteins, while contractile bacteriophages and pyocins encode only one. Using targeted mutagenesis, transmission electron microscopy (TEM), and pulldown studies, we interrogated the roles of the three sheath proteins (Afp2, Afp3, and Afp4) in Afp assembly, in particular the interaction between the two sequence-related helical-sheath-forming proteins Afp2 and Afp3 and their cross talk with the tail termination sheath capping protein (TrP) Afp16 in the sheath maturation process. The expressed assemblies for the -deficient mutant were mostly a mixture of isolated tail fibers, detached baseplates without tail fibers, and sheathless inner tube baseplate complexes (TBCs) with a length similar to that of mature Afp, which were surrounded in many cases by fibrillar polymerized material. In the -deficient mutant, variable-length TBCs with similar but shorter fibrillar polymerized material, largely bereft of tail fibers, were observed, while only detached baseplate assemblies were seen for the -deficient mutant. Furthermore, we found that (i) only complementation of with its mutated counterpart restored mature Afp particles with full biological activity, (ii) purified Afp3 pulled down Afp2 by forming a sodium dodecyl sulfate (SDS)-resistant complex but not vice versa, (iii) Afp16 had a higher affinity for binding Afp2 or Afp3 than Afp4, and (iv) Afp4 is required for the association of the polymerized sheath on the baseplate via Afp2. A proposed model for sheath maturation and assembly in Afp is presented. Members of the contractile bacteriophage-related but evolutionarily divergent eCIS contain not one but three sheath proteins, two of which, namely, Afp2 and Afp3 in the Afp, arranged as alternate hexameric stacks constitute the helical sheath. We revealed that Afp2 and Afp3, even though they are highly similar, possess markedly distinct, crucial roles in Afp assembly. We find that Afp3, by virtue of its interaction with the tail-terminating protein Afp16, regulates tube and sheath length, while Afp2 is critical for proper sheath polymerization and the assembly of the baseplate. The resulting model for the Afp assembly will further guide the manipulation of Afp and its related eCISs as nanodelivery vehicles for pest control and phage therapy.
昆虫气单胞菌的抗食噬菌体（Afp）是典型的外部收缩注射系统（eCIS）。Afp 及其同源物的特征是有三个鞘蛋白，而收缩噬菌体和 pyocins 只编码一个。通过靶向诱变、透射电子显微镜（TEM）和下拉研究，我们研究了三个鞘蛋白（Afp2、Afp3 和 Afp4）在 Afp 组装中的作用，特别是两个序列相关的螺旋鞘形成蛋白 Afp2 和 Afp3 之间的相互作用及其与尾部终止鞘帽蛋白（TrP）Afp16 在鞘成熟过程中的相互作用。-缺陷突变体的表达组装物主要是由分离的尾部纤维、无尾部纤维的脱落基板和鞘内管基板复合物（TBC）组成，其长度与成熟 Afp 相似，在许多情况下，这些复合物被纤维状聚合材料包围。在-缺陷突变体中，观察到具有相似但较短纤维状聚合材料的可变长度 TBC，几乎没有尾部纤维，而-缺陷突变体只观察到脱落的基板组装物。此外，我们发现（i）只有用突变体互补才能恢复具有完整生物活性的成熟 Afp 颗粒，（ii）纯化的 Afp3 通过形成不溶于十二烷基硫酸钠（SDS）的复合物下拉 Afp2，但反之则不行，（iii）Afp16 与 Afp2 或 Afp3 的结合亲和力高于 Afp4，以及（iv）Afp4 是通过 Afp2 将聚合鞘与基板结合所必需的。提出了 Afp 鞘成熟和组装的模型。收缩噬菌体相关但进化上不同的 eCIS 的成员包含的鞘蛋白不是一个，而是三个，其中 Afp2 和 Afp3 在 Afp 中排列为交替的六聚体堆叠构成螺旋鞘。我们揭示了 Afp2 和 Afp3，尽管它们非常相似，但在 Afp 组装中具有明显不同且至关重要的作用。我们发现，Afp3 凭借其与尾部终止蛋白 Afp16 的相互作用，调节管和鞘的长度，而 Afp2 对适当的鞘聚合和基板组装至关重要。Afp 组装的模型将进一步指导 Afp 及其相关 eCIS 作为害虫控制和噬菌体治疗的纳米载体的操作。
