Abstract
BackgroundLeishmania infantum is the etiological agent of zoonotical visceral leishmaniasis in the Mediterranean basin. A recent outbreak in humans has been recently reported in central Spain. Leishmania spp. parasites are transmitted to the mammalian host by the bite of sand flies. The primary vector of L. infantum in Spain is Phlebotomus perniciosus. For decades, research on these parasites has involved the axenic culture model of the promastigote stage including gene expression profiling studies performed in the post-genome era. Unlike the controversial axenic culturing of amastigotes, promastigote cultures are generally accepted and used, although with the precaution of avoiding excessive culture passage.The primary objective of this differentiation study is to compare the gene expression profiles of promastigotes isolated from the foregut of the sand fly and amastigotes. For this purpose, P. perniciosus sand flies were infected with L. infantum and differentiated promastigotes were extracted by dissection of the foreguts. Shotgun DNA microarray hybridization analyses allowed for transcriptome comparison of these promastigotes with amastigotes obtained by infection of the U937 cell line. The results have been compared with those described in published expression analyses using axenic promastigotes.ResultsA total of 277 up-regulated genes were found through this hybridization experiment. The comparison of these particular results with published gene expression profile analyses performed using the same experimental procedure to study cultured promastigotes in stationary phase versus amastigotes revealed considerable differences (approximately 95% of the up-regulated genes were different). We found that the up-regulation rate is lower in amastigotes than in sand fly-derived promastigotes, which is in agreement with the over-expression of genes involved in gene expression regulation and signaling in those promastigote populations.ConclusionsThe up-regulation rate is lower in intracellular amastigotes than in promastigotes obtained from the sand fly gut. This was also reported by us using the promastigote culture model and is an evidence for the hypothesis of promastigote preadaptation towards life in the intracellular environment. Regarding transcript abundance, the set of differentially regulated genes is notably different when using promastigotes from the sand fly foregut instead of axenic cultures.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-849) contains supplementary material, which is available to authorized users.
Highlights
Leishmania infantum is the etiological agent of zoonotical visceral leishmaniasis in the Mediterranean basin
Results and discussion mRNA amplification and microarray hybridization analysis of metacyclic promastigotes isolated from P. perniciosus and amastigotes The total amounts of RNA obtained from Pro-Pper replicates were comprised between 20 and 25 ng and after the first amplification round, 200–250 ng of aRNA were obtained
The number of differences in gene expression found between Pro-Pper and amastigotes is 277 (Figure 2, Table 1), which is comparable to stage-specific gene expression regulation between logarithmic phase promastigotes and amastigotes and higher than between stationary phase promastigotes and amastigotes [23]
Summary
Leishmania infantum is the etiological agent of zoonotical visceral leishmaniasis in the Mediterranean basin. The primary objective of this differentiation study is to compare the gene expression profiles of promastigotes isolated from the foregut of the sand fly and amastigotes. For this purpose, P. perniciosus sand flies were infected with L. infantum and differentiated promastigotes were extracted by dissection of the foreguts. L. infantum is the ethiological agent of zoonotic visceral leishmaniasis in the Mediterranean basin and this species acts as an opportunistic pathogen, as indicated by the increase in co-infections with HIV [3,4]. Phlebotomus perniciosus and P. ariasi are the proven vectors of L. infantum in Spain [7] and P. perniciosus is the major vector of L. infantum in the central and western Mediterranean basin [8]
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