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Effect of L. infantum infection in transcriptome of human neutrophils in vitro [RNA-seq]

ABSTRACT: To understand the expression patterns of coding and non-coding transcripts during acute in vitro infection of human neutrophils by L. infantum we performed a differential gene expression analysis of RNA-Seq data. We isolated neutrophils from whole blood of healthy male donors; cells from each subject (n=5) were split into groups: 1) infected with L. infantum (MOI = 5:1), and 2) uninfected controls. After 3 hours of exposure of infected group to promastigotes of L. infantum, followed by 17 hours of incubation, total RNA was extracted and total RNA-Seq were performed.

ORGANISM(S): Homo sapiens

PROVIDER: GSE230014 | GEO | 2024/06/27

REPOSITORIES: GEO

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Transcriptome analysis of Leishmania infantum PNA+ and PNA- promastigotes by complete shotgun DNA microarrays

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

Differential gene expression in DEVH1 knock-in Leishmania infantum promastigotes

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

In vitro infection of murine osteoclast by the parasite Leishmania infantum

Project description:To investigate the effect of infection of osteoclast by Leishmania infantum, we infected diffentiating bone marrow derived osteoclast with the parasite at day 2 of differentiation We then performed gene expression profiling analysis using data obtained from RNA-seq of 4 different cultures of cells infected for 24 hours compared to 4 control cultures.

2022-10-26 | GSE216141 | GEO

Comparison of cultured and sand fly-derived promastigotes by means of RNA-Seq

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.

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Effect of L. infantum infection in microtranscriptome of human neutrophils in vitro [Expression]

Project description:To understand the expression patterns of miRNAs expression during acute in vitro infection of human neutrophils by L. infantum we performed a microarray.

2024-06-27 | GSE230017 | GEO

Leishmania infantum

Project description:Transcriptome analysis of Leishmania infantum PNA+ and PNA- promastigotes by complete shotgun DNA microarrays

| PRJNA106671 | ENA

Application of Serial Analysis of Gene Expression to the study of the gene expression profile of Leishmania infantum chagasi promastigote

Project description:This study describes the application of the LongSAGE methodology to study the gene expression profile in Leishmania infantum chagasi. A tag library was created using the LongSAGE method and consisted of 14,208 tags of 17 bases. Of these, 8,427 (59.3%) were distinct. BLAST research of the 1,645 most abundant tags showed that 12.8% of them identified the coding sequences of genes, while 82% (1,349/1,645) identified one or more genomic sequences that did not correspond with open reading frames. Only 5.2% (84/1,645) of the tags were not aligned to any position in the L. infantum genome. The UTR size of Leishmania and the lack of CATG sites in some transcripts were decisive for the generation of tags in these regions. LongSAGE revealed the gene expression profile in promastigotes of L. i. chagasi. Additional analysis will allow a better understanding of the expression profile and discovering the key genes in this life cycle.

2011-08-10 | GSE29369 | GEO

Comparative membrane-associated proteome of Leishmania (Leishmania) infantum and L.(L.) amazonensis

Project description:Protozoan parasites of the genus Leishmania are causative agents of leishmaniasis, a wide range of diseases affecting 12 million people worldwide. The species L. (L.) infantum and L. (L.) amazonensis are causative agents of visceral and cutaneous leishmaniasis, respectively. Most proteome analyses of Leishmania have been carried out on whole-cell extracts. However, this approach tends to underrepresent membrane-associated proteins because of their high hydrophobicity and low solubility. Due to the great importance of membrane-associated proteins in biological processes, including host–parasite interactions, virulence and invasiveness, this study applied label-free shotgun proteomics to characterize and evaluate abundance levels of plasma membrane sub-proteome of promastigotes life-stage. The total number of proteins identified in L. (L.) infantum and L. (L.) amazonensis was 2033 and 2243, respectively. Both species shared 1908 of these quantified proteins. After cell localization prediction of all identified proteins, 394 proteins were described as plasma membrane-associated proteins and their majority (320 proteins) was presented in both species. Considering only exclusive proteins, 18 proteins were detected only in L. (L.) infantum and 56 proteins in L. (L.) amazonensis. We used two criteria to define “regulated” proteins; i) proteins with p-value < 0.05 after One-Way ANOVA analysis (quantitative analysis) and proteins detected only in L. (L.) infantum or L. (L.) amazonensis (qualitative analysis).

2022-02-11 | PXD013087 | Pride

Comparative high-throughput gene expression analysis between cultured and sand fly vector-derived Leishmania infantum promastigotes.

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

Neutrophils infected with Leishmania donovani

Project description:In the context of studying visceral leishmaniasis, neutrophils infected with Leishmania donovani have been compared to uninfected neutrophils. Compared time points are 0, 6 and 24 hours post infection. Neutrophils of three human donors have been used. Overall 6 samples for infected neutrophils at time point 6 hours and 6 samples for infected neutrophils at time point 24 hours exist, including three biological samples and two technical samples. Uninfected neutrophils represent 3 samples at time point 0 hours, 3 samples at time point 6 hours and 3 samples at time point 24 hours. Transcriptome of Leishmania donovani culture has been assessed in two replicates.

| EGAS00001004912 | EGA

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