Cryo-EM structures of type IV pili complexed with nanobodies reveal immune escape mechanisms

Type IV pili (T4P) are prevalent, polymeric surface structures in pathogenic bacteria, making them ideal targets for effective vaccines. However, bacteria have evolved efficient strategies to evade type IV pili-directed antibody responses. Neisseria meningitidis are prototypical type IV pili-expressing Gram-negative bacteria responsible for life threatening sepsis and meningitis. This species has evolved several genetic strategies to modify the surface of its type IV pili based on recombination, phase variation and the presence of different alleles of genes involved in posttranslational modifications. This results in changes in pilin subunit amino acid sequence, nature of glycosylation and phosphoform modification, but how these different processes affect antibody binding at the structural level is still unknown. To explore this question, we provide cryo-electron microscopy structures of pili of different sequence types with sufficiently high resolution to visualize posttranslational modifications. We then generated nanobodies directed against type IV pili which alter pilus function in vitro and vivo; and determined their structures complexed with their pilus target. We also determined how the different types of pili surface modifications alter nanobody binding. These different structures shed light on the impressive complementarity between the different strategies used by bacteria to avoid antibody binding. Importantly, we also show that structural information can be used to make informed modifications in nanobodies as countermeasures to these immune evasion mechanisms.

Ⅳ型菌毛（Type IV pili, T4P）是病原菌中广泛存在的聚合性表面结构，因此成为高效疫苗的理想靶点。然而，细菌已演化出高效策略以逃避靶向Ⅳ型菌毛的抗体免疫应答。脑膜炎奈瑟菌（Neisseria meningitidis）是典型的表达Ⅳ型菌毛的革兰氏阴性病原菌，可引发致死性败血症与脑膜炎。该物种已演化出多种遗传策略，通过基因重组、相变异以及参与翻译后修饰（posttranslational modifications）的基因携带不同等位基因的特性，来修饰其Ⅳ型菌毛的表面。这会导致菌毛蛋白亚基的氨基酸序列、糖基化类型以及磷酸化修饰形式发生改变，但目前尚不清楚这些不同过程在结构层面如何影响抗体结合。为探究这一科学问题，我们解析了不同序列类型菌毛的高分辨率冷冻电子显微镜（cryo-electron microscopy）结构，足以清晰观测其翻译后修饰。随后我们制备了靶向Ⅳ型菌毛的纳米抗体（nanobodies），这类纳米抗体可在体外与体内改变菌毛功能，并解析了它们与其菌毛靶标结合的复合物结构。我们还明确了不同类型的菌毛表面修饰如何影响纳米抗体的结合。这些不同的结构揭示了细菌用于逃避抗体结合的多种策略之间存在精妙的互补性。重要的是，我们还证明了可利用结构信息对纳米抗体进行定向改造，以此作为应对这些免疫逃逸机制的有效对策。