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: 9354781
• 负责人: Jesus Valenzuela
• 金额: $ 106.06万
• 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/9354781
• 关键词: Americas; Animals; Anticoagulants; Basic Science; Biological; Biological Markers; Bite; Blocking Antibodies; Blood; Canis familiaris; Cataloging; Catalogs; Central America; Chronic; Classical Complement Pathway; Clinical; Clinical Protocols; Complement; Complement 4b; Complement Inactivators; Complement component C1s; Controlled Study; Cutaneous; Cutaneous Leishmaniasis; Data; Deposition; Development; Disease; Distal; Dog Diseases; Event; Evolution; Factor Xa; Human; Hypersensitivity skin testing; Immune response; Infection; Insecta; Institution; Interferons; Interleukin-10; Interleukin-2; Kininogenase; Laboratories; Lectin; Leishmania; Leishmania infantum; Leishmania mexicana; Leishmaniasis; Lesion; Life; Lutzomyia genus; Mediating; Mexico; Modeling; Molecular; Molecular Biology; Needles; Nonprofit Organizations; Organism; Parasites; Pathogenesis; Pathway interactions; Peptide Hydrolases; Plasmin; Property; Proteins; Public Health; Recombinant Proteins; Recombinants; Reporting; Research; Rodent; Role; Route; Saliva; Salivary; Salivary Glands; Salivary Proteins; Sand Flies; Sequence Analysis; Site; Skin; Source; Specificity; Spleen; Testing; Therapeutic; Thrombin; Time; Transcript; Translating; Trypsin; Update; Vaccinated; Vaccines; Vector-transmitted infectious disease; Visceral; Visceral Leishmaniasis; Work; adaptive immunity; animal facility; base; cDNA Library; comparative; complement pathway; disease transmission; feeding; fundamental research; human disease; immunogenic; immunogenicity; inhibitor/antagonist; neglect; nonhuman primate; novel; pathogen; research study; transcriptome; transcriptomics; transmission process; vaccine candidate; vector; vector transmission

The accomplishments of the section are: 1. We developed a natural model of canine visceral leishmaniasis and identified the skin of the animal as a site where parasites stay for a log period of time irrespective of disease status, and become accessible for disease transmission. Canine leishmaniasis (CanL) is a chronic fatal disease of dogs and a major source of human infection through propagation of parasites in vectors. Here, we infected 8 beagles through multiple experimental vector transmissions with Leishmania infantuminfected Lutzomyia longipalpis. CanL clinical signs varied, although live parasites were recovered from all dog spleens. Splenic parasite burdens correlated positively with Leishmania-specific interleukin 10 levels, negatively with Leishmania-specific interferon and interleukin 2 levels, and negatively with Leishmania skin test reactivity. A key finding was parasite persistence for 6 months in lesions observed at the bite sites in all dogs. These recrudesced following a second transmission performed at a distal site. Notably, sand flies efficiently acquired parasites after feeding on lesions at the primary bite site. In this study, controlled vector transmissions identify a potentially unappreciated role for skin at infectious bite sites in dogs with CanL, providing a new perspective regarding the mechanism of Leishmania transmissibility to vector sand flies. 2. We discovered and characterized the sand fly salivary anticomplement protein specific for the classical pathway of complement. Blood-feeding insects inject potent salivary components including complement inhibitors into their host's skin to acquire a blood meal. Sand fly saliva was shown to inhibit the classical pathway of complement; however, the molecular identity of the inhibitor remains unknown. Here, we identified SALO as the classical pathway complement inhibitor. SALO, an 11kDa protein, has no homology to proteins of any other organism apart from New World sand flies. rSALO anti-complement activity has the same chromatographic properties as the Lu. longipalpis salivary gland homogenate (SGH)counterparts and anti-rSALO antibodies blocked the classical pathway complement activity of rSALO and SGH. Both rSALO and SGH inhibited C4b deposition and cleavage of C4. rSALO, however, did not inhibit the protease activity of C1s nor the enzymatic activity of factor Xa, uPA, thrombin, kallikrein, trypsin and plasmin. Importantly, rSALO did not inhibit the alternative or the lectin pathway of complement. In conclusion our data shows that SALO is a specific classical pathway complement inhibitor present in the saliva of Lu. longipalpis. Importantly, due to its small size and specificity, SALO may offer a therapeutic alternative for complement classical pathway-mediated pathogenic effects in human diseases. 3. We characterized the first salivary gland transcriptome of a sand fly from Mesoamerica. Sand fly saliva has been shown to have proteins with potent biological activities, salivary proteins that can be used as biomarkers of vector exposure, and salivary proteins that are candidate vaccines against different forms of leishmaniasis. Sand fly salivary gland transcriptomic approach has contributed significantly to the identification and characterization of many of these salivary proteins from important Leishmania vectors; however, sand fly vectors in some regions of the world are still neglected, as Bichromomyia olmeca (formerly known as Lutzomyia olmeca olmeca), a proven vector of Leishmania mexicana in Mexico and Central America. Despite the importance of this vector in transmitting Leishmania parasite in Mesoamerica there is no information on the repertoire of B. olmeca salivary proteins and their relationship to salivary proteins from other sand fly species. A cDNA library of the salivary glands of wild-caught B. olmeca was constructed, sequenced, and analyzed. We identified transcripts encoding for novel salivary proteins from this sand fly species and performed a comparative analysis between B. olmeca salivary proteins and those from other sand fly species. With this new information we present an updated catalog of the salivary proteins specific to New World sand flies and salivary proteins common to all sand fly species. We also report in this work the anti-Factor Xa activity of Lofaxin, a salivary anticoagulant protein present in this sand fly species. This study provides information on the first transcriptome of a sand fly from Mesoamerica and adds information to the limited repertoire of salivary transcriptomes from the Americas. This comparative analysis also shows a fast degree of evolution in salivary proteins from New World sand flies as compared with Old World sand flies.

本节的成就是： 1。我们开发了一种自然模型的内脏利什曼病，并确定了动物的皮肤是寄生虫在对数周期内保持对数期的部位，无论疾病状况如何，并且可以用于疾病传播。 犬利什曼病（CANL）是狗的慢性致命疾病，是通过载体中寄生虫传播的主要人类感染来源。在这里，我们通过多种实验载体的传输感染了8个小蛋白蛋糕。尽管从所有狗脾脏中回收了活寄生虫，但CANL临床体征各不相同。脾寄生虫负担与利什曼尼亚特异性白介素10级呈正相关，与利什曼原虫特异性干扰素和白介素2级别负相关，与利什曼尼亚皮肤测试的反应性负相关。一个关键发现是在所有狗的咬伤部位观察到的病变中寄生虫持久性6个月。这些在远端进行了第二次传输之后进行的这些转化。值得注意的是，在主要咬合部位以病变为食后，沙子有效地获得了寄生虫。在这项研究中，受控的载体传输确定了具有canl的狗的感染性咬合部位的皮肤的潜在不欣赏的作用，从而提供了有关利什曼原虫对载体沙蝇传播机制的新观点。 2。我们发现并表征了对经典补体途径特有的沙蝇唾液抗结蛋白。 喂食昆虫注入有效的唾液成分，包括补充抑制剂到宿主的皮肤中以获取血液粉。 Sand Fly Saliva被证明可以抑制经典的补体途径。但是，抑制剂的分子身份仍然未知。在这里，我们将salo确定为经典途径补体抑制剂。 Salo是一种11KDA蛋白，除了新世界沙蝇外，与其他生物的蛋白质没有同源性。 RSalo抗补充活性具有与LU相同的色谱特性。 longipalpis唾液腺匀浆（SGH）对应物和抗rsalo抗体阻止了Rsalo和SGH的经典途径补体活性。 Rsalo和SGH都抑制了C4的C4B沉积和裂解。然而，RSalo并未抑制C1s的蛋白酶活性或因子Xa，UPA，凝血酶，Kallikrein，Kallikrein，胰蛋白酶和纤溶酶的酶活性。重要的是，rsalo没有抑制补体的替代或凝集素途径。总之，我们的数据表明，salo是LU唾液中存在的特定经典途径补体抑制剂。 longipalpis。重要的是，由于其尺寸较小和特异性，Salo可能会为人类疾病中经典途径介导的致病作用提供治疗替代方案。 3。我们表征了来自中美洲的沙蝇的第一个唾液腺转录组。 已证明沙蝇唾液具有具有有效生物学活性的蛋白质，可以用作载体暴露的生物标志物的唾液蛋白，以及针对不同形式的利什曼病的候选疫苗的唾液蛋白。沙蝇唾液腺转录组方法对来自重要利什曼原虫载体的许多唾液蛋白的鉴定和表征做出了重大贡献。但是，世界上某些地区的沙蝇载体仍被忽略，因为墨西哥和中美洲利什曼尼亚墨西哥利什曼尼亚墨西哥人（Leishmania of Mexicana of Mexicana of Mexicana of Mexicana of Mexicana of Mexicana of Mexicana of Leishmania of Mexicana of Bichromomyia Olmeca（以前称为Lutzomyia Olmeca Olmeca）。尽管该载体在中美洲传输利什曼原虫寄生虫的重要性，但没有关于牛b。ermeca唾液蛋白的曲目及其与其他沙蝇物种的唾液蛋白的关系。构建，测序和分析了野生捕获的B. Olmeca的唾液腺的cDNA文库。我们确定了从这种沙蝇物种中编码新型唾液蛋白的转录本，并在Olmeca唾液蛋白和其他砂蝇物种的葡萄球菌蛋白质中进行了比较分析。有了这些新信息，我们介绍了针对新世界沙蝇和所有沙蝇种类共有的唾液蛋白的更新目录。我们还在这项工作中报告了lofaxin的抗因子Xa活性，lofaxin是这种沙蝇物种中存在的一种唾液抗凝蛋白。这项研究提供了有关中美洲砂飞的第一个转录组的信息，并将信息添加到来自美洲唾液转录组有限的曲目中。这种比较分析还表明，与旧世界的沙蝇相比，新世界沙蝇的唾液蛋白的快速发展。