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oai:repisalud.isciii.es:20.500.12105/69572024-09-27T20:47:00Zcom_20.500.12105_2052com_20.500.12105_2051col_20.500.12105_19609

00925njm 22002777a 4500 dc Alcolea, Pedro J author Alonso, Ana author Dominguez-Rodriguez, Mercedes author Parro, Víctor author Jimenez, Maribel author Molina, Ricardo author Larraga, Vicente author 2016-05-10 Zoonotic visceral leishmaniasis is a vector-borne disease caused by Leishmania infantum in the Mediterranean Basin, where domestic dogs and wild canids are the main reservoirs. The promastigote stage replicates and develops within the gut of blood-sucking phlebotomine sand flies. Mature promastigotes are injected in the dermis of the mammalian host and differentiate into the amastigote stage within parasitophorous vacuoles of phagocytic cells. The major vector of L. infantum in Spain is Phlebotomus perniciosus. Promastigotes are routinely axenized and cultured to mimic in vitro the conditions inside the insect gut, which allows for most molecular, cellular, immunological and therapeutical studies otherwise inviable. Culture passages are known to decrease infectivity, which is restored by passage through laboratory animals. The most appropriate source of promastigotes is the gut of the vector host but isolation of the parasite is technically challenging. In fact, this option is not viable unless small samples are sufficient for downstream applications like promastigote cultures and nucleic acid amplification. In this study, in vitro infectivity and differential gene expression have been studied in cultured promastigotes at the stationary phase and in promastigotes isolated from the stomodeal valve of the sand fly P. perniciosus. About 20 ng RNA per sample could be isolated. Each sample contained L. infantum promastigotes from 20 sand flies. RNA was successfully amplified and processed for shotgun genome microarray hybridization analysis. Most differentially regulated genes are involved in regulation of gene expression, intracellular signaling, amino acid metabolism and biosynthesis of surface molecules. Interestingly, meta-analysis by hierarchical clustering supports that up-regulation of 22.4% of the differentially regulated genes is specifically enhanced by the microenvironment (i.e. sand fly gut or culture). The correlation between cultured and naturally developed promastigotes is strong but not very high (Pearson coefficient R2 = 0.727). Therefore, the influence of promastigote culturing should be evaluated case-by-case in experimentation. PLoS Negl Trop Dis. 2016 May 10;10(5):e0004693. 1935-2735 http://hdl.handle.net/20.500.12105/6957 27163123 10.1371/journal.pntd.0004693 1935-2735 PLoS neglected tropical diseases Influence of the Microenvironment in the Transcriptome of Leishmania infantum Promastigotes: Sand Fly versus Culture