title: Influence of the Microenvironment in the Transcriptome of Leishmania infantum Promastigotes: Sand Fly versus Culture.
abstract: 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.
authors.affiliation: Laboratorio de Parasitología Molecular, Departamento de Microbiología Molecular y Biología de las Infecciones, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: Pedro J
authors.initials: PJ
authors.lastname: Alcolea
authors.affiliation: Laboratorio de Parasitología Molecular, Departamento de Microbiología Molecular y Biología de las Infecciones, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Alonso
authors.affiliation: Unidad de Inmunología Microbiana, Centro Nacional de Microbiología, Virología e Inmunología Sanitarias, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
authors.forename: Mercedes
authors.initials: M
authors.lastname: Domínguez
authors.affiliation: Laboratorio de Ecología Molecular, Centro de Astrobiología, Instituto Nacional de Técnica Aeroespacial "Esteban Terradas"-Consejo Superior de Investigaciones Científicas, Torrejón de Ardoz, Madrid, Spain.
authors.forename: Víctor
authors.initials: V
authors.lastname: Parro
authors.affiliation: Unidad de Entomología Médica, Servicio de Parasitología, Centro Nacional de Microbiología, Virología e Inmunología Sanitarias, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
authors.forename: Maribel
authors.initials: M
authors.lastname: Jiménez
authors.affiliation: Unidad de Entomología Médica, Servicio de Parasitología, Centro Nacional de Microbiología, Virología e Inmunología Sanitarias, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Molina
authors.affiliation: Laboratorio de Parasitología Molecular, Departamento de Microbiología Molecular y Biología de las Infecciones, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors.forename: Vicente
authors.initials: V
authors.lastname: Larraga
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004260:DNA Repair; D005786:Gene Expression Regulation; D006706:Homeostasis; D006801:Humans; D018314:Leishmania infantum / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D007898:Leishmaniasis, Visceral / Q000635:transmission*; D005810:Multigene Family; D011576:Psychodidae / Q000469:parasitology*; D015398:Signal Transduction; D059467:Transcriptome