Virulent Rickettsia species utilize the CD300f phosphatidylserine-binding receptor on macrophages for host colonization and pathogenesis

强毒立克次体利用巨噬细胞上的 CD300f 磷脂酰丝氨酸结合受体进行宿主定植和发病机制

• 批准号: 10674996
• 负责人: Mohammed Sayeedur Rahman
• 金额: $ 19.31万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10674996
• 关键词: ANXA5 gene; ARHGEF5 gene; Address; Affinity; Africa; Animals; Anti-Inflammatory Agents; Antibodies; Apoptotic; Appearance; Arthropod Vectors; Arthropods; Bacteria; Binding; Biological Assay; Bone Marrow; Boutonneuse Fever; Cell Surface Receptors; Cell membrane; Cells; Central America; Data; Dendritic Cells; Dermis; Development; Dichloromethylene Diphosphonate; Disease Outbreaks; Distant; Eating; Endemic Flea-Borne Typhus; Endothelial Cells; Etiology; Europe; Excision; Extracellular Domain; Glycerophospholipids; Goals; Grant; Homeostasis; Host Defense; Human; Immune Evasion; Immunoglobulin Variable Region; Immunologic Surveillance; Impairment; Individual; Infection; Invaded; Knowledge; Lethal Dose 50; Life Cycle Stages; Ligands; Link; Lipopolysaccharides; Liposomes; Macrophage; Mediating; Membrane; Membrane Proteins; Middle East; Morbidity - disease rate; Mus; Organ; Parasites; Pathogenesis; Pathogenicity; Phagocytes; Phagocytosis; Phagocytosis Induction; Phosphatidylserines; Process; Recombinants; Reporting; Research; Research Project Grants; Rickettsia; Rickettsia Infections; Rickettsia conorii; Rickettsia rickettsii; Rickettsia typhi; Rocky Mountain Spotted Fever; Role; Signal Transduction; South America; Surface; Surface Antigens; System; TFF1 gene; Testing; Typhus; Vaccines; Virulence; Virulent; Virus; Work; arthropod-borne; fighting; host colonization; human disease; in vivo; member; mortality; mouse model; novel; pathogen; pathogenic bacteria; phosphatidylserine receptor; prevent; programs; receptor; receptor binding; spotted fever; therapeutically effective

PROJECT SUMMARY Rickettsia species are arthropod-borne obligate intracellular bacteria with both symbiotic and pathogenic lifecycles. The global impact of rickettsial infections is highlighted by the resurgence of human infections with R. rickettsii (etiologic agent of Rocky Mountain Spotted Fever) in Central and South America, reappearance of R. conorii (etiologic agent of Boutonneuse Fever) in Europe, Middle East, and Africa9 and the recent outbreaks of R. rickettsii and R. typhi (etiologic agent of murine typhus) in the USA. Unfortunately, our inadequate understanding of Rickettsia-host interaction, in particular at the level on rickettsial engulfment via receptor-ligand- mediated phagocytosis on professional host defense cells, like macrophages (MΦ), has significantly impaired the development of effective therapeutics against these pathogens. To address these knowledge gaps, we propose to: i) characterize the specific rickettsial outer membrane glycerophospholipid ligand(s) involved in the phagocytic process of SFG and TG rickettsiae by MΦ, ii) decipher the role of efferocytic receptor, CD300f, in facilitating host colonization of virulent Rickettsia species. Our preliminary work identified phosphatidylserine (PS), a well-characterized “eat-me” signal involved in the phagocytosis of apoptotic cells (aka efferocytosis), as a putative ligand on outer membrane of rickettsiae. We further validated PS as crucial ligand for engulfment of R. typhi and R. rickettsii [Shelia Smith], two virulent bacteria representing the TG and SFG, respectively, by using recombinant Annexin V, a molecule known to block PS-mediated efferocytosis, in bone marrow-derived macrophages (BMMΦ). As PS-dependent phagocytosis requires PS-receptor recognition, we focused on CD300f, a type I transmembrane cell surface receptors with a high binding affinity to PS. Using WT and CD300f- /- BMMΦ, we showed that engulfment of R. typhi and R. rickettsii was dependent on CD300f expression, which was further confirmed by αCD300f antibody-mediated neutralization assays on WT BMMΦ. We tested the role of CD300f in vivo, by establishing mouse models of rickettsiosis with ~LD50 for both R. typhi and R. rickettsii species in C57BL/6J WT mice and showed that, unlike WT mice, CD300f-/- animals were protected against Rickettsia-induced lethality. Furthermore, in vivo depletion of MΦ, via clodronate-liposomes, suggest that CD300f-expression on MΦ contributed to the protection against Rickettsia-induced lethality. Hence, we will test the hypothesis, that both SFG and TGs Rickettsia exploit efferocytic signaling to invade MΦ, using the following Aims: to identify rickettsial outer membrane glycerophospholipid [phosphatidylserine (PS) or other] ligand(s) involved in the engulfment and colonization of pathogenic Rickettsia by MΦ (Aim 1) and define the contributing role of glycerophospholipid-binding receptor, CD300f, in promoting engulfment and colonization of the host by pathogenic Rickettsia in MΦ (Aim 2). Under this “Exploratory/Developmental Research Grant Program” we will study the mechanisms by which rickettsiae from both SFG and TG exploit the normally efferocytic CD300f-ligand systems on MΦ to successfully colonize the host. In sum, the purpose of this grant is to unveil mechanisms of rickettsial invasion with the goal to identify more proficient targets for anti-virulence therapy.

项目概要 立克次体是节肢动物传播的专性细胞内细菌，具有共生性和致病性 人类感染立克次体的死灰复燃凸显了立克次体感染的全球影响。 立克次体（落基山斑疹热的病原体）在中美洲和南美洲再次出现。 欧洲、中东和非洲 9 的 conorii（布通纽热病的病原体）以及最近爆发的 不幸的是，美国的立克次体和伤寒杆菌（鼠伤寒病原体）我们的能力不足。 了解立克次体与宿主相互作用，特别是在立克次体通过受体-配体吞噬的水平上 巨噬细胞（MΦ）等专业宿主防御细胞介导的吞噬作用显着受损 为了解决这些知识差距，我们开发针对这些病原体的有效疗法。 提议： i) 表征参与以下过程的特定立克次体外膜甘油磷脂配体： MΦ 对 SFG 和 TG 立克次体的吞噬过程，ii) 破译 efferocytic 受体 CD300f 在 我们的初步工作确定了磷脂酰丝氨酸。 (PS)，一种典型的“吃我”信号，参与凋亡细胞的吞噬作用（又名胞吞作用）， 我们进一步验证了 PS 是立克次体外膜上的假定配体。 R. 伤寒菌和 R. rickettsii [Shelia Smith]，两种毒力细菌，分别代表 TG 和 SFG，通过使用 重组膜联蛋白 V，一种已知可阻断骨髓来源的 PS 介导的胞吞作用的分子 由于 PS 依赖性吞噬作用需要 PS 受体识别，因此我们重点关注巨噬细胞 (BMMΦ)。 CD300f，一种与 PS 具有高结合亲和力的 I 型跨膜细胞表面受体。使用 WT 和 CD300f-。 /- BMMΦ，我们发现伤寒杆菌和立克次氏杆菌的吞噬依赖于 CD300f 的表达，这 我们测试了 WT BMMΦ 上的 αCD300f 抗体介导的中和测定进一步证实了这一作用。 通过建立伤寒立克次体病小鼠模型，对伤寒立克次体和立克次氏体的LD50均约为~LD50 C57BL/6J WT 小鼠中的物种，表明与 WT 小鼠不同，CD300f-/- 动物受到保护 此外，通过氯膦酸盐脂质体体内 MΦ 的消耗表明，立克次体引起的致死率。 MΦ 上的 CD300f 表达有助于防止立克次体引起的致死性，因此，我们将进行测试。 假设，SFG 和 TG 立克次氏体均利用细胞信号传导侵入 MΦ，使用以下方法 目的：鉴定立克次体外膜甘油磷脂[磷脂酰丝氨酸（PS）或其他]配体 参与 MΦ 吞噬和定植致病性立克次体（目标 1）并确定贡献因素 甘油磷脂结合受体 CD300f 在促进宿主吞噬和定植中的作用 MΦ 中的致病性立克次体（目标 2）根据该“探索性/发展性研究资助计划”，我们将 研究来自 SFG 和 TG 的立克次体利用正常细胞 CD300f 配体的机制 总之，这笔资助的目的是揭示 MΦ 上的系统成功殖民宿主的机制。 立克次体入侵的目标是确定更有效的抗毒力治疗靶点。