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Stage-specific differential gene expression in Leishmania infantum: from the foregut of Phlebotomus perniciosus to the human phagocyte.

ABSTRACT: BACKGROUND: Leishmania 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. RESULTS: A 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. CONCLUSIONS: The 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.

SUBMITTER: Alcolea PJ

PROVIDER: S-EPMC4203910 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Stage-specific differential gene expression in Leishmania infantum: from the foregut of Phlebotomus perniciosus to the human phagocyte.

Alcolea Pedro J PJ Alonso Ana A Gómez Manuel J MJ Postigo Marina M Molina Ricardo R Jiménez Maribel M Larraga Vicente V

BMC genomics 20141003

<h4>Background</h4>Leishmania 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 profi ...[more]

PMID: 25281593

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Project description:Phlebotomus perniciosus is one of the major vectors of Leishmania infantum in the Mediterranean basin. The aim of this work was (i) to provide information about abundance and temporal dynamics of this Larroussius species in a hot spot area of visceral leishmaniasis in Tunisia, (ii) to detect L. infantum DNA in wild caught female sandflies and (iii) to measure Phlebotomus perniciosus infection rate throughout the active season. Sandflies were collected monthly during one year using CDC miniature light-traps in house and in animal shelters. Male specimens were identified at species level according to morphological characters. Female specimens were conserved individually for molecular study. Leishmania infection was tested by kinetoplast DNA real-time PCR and ITS-1 PCR-sequencing. Subsequent sandfly species identification of infected specimens was done by mitochondrial cytochrome b sequencing. In one year period, overall 4,441 specimens (2230 males and 2211 females) were collected. Sandfly activity started in end-April and ended in early-November. Mean sandfly density in house was significantly lower than in animal shelters (51 ± 50 versus 504 ± 460 sandflies /CDC night, p<0.05). However, a higher proportion of females was found in house (58.4% versus 49.2%, p<0.001). Based on species identification of male specimens, Phlebotomus perniciosus was the dominant species (56% of the whole male sandfly fauna, p<0.0001). It showed two peaks of density in the active season, a sharp one in early May and a higher long lasting one from end-July to end-September. DNA was extracted from 190 female specimens randomly sampled and corresponding to 96 specimens from house and 94 from animal shelters. Twenty four female sandfly were infected by Leishmania infantum. All infected specimens were recognized as Phlebotomus perniciosus. Leishmania infantum infection rate in female sandflies was 2.3 fold higher in house than in animal shelters (17.7% versus 7.4%, p<0.05). In house, estimated number of infected specimens was the highest at the end of the active season. Abundance, dynamics of density and Leishmania infantum infection prevalence of Phlebotomus perniciosus in Tunisian hot spot of visceral leishmaniasis highlight the major role of this Phlebotominae species in L. infantum transmission.

Project description:BackgroundParasite-vector interactions are fundamental in the transmission of vector-borne diseases such as leishmaniasis. Leishmania development in the vector sand fly is confined to the digestive tract, where sand fly midgut molecules interact with the parasites. In this work we sequenced and analyzed two midgut-specific cDNA libraries from sugar fed and blood fed female Phlebotomus perniciosus and compared the transcript expression profiles.ResultsA total of 4111 high quality sequences were obtained from the two libraries and assembled into 370 contigs and 1085 singletons. Molecules with putative roles in blood meal digestion, peritrophic matrix formation, immunity and response to oxidative stress were identified, including proteins that were not previously reported in sand flies. These molecules were evaluated relative to other published sand fly transcripts. Comparative analysis of the two libraries revealed transcripts differentially expressed in response to blood feeding. Molecules up regulated by blood feeding include a putative peritrophin (PperPer1), two chymotrypsin-like proteins (PperChym1 and PperChym2), a putative trypsin (PperTryp3) and four putative microvillar proteins (PperMVP1, 2, 4 and 5). Additionally, several transcripts were more abundant in the sugar fed midgut, such as two putative trypsins (PperTryp1 and PperTryp2), a chymotrypsin (PperChym3) and a microvillar protein (PperMVP3). We performed a detailed temporal expression profile analysis of the putative trypsin transcripts using qPCR and confirmed the expression of blood-induced and blood-repressed trypsins. Trypsin expression was measured in Leishmania infantum-infected and uninfected sand flies, which identified the L. infantum-induced down regulation of PperTryp3 at 24 hours post-blood meal.ConclusionThis midgut tissue-specific transcriptome provides insight into the molecules expressed in the midgut of P. perniciosus, an important vector of visceral leishmaniasis in the Old World. Through the comparative analysis of the libraries we identified molecules differentially expressed during blood meal digestion. Additionally, this study provides a detailed comparison to transcripts of other sand flies. Moreover, our analysis of putative trypsins demonstrated that L. infantum infection can reduce the transcript abundance of trypsin PperTryp3 in the midgut of P. perniciosus.

Dataset Information

Stage-specific differential gene expression in Leishmania infantum: from the foregut of Phlebotomus perniciosus to the human phagocyte.

Publications

Stage-specific differential gene expression in Leishmania infantum: from the foregut of Phlebotomus perniciosus to the human phagocyte.

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