Project description:Leishmania RNA virus 1 (LRV1) is a double stranded RNA (dsRNA) virus found in some strains of the human protozoan parasite Leishmania, the causative agent of leishmaniasis, a neglected tropical disease. Interestingly, the presence of LRV1 inside Leishmania constitutes an important virulence factor which worsens leishmaniasis outcome in a type I interferon (type I IFN) dependent manner and contributes to treatment failure. Understanding how macrophages respond towards Leishmania alone or in combination with LRV1 as well as the role that type I IFNs may play during infection is fundamental to oversee new therapeutic strategies. In order to dissect the macrophage response towards infection, RNA Sequencing (RNA-Seq) was performed on murine wild-type (WT) bone marrow derived macrophages infected with Leishmania guyanensis (Lgy) devoid or not of LRV1 (LgyLRV1- and LgyLRV1+ respectively) or co-infected with LgyLRV1- and Lymphocytic choriomeningitis virus (LCMV) for 8 and 24 hours. Additionally, macrophages were treated with type I IFN (IFNα or IFNβ) after 6 hours of infection.
Project description:Leishmania (Viannia) braziliensis is a parasite prevalent in Brazil and associated with tegumentary leishmaniasis (TL), including cutaneous (CL) and mucosal (ML) forms. The mechanisms of pathogenesis of TL are not fully understood, including some factors related to the host and parasite interaction in response to infection, and especially about Leishmania RNA Virus 1 (LRV1), an endosymbiont virus parasitizing Leishmania species, particularly triggers ML. Molecular approaches are usually applied to compare situations and to understand these interactions. Here, microarray analysis identified 162 differentially expressed genes in LbLRV1+ vs. LbLRV1- infection, with 126 upregulated genes related to IFN signaling, OAS/RNAse L, vitamin D3, and RIG-I type receptors. Additionally, 36 down-regulated genes were observed. Then, two validation assays were performed to confirm these results (RT-qPCR and Cytometric Bead Array). The main results comprise the differential gene expression in cells infected with LbLRV1+ compared to LbLRV1- and control, with overexpression of various genes in LbLRV1+ cells. Cytokine levels showed no significant differences between LbLRV1+ and LbLRV1-. This study highlighted the activation of the OAS/RNase L signaling pathway and the non-genomic actions of vitamin D3 in LbLRV1+ infection compared to LbLRV1- and control. This research contributes to our understanding of the immune response and molecular pathways involved in Leishmania infections, particularly in the presence of LRV1. We used microarrays to detail the global gene expression program underlying infection of human mnocyte-derived macrophages with LbLRV1-, LbLRV1+ and identified distinct classes of genes upregulated during this process.
Project description:Transcriptome analysis of Wigglesworthia glossinidia endosymbiont derived from uninfected and infected samples at 3 time points (3, 10 and 20 days). Expression profiling by array - Wigglesworthia glossinidia endosymbiont of Glossina morsitans morsitans
Project description:Infection with antimony resistant (SbR) but not with sentitive (SbS) Leishmania donovani (LD) gives rise to aggressive pathology in mammalian hosts, the cause of which is far from clear. Some intracellular pathogens exploit autophagy for their own benefit. Here we show that induction of autophagy in normal macrophages (MF) by pharmacological mediators prior to infection with SbRLD (SbRLD-MF) enhanced their growth as compared to untreated MF, unlike SbSLD-MF. Autophagy was evident in SbRLD-MF from electron microscopical studies showing double membrane-bound compartment around amastigote. In SbRLD-MF there is induction of beclin 1, which forms the platform to recruit other interacting molecules to initiate autophagy. Knocking down the beclin 1 transcription factor Nrf2 and subsequent infection with SbRLD showed significantly lower organ parasites as compared to wild type BALB/c mice. Cessation of autophagy in SbRLD-MF at the later stage of infection is coupled with induction of miR-30a, whose binding to 3'UTR of beclin 1 leads to its post-transcriptional attenuation followed by rise in intracellular Ca++ and apoptosis. SbRLD mediated translocation of AP-1 transcription factor to the nucleus induce pri-miR-30a over-expression. Rise in Ca++ causes caspase 8 activtion leading to the cleavage of beclin 1 and initiation of apoptosis in SbRLD-MF. Apoptosis may favor parasite egress for cell to cell transmission. We also found that beclin 1 expression is present in splenocytes of kala-azar patients harbouring SbRLD but not SbSLD. Our results suggest that SbRLD has evolved a unique mechanism for its own benefit which explains, in part, the cause of aggressive pathology. Peritoneal exudate macrophages were isolated from mouse, grown in 60mm plates and infected with Leishmania donovani and total RNA was isolated from cells at 12, 18 and 24 hrs post infection. Leishmania infected macrophage miRNA expression signature was generated. Cells grown on 60mm plates and infected with Leishmania. The main objective of the microarray analysis of mmu-miRNA in antimony resistant and antimony sensitive Leishmania donovani infected macrophages are as follows: 1. To study how the expression of miRNA varies in either antimony resistant or antimony sensitive Leishmania infected macrophages as compared to the normal macrophages as a function of time. LPS was used as control. 2. To study the expression of those miRNAs which are differentially expressed in antimony resistant and antimony sensitive Leishmania infected macrophages at each time point post infection. 3. To identify those miRNAs which are responsible for degradation of autophagy initiating protein beclin 1 mRNA
Project description:Transcriptome analysis of Wigglesworthia glossinidia endosymbiont derived from control samples with or without parasite contact at 10 days. Expression profiling by array - Wigglesworthia glossinidia endosymbiont of Glossina morsitans morsitans
Project description:Transcriptome analysis of Wigglesworthia glossinidia endosymbiont derived from control samples with or without parasite contact at 10 days. Expression profiling by array - Wigglesworthia glossinidia endosymbiont of Glossina morsitans morsitans RNAs are a mix of Wigglesworthia, Sodalis and glossina. RNAs were extracted from 8 samples including 2 conditions (with 4 replicates per condition).
Project description:We report the application of Cappable-seq to selectively enrich prokaryotic endosymbiont transcripts from mixed host-symbiont total RNA.
Project description:The genomic DNAs of strains JPCM5 and 263 of L. infantum, strains LV39 and Friedlin of L. major and strains Parrot-TarII and S125 of L. tarentolae were used in comparative genomic hybridizations to reveal the intra-species and inter-species gene content, and to validate L. tarentolae Parrot-TarII genome sequencing results. Leishmania (Sauroleishmania) tarentolae was first isolated in the lizard Tarentola mauritanica. This species is not known to be pathogenic to humans but is often used as a model organism for molecular analyses or protein overproduction. The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved by high-throughput sequencing technologies. The L. tarentolae genome was first assembled de novo and then aligned against the reference L. major Friedlin genome to facilitate contig positioning and annotation, providing a 23-fold coverage of the genome. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described, and it provides an opportunity for comparison with the completed genomes of the pathogenic Leishmania species. A high synteny was observed in de novo assembled contigs between all sequenced Leishmania species. A number of limited chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic with L. major. Globally, over 90% of the L. tarentolae gene content was shared with the other Leishmania species. There were 250 L. major genes absent from L. tarentolae, and interestingly these missing genes were primarily expressed in the intracellular amastigote stage of the pathogenic parasites. This implies that L. tarentolae may have impaired ability to survive as an intracellular parasite. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the leishmanolysin (GP63) and a gene related to the promastigote surface antigen (PSA31C). Overall, L. tarentolae appears to have a gene content more adapted to the insect stage rather than the mammalian one. This may partly explain its inability to replicate within mammalian macrophages and its suspected preferred life style as promastigote in the lizards.
Project description:We sequenced total RNA from Dirofilaria immitis in order to generate the first tissue-specific gene expression profile of a filarial nematode and its Wolbachia endosymbiont.
Project description:Mouse models were infected with Leishmania donovani and the effects were observed over multiple passages to observe the macrophage-parasite interaction