Stenotrophomonas maltophilia TfcA and TfcB: Antibacterial T4SS effectors from an emerging human pathogen

项目总结 嗜麦芽窄食单胞菌是一种新出现的多重耐药病原菌。发生SM感染 遍及全身，但最常表现为肺炎。SM在囊性纤维化(CF)患者中显著， 通常会增加肺部恶化的风险，最近关于Sm的报道进一步增加了它的重要性 感染新冠肺炎患者。尽管具有重要的临床意义，但对Sm的认识仍然相对有限。 最近，我们发现Sm编码一种IV型分泌系统(T4SS)，该系统在接触依赖的情况下促进 诱导巨噬细胞凋亡，钝化肺上皮细胞的凋亡。然而，也许更多 重要的是，我们发现Sm T4SS以接触依赖的方式介导对大肠杆菌(EC)的杀灭和 多种临床分离的铜绿假单胞菌(PA)，包括来自CF患者的菌株。因此，我们假设 Sm T4SS的抗菌作用影响人类感染，因为Sm和Pa经常在水中共存 在医院和各种类型的感染中，特别是在(慢性阻塞性肺疾病)肺中。在分析的基础上 突变体及其补体，我们确定了两个假定的效应子(TfCA和TfcB)是 Sm T4SS的杀菌活性。缺乏这两种蛋白质的突变体与缺乏这两种蛋白质的突变体一样受损。 T4SS装置，表明TFCA和TfcB解释了(几乎所有)所看到的杀灭效果。同时使用 测定与T4SS偶联蛋白结合和细菌间蛋白易位的2-杂交法 实验证明，TFCA和TfcB是T4SS的真实底物，这一结果得到了测试的证实 缺乏T4SS装置和单个效应器的突变体。将克隆的TFCA(单独)送入 周质导致了对目标细菌的杀灭，这表明这个效应器是必要的和充分的。 用于杀菌活性。生物信息学表明，TFCA代表了一种大型但未定性的 微生物脂肪酶的分支，而TfcB似乎表示一大群但定义不清的溶菌酶样组 酵素。与影响哺乳动物宿主的海量数据相比，T4SS和许多关于 对于VI型分泌系统的抗菌作用，对T4SS的抗菌作用的了解非常少。 事实上，Sm T4SS是迄今为止报道的唯一针对人类病原体的抗菌T4SS，TFCA和TfcB是其 只有记录在案的杀菌效应器。因此，我们建议：1)定义TFCA编码的酶活性 和TfcB，ii)辨别这些活动是否促进杀死PA和EC，以及iii)探索Sm T4SS，TFCA和TfcB 也会杀死其他与CF型肺病同居的细菌，从其他新出现的革兰氏菌到经典的革兰氏阳性菌 病原体对真菌的危害。除了提高我们对Sm的了解，所获得的数据将对以下方面产生广泛的影响 其他T4SS的作用以及T4SS效应器和抗微生物治疗之间可能的新联系。

PROJECT SUMMARY Gram- Stenotrophomonas maltophilia (Sm) is an emergent, multi-drug resistant pathogen. Sm infection occurs throughout the body but is most often manifest as pneumonia. Sm is notable in cystic fibrosis (CF) patients, often heightening risk of lung exacerbations, and its significance is increased further by recent reports of Sm infecting COVID-19 patients. Despite this clinical importance, knowledge of Sm is still relatively limited. Recently, we found that Sm encodes a type IV secretion system (T4SS) that promotes, in a contact-dependent manner, apoptosis of macrophages, while blunting apoptosis in lung epithelial cells. Yet, perhaps even more significant was our finding that the Sm T4SS mediates, in contact-dependent fashion, killing of E. coli (Ec) and multiple clinical isolates of Pseudomonas aeruginosa (Pa), including those from CF patients. Thus, we posited that the antibacterial effect of Sm T4SS impacts human infection, as Sm and Pa are often together in water systems in hospitals and in various types of infections, especially in the (CF) lungs. Based on the analysis of mutants and their complements, we identified two putative effectors (TfcA and TfcB) as being required for the bactericidal activity of the Sm T4SS. A mutant lacking both these proteins was as impaired as a mutant lacking the T4SS apparatus, indicating that TfcA and TfcB account for (nearly all) the killing effects seen. Using both a 2-hybrid assay that measures binding to the T4SS coupling protein and an interbacterial protein translocation assay, we determined that TfcA and TfcB are bona fide substrates of the T4SS, a result confirmed by testing mutants lacking both the T4SS apparatus and the individual effectors. Delivery of cloned TfcA (alone) into the periplasm resulted in the killing of target bacteria, indicating that this effector is both necessary and sufficient for bactericidal activity. Bioinformatics suggested that TfcA is representative of a large but uncharacterized branch of microbial lipases, whereas TfcB appears to signify a large but ill-defined group of lysozyme-like enzymes. In contrast to the vast data re T4SSs impacting mammalian hosts and the many studies on the antibacterial role of type VI secretion systems, knowledge of the antibacterial role of T4SSs is very minimal. Indeed, Sm T4SS is thus far the only antibacterial T4SS reported for a human pathogen, and TfcA and TfcB its only documented bactericidal effectors. Thus, we propose to i) define the enzyme activities encoded by TfcA and TfcB, ii) discern if those activities promote killing of Pa and Ec, and iii) explore if Sm T4SS, TfcA, and TfcB also kill other co-inhabitants of the CF lung, ranging from other emerging Gram- bacteria to classic Gram+ pathogens to fungi. Besides improving our knowledge of Sm, the data obtained will have broad implications for the roles of other T4SSs and possible new links between T4SS effectors and anti-microbial therapies.