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

嗜麦芽寡养单胞菌 TfcA 和 TfcB：来自新兴人类病原体的抗菌 T4SS 效应子

• 批准号: 10661253
• 负责人: NICHOLAS P CIANCIOTTO
• 金额: $ 23.03万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-17 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10661253
• 关键词: Achromobacter xylosoxidans; Anti-Bacterial Agents; Antibiotics; Apoptosis; Aspergillus fumigatus; Bacteria; Binding; Bioinformatics; Biological Assay; Burkholderia cepacia; C-terminal; COVID-19 patient; Cell Death; Cells; Cessation of life; Clinical; Complement; Consensus Sequence; Coupling; Data; Development; Enzymes; Epithelial Cells; Escherichia coli; Genes; Haemophilus influenzae; Hospitals; Human; Hybrids; Immunity; Impairment; Individual; Infection; Knowledge; Link; Lipase; Lipids; Lung; Macrophage; Measures; Mediating; Medical; Membrane; Microbe; Muramidase; N-Acetylmuramoyl-L-alanine Amidase; Pathogenesis; Peptidoglycan; Pneumonia; Protein Secretion; Protein translocation; Proteins; Pseudomonas aeruginosa; Pulmonary Cystic Fibrosis; Ralstonia; Reporting; Research Project Grants; Resistance; Risk; Role; Staphylococcus aureus; Stenotrophomonas maltophilia; System; Testing; Type IV Secretion System Pathway; United States National Institutes of Health; Water; Work; antimicrobial; bactericide; cystic fibrosis patients; disorder control; emerging human pathogen; enzyme activity; fungus; human pathogen; improved; microbial; microorganism interaction; multi-drug resistant pathogen; mutant; novel; pathogen; periplasm; three dimensional structure; tool

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.

项目摘要 施加性促嗜性嗜性嗜性（SM）是一种新兴的多药耐药性病原体。 SM感染发生 在整个身体中，但通常表现为肺炎。 SM在囊性纤维化（CF）患者中是著名的， 通过SM的最新报道，通常会增加肺部恶化的风险及其意义 感染Covid-19患者。尽管临床重要性，SM的知识仍然相对有限。 最近，我们发现SM编码了IV型分泌系统（T4SS），该系统在接触依赖性中促进 方式，巨噬细胞的凋亡，同时使肺上皮细胞的凋亡钝化。但是，也许更多 我们的发现是，SM T4SS以接触依赖性的方式进行了介导，杀死大肠杆菌（EC）和 铜绿假单胞菌（PA）的多种临床分离株，包括来自CF患者的临床分离株。因此，我们提出了 SM T4SS的抗菌作用会影响人类的感染，因为SM和PA经常在水中在一起 医院和各种类型感染的系统，尤其是（CF）肺部。基于分析 突变体及其补充，我们确定了两个推定的效应子（TFCA和TFCB）是必需的 SM T4SS的杀菌活性。缺乏这两种蛋白质的突变体与缺乏突变体一样受损 T4SS设备表明TFCA和TFCB（几乎全部）所看到的杀人效应。使用两个 2杂交测定法，测量与T4SS耦合蛋白和细菌间蛋白质易位结合的测定法 测定，我们确定TFCA和TFCB是T4SS的真正基板，这是通过测试证实的结果 缺乏T4SS机构和个体效应子的突变体。将克隆的TFCA（单独）传递到 pariplasm导致靶细菌杀死，表明该效应器既需要又足够 用于杀菌活性。生物信息学表明，TFCA代表了大型但未表征的 微生物脂肪酶的分支，而TFCB似乎表示一组较大但不确定的溶菌酶样 酶。与影响哺乳动物宿主的大量数据相比 VI型分泌系统的抗菌作用，对T4SS的抗菌作用的了解非常小。 实际上，迄今为止，SM T4S是唯一报道了人类病原体的抗菌T4SS，而TFCA和TFCB ITS 仅记录了杀菌效应子。因此，我们建议i）定义由TFCA编码的酶活性 tfcb，ii）辨别这些活动是否促进了PA和EC的杀害，以及iii）探索SM T4SS，TFCA和TFCB是否 还杀死CF肺的其他共同居民，从其他新兴革兰细菌到经典革兰氏+ 真菌的病原体。除了提高我们对SM的了解外，获得的数据还将对 其他T4SS的作用以及T4SS效应子与抗微生物疗法之间可能的新联系。