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 嗜麦芽窄食单胞菌（Sm）是一种新发现的多重耐药病原菌. Sm感染发生 但最常表现为肺炎。Sm在囊性纤维化（CF）患者中是显著的， 通常会增加肺恶化的风险，其重要性因最近的Sm 感染新冠肺炎患者。尽管这种临床重要性，知识的Sm仍然相对有限。 最近，我们发现Sm编码一种IV型分泌系统（T4SS），该系统以接触依赖的方式促进细胞的增殖。 的方式，巨噬细胞的凋亡，而钝化肺上皮细胞的凋亡。然而，也许更多的是， 我们发现Sm T4SS以接触依赖的方式介导E.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患者的分离株。因此，我们假设 Sm T4SS的抗菌作用影响人类感染，因为Sm和Pa通常在水中一起 在医院和各种类型的感染，特别是在（CF）肺系统。通过分析 突变体及其互补物，我们确定了两个假定的效应子（TfcA和TfcB）作为所需的 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）所见的杀伤作用。同时使用 2-测量与T4SS偶联蛋白的结合和细菌间蛋白质易位的杂交测定 通过检测，我们确定TfcA和TfcB是T4SS的真正底物，这一结果通过测试证实 缺乏T4SS装置和单个效应子的突变体。将克隆的TfcA（单独）递送到 周质导致靶细菌的杀伤，表明这种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是一个大的，但未表征的代表。 TfcB是微生物脂肪酶的一个分支，而TfcB似乎意味着一个庞大但定义不清的溶菌酶样脂肪酶组。 内切酶与T4SS影响哺乳动物宿主的大量数据和许多关于T4SS的研究相反， 尽管关于VI型分泌系统的抗细菌作用的知识很少，但是关于T4SS的抗细菌作用的知识非常少。 事实上，Sm T4SS是迄今为止报道的针对人类病原体的唯一抗菌T4SS，并且TfcA和TfcB是其唯一的抗菌T4SS。 只有记录的杀菌效应。因此，我们建议i）定义由TfcA编码的酶活性 ii）辨别这些活性是否促进Pa和Ec的杀伤，以及iii）探索Sm T4SS、TfcA和TfcB是否 也杀死CF肺的其他共同居民，从其他新兴的革兰氏细菌到经典的革兰氏+ 病原体到真菌。除了提高我们对Sm的认识外，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效应子与抗微生物疗法之间可能的新联系。