Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes.
Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes.
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DOI：
10.1371/journal.ppat.1005296
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发表时间：
2015-12
期刊：
影响因子：
6.7
通讯作者：
Teixeira SM
中科院分区：
文献类型：
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作者：
de Paiva RM;Grazielle-Silva V;Cardoso MS;Nakagaki BN;Mendonça-Neto RP;Canavaci AM;Souza Melo N;Martinelli PM;Fernandes AP;daRocha WD;Teixeira SM
利什曼病是一种人类寄生虫病，其表现从皮肤溃疡到致命的内脏感染，由几种利什曼原虫引起。这些原生动物寄生虫在沙蝇媒介的肠道内以细胞外鞭毛前鞭毛体的形式复制，在脊椎动物宿主巨噬细胞的寄生液泡内作为无鞭毛体复制。阿马斯汀是存在于几种锥虫基因组中的大基因家族编码的表面糖蛋白，在克氏锥虫和利什曼原虫的胞内无鞭毛期高表达。在本研究中，我们发现巴西乳杆菌基因组中包含52个属于上述四个淀粉蛋白亚家族的淀粉素基因，并且在巴西乳杆菌无鞭毛体中，所有亚家族成员的表达均上调。虽然一级序列比对没有显示出与任何已知蛋白质序列的同源性，但基于二级结构预测的同源性搜索表明，amastins与Claudins相关，Claudins是真核紧密连接复合体的一组蛋白质。通过下调δ-amastins在巴西乳杆菌中的表达，它们在感染过程中的重要作用变得明显。δ-amastin基因敲除的寄生虫在体外感染小鼠巨噬细胞后表现出生长障碍，当接种到BALB/c小鼠时完全不能产生感染，这种减弱的表型可通过RNAi抗性的amastin基因的重新表达而逆转。进一步强调了它们在宿主-寄生虫相互作用中的重要作用，对感染阿马斯汀击倒寄生虫的巨噬细胞的电子显微镜分析显示，通常观察到的野生型无鞭毛体表面与寄生虫液泡膜之间的紧密接触发生了显著变化。利什曼病是一种由20多种利什曼原虫引起的寄生虫病，全世界约有1200万人受到影响，目前还没有有效的疫苗。根据利什曼原虫种类的不同，这种疾病的临床表现也不同，从自我解决的皮肤损害到威胁生命的内脏疾病。除了现有药物的毒性、疗程长和疗效有限外，一个主要的担忧是抗药性寄生虫和更具毒力的变种的开发。除了迫切需要开发对这种寄生虫更有效的新药和预防新感染的疫苗外，还必须更好地了解决定利什曼原虫毒力的因素。在这里，我们描述了编码利什曼原虫哺乳动物阶段优先表达的表面蛋白的基因家族的特征，该基因家族可能直接参与了细胞内寄生虫和宿主细胞膜之间建立的密切相互作用。通过在感染的小鼠模型中抑制巴西乳杆菌中阿马斯汀基因的表达，我们证明了这些蛋白对于细胞内寄生虫的生存是必不可少的。
Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several Leishmania species. These protozoan parasites replicate as extracellular, flagellated promastigotes in the gut of a sandfly vector and as amastigotes inside the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several trypanosomatids and highly expressed in the intracellular amastigote stages of Trypanosoma cruzi and Leishmania spp. Here, we showed that the genome of L. braziliensis contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in L. braziliensis amastigotes. Although primary sequence alignments showed no homology to any known protein sequence, homology searches based on secondary structure predictions indicate that amastins are related to claudins, a group of proteins that are components of eukaryotic tight junction complexes. By knocking-down the expression of δ-amastins in L. braziliensis, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after in vitro infection of mouse macrophages and completely failed to produce infection when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-parasite interactions, electron microscopy analyses of macrophages infected with amastin knockdown parasites showed significant alterations in the tight contact that is normally observed between the surface of wild type amastigotes and the membrane of the parasitophorous vacuole. Leishmaniasis is a parasitic disease caused by more than 20 species of the genus Leishmania that affects about 12 million people throughout the world and for which there is not an effective vaccine. Depending on the Leishmania species, clinical manifestation of the disease varies from self-resolving skin lesions to life-threatening visceralizing diseases. In addition to the toxicity of currently available drugs, their long treatment course, and limited efficacy, a major concern is the development of drug resistant parasite and more virulent variants. Together with the urgent need to develop new drugs that are more effective against this parasite as well as a vaccine to prevent new infections, it is also imperative to develop a better understanding of the factors that determine Leishmania virulence. Here, we describe the characterization of a gene family encoding surface proteins preferentially expressed in the mammalian stage of Leishmania that may be directly involved with the close interaction that is established between the intracellular parasite and host cell membranes. By inhibiting amastin gene expression in L. braziliensis in a mouse model of infection, we showed that these proteins are essential for intracellular parasite survival.