Differential gene expression in DEVH1 knock-in Leishmania infantum promastigotes
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PROVIDER: PRJNA836924 | ENA |
REPOSITORIES: ENA
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Project description:The DEAD/H RNA helicase LINF_220021200 (DEVH1) gene from Leishmania infantum (Kinetoplastida:Trypanosomatidae) was cloned in the pTEX expression plasmid vector for trypanosomatids. Leishmania infantunm promastigotes were transfected and a knock-in L. infantum promastigote cell line was selected with geneticin (G418). A pTEX control promastigote line was also generated. Then, three independent biological replicate cultures of each pTEX-DEVH1 and pTEX promastigote lines were performed in the presence of the selective agent. The parasites were harvested on day 7 (stationary phase). Total mRNA samples were obtained. Cyanine dye-labelled samples were obtained from the knock-in and the control line (Cy5 and Cy3, respectively) and they were hybridized with custom whole-genome L. infantum DNA microarrays. This platform is included in GEO (GPL6781) and has also been repeatedly used in different experiments from 2009. Hybridization analysis allowed for finding differentially expressed genes due to the effect of induced over-expression of the DEVH1-encoding gene in the knock-in promastigote line compared to the control line. Genes involved in parasite infectivity and survival such as the HASP/SHERP gene cluster and an amastin gene or redox homeostasis genes are significantly down-regulated in the pTEX-DEVH1 knock-in promastigote line, whereas genes related to growth are up-regulated. This is in agreement with previous experimental data supporting that L. infantum DEVH1 knock-in promastigotes are able to recover the growth rate when stress conditions are removed.
2022-08-03 | GSE202674 | GEO
Project description:Leishmania infantum is the causative agent of zoonotic visceral leishmaniasis in Mediterranean areas and also acts as an opportunistic parasite in HIV patients. Metacyclic promastigotes are transmitted during bloodmeals of the sand-fly host after development. Metacyclogenesis can be micmiked in axenic cultures and peanut lectin (PNA) agglutination followed by two-step centrifugation allows the separation of procyclic and metacyclic promastigotes in L. major. The purpose of this study is to isolate both fractions simultaneously from the same population of L. infantum in stationary phase of axenic culture and compare their expression profiles through DNA microarrays, specially focusing on metacyclic promastigotes. Whole-genome shotgun DNA microarrays were constructed and used to analyse the stationary-phase procyclic and metacyclic expression profiles. Four biological replicates of the experiment were performed and analysed, so that 322 clones with meaningful values of stage-specific regulation were selected. We found several genes dealing with primary metabolism, differentiation in procyclic promastigotes and with development of infectivity in metacyclic promastigotes. The differences we have found between the procyclic (PNA+) and metacyclic (PNA-) transcriptomes demonstrate that negative selection of metacyclic promastigotes through PNA agglutination is suitable in L. infantum and both fractions can be isolated. In addition, up-regulation of genes implied in lipophosphoglycan (LPG), proteophosphoglycan (PPG) and glycoprotein biosynthesis indicate that metacyclic promastigotes are related with infectivity. Keywords: comparative hybridization between cDNAs from procyclic PNA+ and metacyclic PNA- promastigotes of L.infantum
2009-05-14 | GSE11269 | GEO
Project description:Leishmania infantum (Kinetoplastida:Trypanosomatidae) is the etiological agent of zoonotic visceral leishmaniasis in the Mediterranean basin. The motile promastigote stage infects the hematophagous sand fly vector host and amastigotes survives and multiplies within phagocytes of the mammalian host. Promastigotes are routinely cultured in liquid undefined media and are considered to mimic the environment within the sand fly gut. We have put this to the test by high-throughput gene expression profiling by shotgun DNA microarrays generated in our laboratory. This has been possible thanks to RNA amplification.
2016-05-05 | GSE70992 | GEO
Project description:Transcriptome analysis of Leishmania infantum PNA+ and PNA- promastigotes by complete shotgun DNA microarrays
| PRJNA106671 | ENA
Project description:Leishmania chagasi is the causative agent of zoonotic visceral leishmaniasis in Brazil, being domestic and stray dogs the main reservoirs. The development of the parasite involves two stages. The promastigote is extracellular and develops within the sand fly gut. The amastigote survives inside the harsh environment of the phagolysosome of mammalian host phagocytes, where pH is acidic, temperature higher than in the sand fly vector and hydrolytic enzymes act. In addition, the host phagocyte displays the nitric oxide defense mechanism against the amastigote. Promastigotes are also able to withstand NO even when they develop within the sand fly gut. This can be explained with the pre-adaptative hypothesis, which has been supported by us and others elsewhere and consists of preparation of promastigotes in advance for development towards the amastigote stage. For this reason, the comparison of NO-resistant and sensitive promastigotes is valuable. The two-dimension electrophoresis-mass spectrometry (2DE-MS/MS) approach has been performed to study differential protein abundance comparing L. chagasi NO-sensitive and resistant promastigotes. This analysis has revealed differential expression of genes directly and indirectly involved in NO-resistance, highlighting up-regulation of the glucose-6-phosphate dehydrogenase (G6PD) in NO-resistant promastigotes and down-regulation of the glutathione peroxidase (GPX) and the arginase (ARG) in NO-sensitive ones. These data are a starting point in the search of vaccine candidates and/or drug targets.
2016-03-31 | PXD002939 | Pride
Project description:Comparative high-throughput gene expression analysis between cultured and sand fly vector-derived Leishmania infantum promastigotes.
| PRJNA290068 | ENA
Project description:A laboratory colony of Phlebotomus perniciosus sand flies was maintained. Sand flies were infected with cultured Leishmania infantum promastigotes in stationary phase. Ten infected sand flies were dissected after 5 days and promastigotes within the gut pooled. The cells were immediately washed in PBS once and lysed in TRIzol reagent (Life Technologies). RNA isolation was completed according to the manufacturer's instructions, obtaining 63ng. RNA-seq libraries were generated using the spliced leader sequence for second strand synthesis (Cuypers et al., 2017; Haydock et al., 2015), thus allowing for specific amplification of sequences from L. infantum promastigotes, thus avoiding contamination with material from the sand fly gut. Single-end sequencing was performed in an Illumina HiSeq2500 instrument and data analysis was conducted using bowtie2, samtools, featureCounts and Geneious. The main findings are: i) substantial differences in differential gene expression between sand fly-derived (sfPro) and cultured (acPro) promastigotes; and ii) over-expression of genes involved in metacyclogenesis in sfPro vs. acPro, including gp63 genes, autophagy genes, etc.
2018-08-15 | GSE102668 | GEO