Molecular approaches to understand vector-host and vector-pathogen interactions

了解载体-宿主和载体-病原体相互作用的分子方法

• 批准号: 8555876
• 负责人: Jesus Valenzuela
• 金额: $ 122.27万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8555876
• 关键词: Accounting; Actins; Activated Partial Thromboplastin Time measurement; Adjuvant; Animals; Antibodies; Anticoagulants; Antigens; Antithrombin III; Apyrase; Arthritis; Binding; Bite; Blood coagulation; Bone Marrow; CD3 Antigens; CD4 Positive T Lymphocytes; Calorimetry; Carotid Arteries; Cell Line; Cells; Chemotaxis; Clinical Research; Coagulation Process; Cutaneous; Dendritic Cells; Development; Disease; Disease Outcome; Edema; Environment; Enzyme-Linked Immunosorbent Assay; Enzymes; Equilibrium; Experimental Arthritis; Factor Xa; Flow Cytometry; GRK; Gene Expression Profile; Helper-Inducer T-Lymphocyte; Hyaluronidase; IL8RB gene; Immigration; Immune response; Immunity; Immunization; Immunomodulators; Inbred BALB C Mice; Infection; Inflammation; Inflammatory; Injection of therapeutic agent; Interferon Type II; Interferons; Interleukin-10; Interleukin-17; Investigation; Leishmania; Leishmania infantum; Leishmania major; Leishmania tropica; Leishmaniasis; Lesion; Lutzomyia genus; Mammalian Cell; Measures; Mechanics; Midgut; Modeling; Molecular; Molecular Biology; Mus; Names; Needles; Ovalbumin; PAR-2 Receptor; Pain; Parasite Control; Parasites; Pathology; Phlebotomus; Physiological; Plasmids; Production; Property; Proteins; Proteome; Proteomics; Prothrombin time assay; Recombinant Proteins; Recombinants; Reporting; Research; SELL gene; Saliva; Salivary; Salivary Glands; Salivary Proteins; Sand Flies; Silicon Dioxide; Surface Plasmon Resonance; T memory cell; TNF gene; Testing; Thromboplastin; Thrombosis; Thrombus; Time; Titrations; Ulcer; Vaccinated; Vaccines; Virulence; Visceral Leishmaniasis; Work; arginyl-glycyl-glutamic acid; human disease; in vivo; inhibitor/antagonist; lymph nodes; migration; neglect; neutrophil; novel; pathogen; polymerization; prevent; protective efficacy; research study; response; stoichiometry; transcriptomics; transmission process; vaccine candidate; vector; vector-based vaccine

The accomplishments of the section are: 1) Immunity to Sand Fly Salivary Protein LJM11 Modulates Host Response to Vector-Transmitted Leishmania Conferring pathology-Free Protection. Leishmania vaccines that protect against needle challenge fail against the potency of a Leishmania-infected sand fly transmission. Here, we demonstrate that intradermal immunization of mice with 500ng of the sand fly salivary recombinant protein LJM11 (rLJM11) from Lutzomyia longipalpis, in the absence of adjuvant, induces long-lasting immunity that results in ulcer-free protection against Leishmania major delivered by vector bites. This protection is antibody independent and abrogated by depletion of CD4(+) T cells. Two weeks after challenge, early induction of IFN-gamma; specifically to rLJM11 correlates to diminished parasite replication in protected animals. At this time point, Leishmania-specific induction of IFN-gamma; in these mice is low in comparison with its high level in non-protected controls. We hypothesize that early control of parasites in a T-cell helper type 1 environment induced by immunity to LJM11 permits the slow development of Leishmania-specific immunity in the absence of open ulcers. Leishmania-specific immunity observed 5 weeks after infection in rLJM11-immunized mice shows a twofold increase over controls in the percentage of IFN-gamma;-producing CD4(+) T cells. We propose LJM11 as an immunomodulator that drives an efficient and controlled protective immune response to a sand fly-transmitted Leishmania somewhat mimicking "leishmanization"-induced protective immunity but without its associated lesions 2) KSAC, a defined Leishmania antigen, plus adjuvant protects against the virulence of L. major transmitted by its natural vector Phlebotomus duboscqi. Recombinant KSAC and L110f are promising Leishmania vaccine candidates. Both antigens protected BALB/c mice against L. major infection following needle challenge. Considering the virulence of vector-transmitted Leishmania infections, we vaccinated BALB/c mice with either KSAC+GLA-SE or L110f+GLA-SE to assess protection against L. major transmitted via its vector Phlebotomus duboscqi. Mice receiving the KSAC or L110f vaccines were challenged by needle or L. major-infected sand flies. Following sand fly challenge, KSAC-vaccinated mice were protected while L110f-vaccinated animals showed partial protection. Protection correlated with the ability of SLA to induce IFN-gamma;-producing CD4(+)CD62L(low)CCR7(low) effector memory T cells pre- and post-sand fly challenge. This study demonstrates the protective efficacy of KSAC+GLA-SE against sand fly challenge; the importance of vector-transmitted challenge in evaluating vaccine candidates against Leishmania infection; and the necessity of a rapid potent Th1 response against Leishmania to attain true protection. 3) Salivary gland transcriptomes and proteomes of Phlebotomus tobbi and Phlebotomus sergenti, vectors of leishmaniasis. Phlebotomus tobbi is a vector of Leishmania infantum, and P. sergenti is a vector of Leishmania tropica. To better understand the components and possible implications of sand fly saliva in leishmaniasis, the transcriptomes of the salivary glands (SGs) of these two sand fly species were sequenced, characterized and compared. The most abundant, secreted putative proteins were categorized as antigen 5-related proteins, apyrases, hyaluronidases, D7-related and PpSP15-like proteins, ParSP25-like proteins, PpSP32-like proteins, yellow-related proteins, the 33-kDa salivary proteins, and the 41.9-kDa superfamily of proteins. This analysis of P. sergenti is the first description of the subgenus Paraphlebotomus salivary components. The investigation of the subgenus Larroussius sand fly P. tobbi expands the repertoire of salivary proteins in vectors of Le. infantum. Although P. tobbi transmits a cutaneous form of leishmaniasis, its salivary proteins are most similar to other Larroussius subgenus species transmitting visceral leishmaniasis. These transcriptomic and proteomic analyses provide a better understanding of sand fly salivary proteins across species and subgenera that will be vital in vector-pathogen and vector-host research. 4) Lufaxin, a Novel Factor Xa Inhibitor From the Salivary Gland of the Sand Fly Lutzomyia longipalpis, Blocks Protease-Activated Receptor 2 Activation and Inhibits Inflammation and Thrombosis In Vivo. The aim of the study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. Several L. longipalpis salivary proteins were expressed in mammalian cells (293 F) and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant of 3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl(3)-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. 5) The protein LJM 111 from Lutzomyia longipalpis salivary gland extract (SGE) accounts for the SGE-inhibitory effects upon inflammatory parameters in experimental arthritis model. 5) The protein LJM 111 from Lutzomyia longipalpis salivary gland extract (SGE) accounts for the SGE-inhibitory effects upon inflammatory parameters in experimental arthritis model. Several studies have pointed out the immunomodulatory properties of the Salivary Gland Extract (SGE) from Lutzomyia longipalpis. We aimed to identify the SGE component (s) responsible for its effect on ovalbumin (OVA)-induced neutrophil migration (NM) and to evaluate the effect of SGE and components in the antigen-induced arthritis (AIA) model. We tested the anti-arthritic activities of SGE and the recombinant LJM111 salivary protein (rLJM111) by measuring the mechanical hypernociception and the NM into synovial cavity. Furthermore, we measured IL-17, TNF- and IFN- released by lymph nodes cells stimulated with mBSA or anti-CD3 using enzyme-linked immunosorbent assay (ELISA). Additionally, we tested the effect of SGE and rLJM111 on co-stimulatory molecules expression (MHC-II and CD-86) by flow cytometry, TNF- and IL-10 production (ELISA) of bone marrow-derived dendritic cells (BMDCs) stimulated with LPS, chemotaxis and actin polymerization from neutrophils. Besides, the effect of SGE on CXCR2 and GRK-2 expression on neutrophils was investigated. We identified one plasmid expressing the protein LJM111 that prevented NM in OVA-challenged immunized mice. Furthermore, both SGE and rLJM111 inhibited NM and pain sensitivity in AIA and reduced IL-17, TNF- and IFN-. SGE and rLJM111 also reduced MHC-II and CD-86 expression and TNF- whereas increased IL-10 release by LPS-stimulated BMDCs. SGE, but not LJM 111, inhibited neutrophils chemotaxis and actin polymerization. Additionally, SGE reduced neutrophil CXCR2 expression and increased GRK-2. Thus, rLJM111 is partially responsible for SGE mechanisms by diminishing DC function and maturation but not chemoattraction of neutrophils.

该科的成果有： 1) 对白蛉唾液蛋白 LJM11 的免疫调节宿主对媒介传播利什曼原虫的反应，从而提供无病理保护。 预防针刺攻击的利什曼原虫疫苗无法抵抗利什曼原虫感染的白蛉传播的效力。在这里，我们证明，在没有佐剂的情况下，用 500 ng 来自长须蝇的白蛉唾液重组蛋白 LJM11 (rLJM11) 对小鼠进行皮内免疫，可诱导持久的免疫，从而通过载体传递针对重大利什曼原虫的无溃疡保护叮咬。这种保护不依赖于抗体，并且通过消除 CD4(+) T 细胞而消除。攻击后两周，早期诱导IFN-γ；特别是 rLJM11 与受保护动物体内寄生虫复制的减少相关。此时，利什曼原虫特异性诱导 IFN-γ；与未受保护的对照组中的高水平相比，这些小鼠中的水平较低。我们假设，在 LJM11 免疫诱导的 T 细胞辅助 1 型环境中早期控制寄生虫，可以在没有开放性溃疡的情况下缓慢发展利什曼原虫特异性免疫。 rLJM11 免疫小鼠感染后 5 周观察到的利什曼原虫特异性免疫显示，产生 IFN-γ 的 CD4(+) T 细胞的百分比比对照组增加两倍。我们建议 LJM11 作为一种免疫调节剂，可驱动针对沙蝇传播的利什曼原虫的有效且受控的保护性免疫反应，有点模仿“利什曼化”诱导的保护性免疫，但没有相关病变 2) KSAC，一种明确的利什曼原虫抗原，加上佐剂可防止由其天然载体杜博斯白蛇传播的利什曼原虫的毒力。 重组 KSAC 和 L110f 是有前途的利什曼原虫候选疫苗。两种抗原均可保护 BALB/c 小鼠在针刺后免受大型利斯特氏菌感染。考虑到媒介传播的利什曼原虫感染的毒力，我们给 BALB/c 小鼠接种 KSAC+GLA-SE 或 L110f+GLA-SE 疫苗，以评估对通过媒介白蛉属杜博斯奇 (Phlebotomus duboscqi) 传播的利什曼原虫的保护作用。 接受 KSAC 或 L110f 疫苗的小鼠受到针叶蝇或 L. Major 感染的白蛉的攻击。白蛉攻击后，接种 KSAC 疫苗的小鼠受到保护，而接种 L110f 疫苗的小鼠则显示出部分保护。在沙蝇攻击前后，保护作用与 SLA 诱导产生 IFN-γ 的 CD4(+)CD62L(低)CCR7(低)效应记忆 T 细胞的能力相关。本研究证明了 KSAC+GLA-SE 对白蛉攻击的保护功效；媒介传播挑战在评估针对利什曼原虫感染的候选疫苗中的重要性；以及针对利什曼原虫快速有效的 Th1 反应的必要性，以获得真正的保护。 3) 利什曼病载体白蛉和塞氏白蛉的唾液腺转录组和蛋白质组。 Phlebotomus tobbi 是婴儿利什曼原虫的媒介，P. sergenti 是热带利什曼原虫的媒介。 为了更好地了解白蛉唾液的成分和对利什曼病的可能影响，对这两种白蛉物种的唾液腺（SG）的转录组进行了测序、表征和比较。最丰富的分泌推定蛋白被分类为抗原 5 相关蛋白、腺苷三磷酸双磷酸酶、透明质酸酶、D7 相关蛋白和 PpSP15 样蛋白、ParSP25 样蛋白、PpSP32 样蛋白、黄色相关蛋白、33 kDa 唾液蛋白，以及 41.9-kDa 蛋白质超家族。对 P. sergenti 的分析是对 Paraphlebotomus 唾液成分的首次描述。对 Larroussius 沙蝇亚属 P. tobbi 的研究扩展了 Le 载体中唾液蛋白的库。婴儿。尽管托比假单胞菌传播皮肤形式的利什曼病，但其唾液蛋白与传播内脏利什曼病的其他拉鲁斯亚属物种最为相似。这些转录组和蛋白质组分析可以更好地了解跨物种和亚属的白蛉唾液蛋白，这对于媒介病原体和媒介宿主研究至关重要。 4) Lufaxin 是一种来自白蛉 Lutzomyia longipis 唾液腺的新型 Xa 因子抑制剂，可阻断蛋白酶激活受体 2 的激活并抑制体内炎症和血栓形成。 这项研究的目的是鉴定白蛉 Lutzomyia longipalis 独特的唾液抗凝剂，这种物质几十年来一直难以捉摸。几种长须乳杆菌唾液蛋白在哺乳动物细胞 (293 F) 中表达，并筛选其对凝血的抑制作用。一种名为 Lufaxin 的新型 32.4 kDa 分子被鉴定为一种缓慢、紧密、非竞争性和可逆的 Xa 因子 (FXa) 抑制剂。值得注意的是，Lufaxin 的一级序列与迄今为止报道的任何生理学或唾液凝血抑制剂不具有相似性。 Lufaxin 对 FXa 具有特异性，不会与 FX、Dansyl-Glu-Gly-Arg-FXa 或 15 种其他酶发生相互作用。此外，Lufaxin 阻断凝血酶原酶并增加凝血酶原时间和活化部分凝血活酶时间。表面等离子共振实验表明，FXa 与 Lufaxin 的平衡常数为 3 nM，等温滴定量热法确定化学计量为 1:1。 Lufaxin 还可以防止 MDA-MB-231 细胞系中 FXa 激活蛋白酶激活受体 2，并消除在小鼠爪子中注射 FXa 引发的水肿形成。此外，Lufaxin 可防止 FeCl(3) 诱导的颈动脉血栓形成，并延长体外活化的部分凝血活酶时间，这意味着它在体内可作为抗凝剂。 5) 来自 Lutzomyia longipis 唾液腺提取物 (SGE) 的蛋白质 LJM 111 解释了 SGE 对实验性关节炎模型中炎症参数的抑制作用。 5) 来自 Lutzomyia longipis 唾液腺提取物 (SGE) 的蛋白质 LJM 111 解释了 SGE 对实验性关节炎模型中炎症参数的抑制作用。 几项研究指出了长须鼠唾液腺提取物 (SGE) 的免疫调节特性。我们的目的是确定对卵清蛋白 (OVA) 诱导的中性粒细胞迁移 (NM) 产生影响的 SGE 成分，并评估 SGE 和成分在抗原诱导的关节炎 (AIA) 模型中的作用。我们通过测量机械性痛觉感受和进入滑膜腔的 NM 来测试 SGE 和重组 LJM111 唾液蛋白 (rLJM111) 的抗关节炎活性。此外，我们使用酶联免疫吸附测定（ELISA）测量了用 mBSA 或抗 CD3 刺激的淋巴结细胞释放的 IL-17、TNF- 和 IFN-。此外，我们通过流式细胞术、刺激的骨髓源性树突状细胞 (BMDC) 的 TNF- 和 IL-10 产生 (ELISA) 测试了 SGE 和 rLJM111 对共刺激分子表达（MHC-II 和 CD-86）的影响与 LPS、趋化性和中性粒细胞的肌动蛋白聚合有关。此外，还研究了SGE对中性粒细胞CXCR2和GRK-2表达的影响。我们鉴定了一种表达 LJM111 蛋白的质粒，该蛋白可预防 OVA 攻击的免疫小鼠中的 NM。此外，SGE 和 rLJM111 都能抑制 AIA 中的 NM 和疼痛敏感性，并减少 IL-17、TNF-和 IFN-。 SGE 和 rLJM111 还降低了 MHC-II 和 CD-86 的表达以及 TNF-α，同时增加了 LPS 刺激的 BMDC 释放的 IL-10。 SGE（而非 LJM 111）抑制中性粒细胞趋化性和肌动蛋白聚合。此外，SGE 降低了中性粒细胞 CXCR2 的表达并增加了 GRK-2 的表达。因此，rLJM111 通过减少 DC 功能和成熟来部分负责 SGE 机制，但不影响中性粒细胞的化学吸引。