Vector Biological Studies in Leishmaniasis

利什曼原虫能合成大量由Gal-Man-PO 4重复单元组成的含磷酸聚糖的分子，包括表面脂磷酸聚糖（LPG）、表面和分泌的蛋白磷酸聚糖（PPG）。 LPG是利什曼原虫前鞭毛体的主要细胞表面分子，已知介导寄生虫附着于载体中肠，这防止了在血粉残留物排泄期间感染的损失。 吸血诱导的中肠消化酶对寄生虫的生存构成另一个潜在的障碍。 用L. duboscqi和Lutzomyia longipalpis进行了介体能力的测定。总磷酸聚糖（LPG 2 KO）或单独LPG（LPG 1 KO）特异性缺陷的主要突变体，沿着它们各自的基因添加回转染子。 我们的结果显示，感染饲料后48-72小时，所有寄生虫发育良好，但LPG 2KO除外，其显示前鞭毛体的存活和生长减少。 消化血粉传代后（7-21天），LPG 1 KO和LPG 2KO寄生虫均不能在杜氏毕赤酵母中存活，尽管LPG 1 KO继续在L.长须的 胰蛋白酶和碱性蛋白酶活性的抑制剂促进了LPG 2KO的早期中肠存活。 磷酸糖基化PPG被证明是赋予对中肠消化酶的抗性的关键分子，因为它阻止了暴露于从血液喂养的苍蝇制备的中肠裂解物的LPG 2KO前鞭毛体的杀伤。 一个主要的努力已被导向量化的自然传播剂量的利什曼原虫哺乳动物宿主在叮咬感染的白蛉，这是目前未知的任何天然白蛉载体。 需要定量数据来确定设计旨在模拟自然传播的实验性针头感染时应使用的寄生虫剂量，并揭示可能影响临床结果的自然剂量范围。 我们开发了一种基于实时PCR的方法，以确定在喂养过程中由一个单一的感染感染的苍蝇（白蛉dubosqui）接种到活小鼠的耳朵利什曼原虫主要寄生虫的数量。 大多数受感染的小鼠接种了低于260个利什曼原虫的低剂量。 然而，四分之一的人接受了高达10万的更高剂量。 为了测试接种物的大小对疾病结果的影响，我们比较了C57 BL/6小鼠耳朵中的高（5000）和低（100）剂量皮内针头感染。 为了模拟自然传播，我们使用白蛉衍生的亚环形式的L。主要和预先暴露注射部位的叮咬未感染的苍蝇。 正如预期，接受较高剂量的小鼠的病理学显著更差，但出乎意料的是，低剂量激发小鼠愈合后持续寄生虫的数量更高。 结果表明，透射剂量影响疾病的严重程度和感染水库的潜力。 沙蝇叮咬传播首次被用于评估实验性疫苗对利什曼病的有效性。 各种非活体、重组蛋白和基于DNA的疫苗已显示在动物模型中赋予保护作用。 值得注意的是，没有实验疫苗在受控的实验室环境中通过白蛉攻击进行评估。 我们最近改进了我们使用感染的白蛉将皮肤利什曼病有效地传播给小鼠的能力，并且已经产生了一组明确的数据，表明赋予针对针攻击的强大保护的非活疫苗对白蛉攻击无效。 由于白蛉叮咬与嗜中性粒细胞大量募集至皮肤中寄生虫沉积部位相关，因此嗜中性粒细胞是亚循环前鞭毛体的初始细胞靶标并被用作沉默寄生虫进入巨噬细胞的手段的累积证据可以解释暴露于感染苍蝇的接种小鼠中免疫力表达受损。 以表达红色荧光蛋白（RFP）的L.我们主要研究了感染后急性时间点在耳真皮中的代谢环前鞭毛体的细胞靶。 我们报告说，75-80%的寄生虫在感染后20小时内包含在中性粒细胞内。 相比之下，在感染后48小时，50%的寄生虫被发现在巨噬细胞内。 受感染的中性粒细胞不会凋亡，并且含有可以在培养物中繁殖的活寄生虫。 分类感染的中性粒细胞在注射到幼稚小鼠后建立感染，类似于培养的寄生虫。 采用双光子显微镜，我们能够可视化寄生虫依赖的中性粒细胞招聘和随后的寄生虫吞噬中性粒细胞。 这些发现有力地表明L. major在急性感染期间优先募集和感染嗜中性粒细胞，并且这些感染的嗜中性粒细胞不能杀死细胞内寄生虫，从而支持急性利什曼原虫感染的特洛伊木马模型，其中L.梅杰利用中性粒细胞作为沉默进入巨噬细胞的手段。

Leishmania synthesize abundant phosphoglycan-containing molecules made up of Gal-Man-PO4 repeating units, including the surface lipophosphoglycan (LPG), and the surface and secreted proteophosphoglycan (PPG). LPG, the major cell surface molecule of Leishmania promastigotes, is known to mediate parasite attachment to the vector midgut, which prevents the loss of infection during excretion of the bloodmeal remnants. Midgut digestive enzymes induced by blood feeding pose another potential barrier to parasite survival. The vector competence of Phlebotomus duboscqi and Lutzomyia longipalpis was tested using L. major mutants specifically deficient in either total phosphoglycans (LPG2KO) or LPG alone (LPG1KO), along with their respective gene add back tranfectants. Our results showed that 48-72 hours after the infective feed, all parasites developed well except the LPG2KO, which showed reduced survival and growth of promastigotes. Following passage of the digested bloodmeal (7-21 days), neither the LPG1KO nor LPG2KO parasites were able to survive in P. duboscqi, although the LPG1KO continued to survive in L. longipalpis. Inhibitors of trypsin and alkaline protease activities promoted the early midgut survival of the LPG2KO. Phosphoglycosylated PPG was shown to be the key molecule conferring resistance to midgut digestive enzymes, as it prevented killing of LPG2KO promastigotes exposed to midgut lysates prepared from bloodfed flies. A major effort has been directed toward quantification of the natural transmission dose of Leishmania to mammalian host during the bite of an infected sand fly, which is currently unknown for any natural sand fly vector. The quantitative data are needed to define the dose of parasites that should be used when designing experimental needle infections intended to mimic natural transmission, and revealing natural dose ranges that may influence clinical outcome. We developed a real-time PCR-based method to determine the number of Leishmania major parasites inoculated into the ears of living mice during feeding by a single infected infected fly (Phlebotomus dubosqui). Most of the infected mice were inoculated with a low dose of fewer than 260 Leishmania parasites. However, one in four received a higher dose of up to 100,000. To test the impact of the size of the innoculum on disease outcome, we compared high (5000) and low (100) dose intradermal needle infections in the ears of C57BL/6 mice. To mimic natural transmission, we used sand fly derived metacyclic forms of L. major and pre-exposed the injection site to the bites of un-infected flies. As expected, pathology was significantly worse in the mice receinving the higher dose, but unexpectedly, the number of persisting parasites following healing was higher in the low dose challenged mice. The results indicate that transmitted dose influences the severity of disease and infection reservoir potential. Transmission by sand fly bite is being used for the first time to evaluate the efficacy of experimental vaccines against leishmaniasis. A variety of non-living, recombinant protein, and DNA based vaccines have been shown to confer protection in animal models. Remarkably, no experimental vaccine has been evaluated by sand fly challenge in a controlled, laboratory setting. We have recently refined our ability to efficiently transmit cutaneous leishmanisis to mice using infected sand flies, and have produced a clear set of data to indicate that the non-living vaccines that confer powerful protection against needle challenge are ineffective against sand fly challenge. As sand fly bites are associated with a massive recruitment of neutrophils to the site of parasite deposition in the skin, the accumulating evidence that neutrophils are the initial cellular target of metacyclic promastigotes and are employed as means of silent parasite entry into macrophages, may explain the compromised expression of immunity in the vaccinated mice exposed to infected flies. Employing a red fluorescent protein (RFP) expressing strain of L. major we investigated the cellular target of metacyclic promastigotes at acute time points post-infection in the ear dermis. We report that 75-80% of parasites are contained within neutrophils at 20 hours post-infection. In contrast, by 48 hours post-infection 50% of parasites are found within macrophages. Infected neutrophils are not apoptotic and contain viable parasites that can be propagated in culture. Sorted infected neutrophils established infection upon injection into nave mice similar to that of cultured parasites. Employing two-photon microscopy we were able to visualize parasite dependent neutrophil recruitment and subsequent parasite phagocytosis by neutrophils. These findings strongly suggest that L. major preferentially recruits and infects neutrophils during acute infection and these infected neutrophils fail to kill intracellular parasites, thereby supporting the Trojan Horse Model of acute Leishmania infection in which L. major employs neutrophils as a means by which to gain silent entry into macrophages.