Salivary Gland Transcriptomes and Proteomes of Phlebotomus tobbi and Phlebotomus sergenti, Vectors of Leishmaniasis

BackgroundPhlebotomus tobbi is a vector of Leishmania infantum, and P. sergenti is a vector of Leishmania tropica. Le. infantum and Le. tropica typically cause visceral or cutaneous leishmaniasis, respectively, but Le. infantum strains transmitted by P. tobbi can cause cutaneous disease. 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. Methodology/Principal FindingscDNA libraries of P. tobbi and P. sergenti female SGs were constructed, sequenced, and analyzed. Clones (1,152) were randomly picked from each library, producing 1,142 high-quality sequences from P. tobbi and 1,090 from P. sergenti. 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. Phylogenetic analyses and multiple sequence alignments of putative proteins were used to elucidate molecular evolution and describe conserved domains, active sites, and catalytic residues. Proteomic analyses of P. tobbi and P. sergenti SGs were used to confirm the identification of 35 full-length sequences (18 in P. tobbi and 17 in P. sergenti). To bridge transcriptomics with biology P. tobbi antigens, glycoproteins, and hyaluronidase activity was characterized. ConclusionsThis 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.【背景】陶氏白蛉（Phlebotomus tobbi）是婴儿利什曼原虫（Leishmania infantum）的传播媒介，而塞氏白蛉（Phlebotomus sergenti，简称P. sergenti）是热带利什曼原虫（Leishmania tropica）的传播媒介。通常情况下，婴儿利什曼原虫和热带利什曼原虫分别引发内脏利什曼病与皮肤利什曼病，但由陶氏白蛉传播的婴儿利什曼原虫毒株可引发皮肤型疾病。为更深入解析白蛉唾液在利什曼病中的组成成分与潜在作用机制，本研究对这两种白蛉的唾液腺（salivary glands, SGs）转录组进行了测序、注释与比较分析。 【研究方法与主要结果】本研究构建并测序分析了塞氏白蛉与陶氏白蛉雌性唾液腺的cDNA文库。从每个文库中随机挑取1152个克隆，最终从陶氏白蛉获得1142条高质量序列，从塞氏白蛉获得1090条高质量序列。丰度最高的分泌型推定蛋白可分为以下几类：抗原5相关蛋白、腺苷三磷酸双磷酸水解酶（apyrases）、透明质酸酶、D7相关蛋白、PpSP15类蛋白、ParSP25类蛋白、PpSP32类蛋白、黄色素相关蛋白、33kDa唾液蛋白以及41.9kDa蛋白超家族。通过对推定蛋白进行系统发育分析与多序列比对，阐明了其分子进化特征，并描述了保守结构域、活性位点与催化残基。本研究还通过蛋白质组学分析，验证了35条全长序列的身份（其中陶氏白蛉18条，塞氏白蛉17条）。为将转录组学研究与生物学功能相衔接，本研究还对陶氏白蛉的抗原、糖蛋白以及透明质酸酶活性进行了表征。 【结论】本研究对塞氏白蛉唾液腺成分的描述，是首次对帕拉白蛉亚属（Paraphlebotomus）唾液组分的报道。针对拉里欧斯亚属（Larroussius）白蛉陶氏白蛉的研究，拓展了婴儿利什曼原虫传播媒介的唾液蛋白组库。尽管陶氏白蛉可传播皮肤型利什曼病，但其唾液蛋白与其他传播内脏利什曼病的拉里欧斯亚属物种的唾液蛋白最为相似。本研究的转录组与蛋白质组学分析，增进了不同物种及亚属白蛉唾液蛋白的认知，可为媒介-病原体互作及媒介-宿主互作研究提供重要支撑。