可收缩注射系统（Contractile injection systems，CISs）是在细菌和古细菌中广泛存在的一种重要的蛋白分泌系统，能够将细菌的毒力因子注射至靶细胞以使细菌获得生存优势。CISs的毒力因子决定着细菌的毒力和致病性，可作用于不同的细胞类型（细菌、真菌和真核细胞），因此获得了研究者的关注和重视。光杆菌毒力基因簇（Photorhabdus Virulence Cassette，PVC）作为典型的细胞外CISs，可被释放到胞外环境并从外部空间攻击宿主细胞，在光杆菌致病过程中发挥了重要作用，其更是被改造成为高效的蛋白递送工具。本论文选取PVC基因簇下游毒力因子PagT1（非共生光杆菌基因毒素1，Photorhabdus asymbiotica genotoxin 1）为研究对象，该毒力因子在现有数据库中不能搜索到类似的同源蛋白，提示其具有新型的作用方式。PagT1毒力机制研究不仅能拓宽对PVC毒力因子的认识，为广泛分布的类似新型毒力因子研究奠定基础，对光杆菌致病性及临床细菌感染性疾病治疗提供重要参考，还可为PVC提供其完美匹配的毒力货物，使其可作为一种高效的特定细胞杀伤工具。此外PVC尾丝蛋白的一个区域能特异性识别细胞受体，通过替换这部分结构域可以用于PVC针对特定小鼠和人类细胞的重定向。通过发现PVC新型毒力因子以及对PVC进行靶向性改造可为肿瘤等疾病的靶向治疗提供全新的治疗工具。本论文选用HER2为靶蛋白验证PVC靶向递送的可行性。目前HER2相关肿瘤药物如抗体、酪氨酸激酶抑制剂（TKIs）治疗仅采用单一的作用机制，即阻断HER2和HER3之间的相互作用，仅能短暂有效地阻断HER2功能，而不会导致细胞凋亡，这是产生耐药性的原因之一，并且由于各种机制，长期的抗体治疗也会产生耐药性，这在今天仍然是一个重大的临床挑战。虽然生物技术和制药行业已经关注了许多其他靶标，但靶向HER2的潜力尚未充分发挥，HER2仍然是开发新型抗肿瘤药物的优秀候选者。设计的锚蛋白重复序列蛋白（Designed ankyrin repeat proteins，DARPins）是一种新型结合蛋白，DARPins是部分合成的蛋白质，由多个堆叠的锚蛋白结构域重复序列组成，旨在模拟天然抗体的结合特性。每个锚蛋白重复模块的表面暴露区域中具有氨基酸多样性，这种内在特性，以及它们天然的高热稳定性和溶解性，使得DARPins易于定制，以实现对多种蛋白质靶点的高亲和力结合。本论文选用一种经验证的可特异结合HER2的DARPin，用来代替PVC尾丝蛋白Pvc13中的受体结合域，希望通过基因融合，实现融合表达HER2结合性DARPin的PVC颗粒特异识别肿瘤细胞表面特定受体HER2，进而达到精准递送基因毒素PagT1、杀伤肿瘤细胞的目的。

本论文结果表明，PagT1是PVC的毒力因子，通过PVC递送PagT1至小鼠及人巨噬细胞，鉴定了PagT1的毒性作用。通过可识别大部分人类细胞的PVCR7递送PagT1后，发现也能造成人肝癌、宫颈癌、肺癌、视网膜神经胶质瘤等多种癌细胞的明显杀伤，提示PagT1具有泛毒性。进一步探究PagT1的毒性机制，本论文发现PagT1为一种基因毒素，可以定位于细胞核造成基因组损伤进而导致细胞的PARP1依赖性细胞死亡。通过CRISPR/Cas9 sgRNA文库筛选和转录组测序，结合基因敲降、使用信号通路抑制剂等发现I型干扰素-JAK-STAT信号通路对PagT1的细胞毒性至关重要。本论文还发现，PagT1蛋白活性位点为D217和E219，将其装载入PVC递送至真核细胞后，PagT1依赖其核定位信号定位于细胞核，并通过其ADP核糖转移酶活性消耗NAD+造成NAD+耗竭，影响PARP1对基因组损伤的修复过程，造成细胞基因组的损伤，引起细胞发生PARP1依赖性细胞死亡Parthanatos，进而通过I型干扰素-JAK-STAT信号通路导致细胞死亡。接下来通过将可结合HER2的DARPin替换PVC尾丝蛋白Pvc13的受体结合域，构建得到靶向HER2的PVCHER2，装载PagT1后，可以特异性杀伤包括胶质瘤、膀胱癌等多种HER2阳性肿瘤细胞系，并进一步在膀胱癌类器官和小鼠原位胶质瘤模型中得到验证。鉴于PVC载体可以进行针对多种类型细胞表面受体的特异性理性改造，本论文初步证明了靶向改造、装载基因毒素PagT1的PVC作为肿瘤精准治疗药物的可行性。

Contractile injection systems (CISs) are an important protein secretion system widely present in bacteria and archaea, capable of injecting bacterial virulence factors into target cells to confer survival advantages to bacteria. The virulence factors of CISs determine the virulence and pathogenicity of bacteria and can act on different cell types (bacteria, fungi, and eukaryotic cells), thus attracting the attention and emphasis of researchers. The Photorhabdus Virulence Cassette (PVC) as a typical extracellular CISs can be released into the extracellular environment and attack host cells from the external space, playing an important role in the pathogenic process of Photorhabdus. It has also been modified into an efficient protein delivery tool. This thesis selects PagT1 (Photorhabdus asymbiotica genotoxin 1), a downstream virulence factor of the PVC gene cluster, as the research object. This virulence factor cannot be searched for similar homologous proteins in existing databases, suggesting that it has a novel mode of action. The study of the virulence mechanism of PagT1 can not only broaden the understanding of PVC virulence factors, lay the foundation for the study of similar novel virulence factors widely distributed, provide important references for the pathogenicity of Photorhabdus and the treatment of clinical bacterial infectious diseases, but also provide a perfect match for the virulence cargo of PVC, making it a highly efficient specific cell-killing tool. In addition, a region of the PVC tail filament protein can specifically recognize cell receptors. By replacing this structural domain, it can be used to redirect PVC to specific mouse and human cells. The discovery of new virulence factors of PVC and the targeted modification of PVC can provide new therapeutic tools for targeted treatment of diseases such as tumors. This thesis selects HER2 as the target protein to verify the feasibility of PVC targeted delivery. Currently, HER2-related tumor drugs such as antibodies and tyrosine kinase inhibitors (TKIs) only use a single mechanism of action, that is, blocking the interaction between HER2 and HER3, which can only temporarily and effectively block the function of HER2 without causing cell apoptosis. This is one of the reasons for the development of drug resistance. Moreover, due to various mechanisms, long-term antibody treatment can also lead to drug resistance, which remains a major clinical challenge today. Although the biotechnology and pharmaceutical industries have focused on many other targets, the potential of targeting HER2 has not been fully exploited, and HER2 remains an excellent candidate for the development of new anti-tumor drugs. Designed ankyrin repeat proteins (DARPins) are a new type of binding protein. DARPins are partially synthetic proteins composed of multiple stacked ankyrin domain repeats, aiming to simulate the binding characteristics of natural antibodies. Each ankyrin repeat module has amino acid diversity in the surface exposed region, and this intrinsic property, along with their natural high thermal stability and solubility, makes DARPins easy to customize to achieve high-affinity binding to various protein targets. This thesis selects a validated DARPin that can specifically bind to HER2 to replace the receptor binding domain of the PVC tail filament protein Pvc13. It is hoped that through gene fusion, the PVC particles expressing the HER2-binding DARPin can specifically recognize the specific receptor HER2 on the surface of tumor cells, thereby achieving the precise delivery of the genotoxin PagT1 and killing tumor cells.

Our results indicate that PagT1 is a virulence factor of PVC. By delivering PagT1 to mouse and human macrophages via PVC, we identified the toxic effects of PagT1. After delivering PagT1 using PVCR7, which can recognize most human cells, we found that it could also cause significant killing of various cancer cells, including human liver cancer, cervical cancer, lung cancer, and retinoblastoma, suggesting that PagT1 has pan-toxicity. Further exploration of the toxicity mechanism of PagT1 revealed that it is a genotoxin that can localize in the nucleus, causing genomic damage and leading to PARP1-dependent cell death. Through CRISPR/Cas9 sgRNA library screening and transcriptome sequencing, combined with gene knockdown and the use of signaling pathway inhibitors, we found that the type I interferon-JAK-STAT signaling pathway is crucial for the cytotoxicity of PagT1. We also discovered that the active sites of PagT1 are D217 and E219. After loading PagT1 into PVC and delivering it to eukaryotic cells, PagT1 localizes in the nucleus depending on its nuclear localization signal and consumes NAD+ through its ADP-ribosyltransferase activity, causing NAD+ depletion and affecting the repair process of genomic damage by PARP1, resulting in genomic damage in cells and triggering PARP1-dependent cell death (Parthanatos). This process further leads to cell death through the type I interferon-JAK-STAT signaling pathway. Next, we replaced the receptor binding domain of the PVC tail fiber protein Pvc13 with a DARPin that can bind to HER2 to construct PVCHER2 targeting HER2. After loading PagT1, it can specifically kill various HER2-positive tumor cell lines, including glioma and bladder cancer, and this was further verified in bladder cancer organoids and mouse orthotopic glioma models. Given that the PVC vector can be rationally modified for specificity towards various types of cell surface receptors, this work initially demonstrates the feasibility of using PVC, which is targeted and loaded with the genotoxin PagT1, as a precision therapeutic drug for tumors.