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.

项目概要 嗜麦芽窄食单胞菌 (Sm) 是一种新出现的多重耐药病原体。发生 Sm 感染 遍布全身，但最常表现为肺炎。 Sm 在囊性纤维化 (CF) 患者中很显着， 通常会增加肺部病情恶化的风险，最近 Sm 的报道进一步增加了其重要性 感染 COVID-19 患者。尽管具有临床重要性，但对 Sm 的了解仍然相对有限。 最近，我们发现 Sm 编码 IV 型分泌系统（T4SS），该系统促进接触依赖性 方式，巨噬细胞凋亡，同时抑制肺上皮细胞凋亡。然而，也许更多 重要的是我们发现 Sm T4SS 以接触依赖性方式介导杀死大肠杆菌 (Ec) 和 铜绿假单胞菌 (Pa) 的多种临床分离株，包括来自 CF 患者的分离株。因此，我们提出 Sm T4SS 的抗菌作用会影响人类感染，因为 Sm 和 Pa 经常一起存在于水中 医院系统和各种类型的感染，特别是（CF）肺部感染。基于以下分析 突变体及其补体，我们确定了两个假定的效应子（TfcA 和 TfcB） 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到损害。 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 测量与 T4SS 偶联蛋白的结合和细菌间蛋白易位的 2 杂交测定 测定中，我们确定 TfcA 和 TfcB 是 T4SS 的真正底物，这一结果通过测试得到证实 缺乏 T4SS 装置和个体效应器的突变体。将克隆的 TfcA（单独）递送至 周质导致目标细菌被杀死，表明该效应器是必要且充分的 用于杀菌活性。生物信息学表明 TfcA 代表了一个大型但未表征的 微生物脂肪酶的一个分支，而 TfcB 似乎表示一大群但定义不明确的溶菌酶样 酶。与 T4SS 影响哺乳动物宿主的大量数据以及关于 T4SS 的许多研究相比， VI 型分泌系统的抗菌作用，但对 T4SS 的抗菌作用的了解非常少。 事实上，Sm T4SS 是迄今为止唯一报道的针对人类病原体的抗菌 T4SS，而 TfcA 和 TfcB 是其唯一的抗菌 T4SS。 仅记录了杀菌效应器。因此，我们建议 i) 定义 TfcA 编码的酶活性 和 TfcB，ii) 辨别这些活动是否促进 Pa 和 Ec 的杀灭，以及 iii) 探索 Sm T4SS、TfcA 和 TfcB 是否 还杀死 CF 肺的其他共生菌，从其他新兴的革兰氏菌到经典的革兰氏+ 病原体为真菌。除了提高我们对 Sm 的了解之外，获得的数据还将对以下方面产生广泛的影响： 其他 T4SS 的作用以及 T4SS 效应器和抗微生物疗法之间可能存在的新联系。