Influence of the microenvironment in the transcriptome of leishmania infantum promastigotes: sand fly versus culture

23 p.-5 fig.-3 tab. 8 tab. supl.-1 text. supl.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.This work has been funded by the Spanish Ministry of Economy and Competitiveness (grant AGL2010-21806-C02-01) and by the Ramón Areces Foundation (contract 050204100014, OTT code 20100338). PJA thanks CSIC for the I3P-BPD2003-1 grant and two contracts of employment for a position included in the A1 group (respectively developed from January 16th to July 23rd 2008 and from October 16th 2008 to April 15th 2009). AA thanks CSIC for the Ph.D. contract 5072160068 W0SC000077 within the A1 group.Peer reviewe