Salmonella-host interaction is mediated by a novel Cdc42 effector and lipidations

沙门氏菌-宿主相互作用是由新型 Cdc42 效应子和脂化介导的

• 批准号: 9896663
• 负责人: Sheila Bandyopadhyay
• 金额: $ 4.37万
• 依托单位: RUTGERS THE STATE UNIV OF NJ NEWARK
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9896663
• 关键词: Ablation; Actins; Address; Affect; Bacteria; Bacterial Infections; Binding; Binding Proteins; Biological Assay; Biotin; COS-1 Cells; Caco-2 Cells; Cell physiology; Cells; Cellular biology; Cessation of life; Cytoskeletal Modeling; Cytoskeletal Proteins; Data; Development; Diarrhea; Disease; Disease susceptibility; Dominant-Negative Mutation; Enzymes; Epithelial; Epithelial Cells; Epithelium; Family member; Fever; Gastrointestinal Diseases; Genetic; Genetic Engineering; Goals; Hospitalization; Human; Impairment; In Vitro; Infection; Invaded; Knockout Mice; Knowledge; Lead; Lipids; Mediating; Modeling; Modification; Molecular; Mus; Pathologic; Polymers; Predisposition; Process; Proteins; Proteomics; RNA Splicing; Role; Salmonella; Salmonella enterica; Salmonella infections; Salmonella typhimurium; Site; Techniques; Therapeutic; Transgenic Mice; United States; Variant; Virulence; Vomiting; commensal bacteria; enteric pathogen; experimental study; gastrointestinal; gut colonization; improved; in vivo; innovation; intestinal epithelium; monolayer; mouse model; mutant; non-typhoidal Salmonella; novel; palmitoylation; pathogen; pathogenic bacteria; prenylation; prevent; recruit; rho GTP-Binding Proteins; therapeutic development

PROJECT SUMMARY Gastrointestinal diseases caused by Salmonella enterica serovar Tymphimurium (S. Typhimurium) lead to numerous illnesses, hospitalizations, and deaths globally every year. S. Typhimurium successfully invades host cells by injecting virulence effectors that modulate the host actin cytoskeletal network, through host cell machinery such as Rho GTPases. The Rho GTPase family member Cdc42 has been identified as a target of S. Typhimurium; however, specific mechanisms for its recruitment and its function in vivo during a Salmonella infection remain unclear. Preliminary studies demonstrate that Cdc42 Effector Protein 1 (Cdc42EP1), a novel binding protein isolated from Cdc42 interactome, localizes to Salmonella entry sites in epithelial cells. Additionally, its interaction with Cdc42 variants deficient of their lipid modifications is reduced. Although S. Typhimurium has been found to utilize host lipidation enzymes to modify its own proteins, it is unknown how the prenylation and palmitoylation modifications of Cdc42 impact Salmonella’s invasive ability. We hypothesize that S. Typhimurium recruits Cdc42 via Cdc42EP1, and that altering Cdc42’s lipid modifications impacts bacterial invasion and disease susceptibility in vivo. Two aims are proposed to determine the cellular function and impact of Cdc42EP1-Cdc42 machinery during Salmonella infection in vitro and in vivo. The specific goal of this project is to understand whether Salmonella exploits Cdc42EP1-Cdc42 machinery to gain entry and survival in host cells, and how this cellular machinery impacts the pathogen’s invasive ability in vivo. Aim 1 will address the molecular mechanisms by which S. Typhimurium manipulate and recruit both Cdc42 and Cdc42EP1 during invasion. The role of Cdc42EP1 during Salmonella invasion, its spatial and temporal relationship with Cdc42 upon infection, and its interaction with lipidation-deficient Cdc42 mutants during infection will all be studied. Aim 2 will utilize our unique genetically engineered Cdc42 mouse models to determine whether altering Cdc42 machinery affects the susceptibility to a Salmonella infection in vivo. Cdc42 knockout mice will be used to determine how deletion of Cdc42 impacts Salmonella invasion. We will also utilize our newly developed transgenic mice that express palmitoylable Cdc42 to understand how this modification may impact a bacterial infection and disease susceptibility. In addition to analysis of bacterial invasion in these mice, we will determine if Salmonella modify the host protein using an acyl biotin exchange assay. Together, these aims will reveal the mechanism of Salmonella-Cdc42 interaction at cell biology and physiology levels.

项目总结 鼠伤寒沙门氏菌引起的胃肠道疾病 每年在全球范围内导致无数的疾病、住院和死亡。鼠伤寒沙门氏菌成功 通过注射调节宿主肌动蛋白细胞骨架网络的毒力效应器来入侵宿主细胞，通过 宿主细胞机制，如Rho GTP酶。Rho GTPase家族成员cdc42已被鉴定为 鼠伤寒沙门氏菌的靶标；然而，其募集的特定机制及其在体内的作用 沙门氏菌感染仍不清楚。初步研究表明，CDC42效应蛋白1 CDC42EP1是一种新的结合蛋白，定位于沙门氏菌进入沙门氏菌的部位。 上皮细胞。此外，它与缺乏脂质修饰的Cdc42变体的相互作用也减少了。 尽管已经发现鼠伤寒沙门氏菌利用宿主脂肪化酶来修饰自己的蛋白质，但它是 尚不清楚CDC42的苯丙基化和棕榈酰化修饰如何影响沙门氏菌的侵袭能力。 我们推测，鼠伤寒沙门氏菌通过CDC42EP1征募CDC42，改变CDC42‘S脂 修饰会影响细菌在体内的侵袭和疾病易感性。提出了两个目标，以 体外检测CDC42EP1-CDC42机制在沙门氏菌感染过程中的细胞功能和影响 在活体内。这个项目的具体目标是了解沙门氏菌是否利用cdc42ep1-cdc42 在宿主细胞中获得进入和生存的机制，以及这种细胞机制如何影响病原体 体内侵袭能力。 目标1将阐述鼠伤寒沙门氏菌操纵和招募两者的分子机制。 CDC42和CDC42EP1在侵袭过程中的表达。CDC42EP1在沙门氏菌入侵中的作用及其空间分布 感染后与CDC42的时间关系及其与脂肪氧化缺陷的CDC42突变体的相互作用 在感染过程中都将被研究。 AIM 2将利用我们独特的基因工程CDC42小鼠模型来确定 改变CdC42的机械结构会影响体内对沙门氏菌感染的敏感性。CDC42基因敲除小鼠将 用来确定CDC42的缺失如何影响沙门氏菌的侵袭。我们还将利用我们新的 开发了表达棕榈酰化的cdc42的转基因小鼠，以了解这种修饰可能产生的影响。 细菌感染和疾病易感性。除了分析这些小鼠的细菌入侵情况外，我们还 将使用酰基生物素交换试验确定沙门氏菌是否修改宿主蛋白。 综上所述，这些目标将揭示沙门氏菌-cdc42相互作用的细胞生物学和 生理水平。