Dataset Information

Proteomic map of the unsequenced kala-azar vector Phlebotomus papatasi

ABSTRACT: The debilitating disease kala-azar or visceral leishmaniasis (VL) is caused by the kinetoplastid protozoan parasite Leishmania donovani. The parasite is transmitted by the hematophagous sandfly vector of the genus Phlebotomus in the old world and Lutzomyia in the new world. The predominant Phlebotomine species associated with transmission of kala-azar are Phlebotomus papatasi and Phlebotomus argentipes. The infected female sandfly transmits the parasite when it takes a blood meal. Understanding the molecular interaction of the sand fly-Leishmania during the development of parasite within the gut of the sandfly is crucial to understanding parasite life cycle. The complete genome sequences of sandfly vectors (Phlebotomus and Lutzomyia) are currently not available and sequencing efforts are underway. Non-availability of genome sequence can hamper identification of proteins in the sandfly vector. In the present study we have carried out proteogenomic analysis of unsequenced sandfly vector P. paptasi cell line using high-resolution mass spectrometry and comparative homology-based searches using related dipteran protein data (mosquitoes and fruit fly). This study resulted in identification of 1,312 proteins from P. papatasi based on homology. Our study demonstrates the power of proteogenomic approaches in mapping the proteomes of unsequenced organisms.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Phlebotomus Papatasi (sandfly)

SUBMITTER: Akhilesh Pandey

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD002115 | Pride | 2015-08-31

REPOSITORIES: Pride

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Publications

A proteomic map of the unsequenced kala-azar vector Phlebotomus papatasi using cell line.

Pawar Harsh H Chavan Sandip S Mahale Kiran K Khobragade Sweta S Kulkarni Aditi A Patil Arun A Chaphekar Deepa D Varriar Pratyasha P Sudeep Anakkathil A Pai Kalpana K Prasad T S K TSK Gowda Harsha H Patole Milind S MS

Acta tropica 20150822

The debilitating disease kala-azar or visceral leishmaniasis is caused by the kinetoplastid protozoan parasite Leishmania donovani. The parasite is transmitted by the hematophagous sand fly vector of the genus Phlebotomus in the old world and Lutzomyia in the new world. The predominant Phlebotomine species associated with the transmission of kala-azar are Phlebotomus papatasi and Phlebotomus argentipes. Understanding the molecular interaction of the sand fly and Leishmania, during the developmen ...[more]

PMID: 26307495

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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:Leishmania donovani, an intracellular protozoan parasite, is the causative agent of visceral leishmaniasis or kala-azar, the most severe form of leishmaniasis in humans. To date, our understanding of the molecular mechanisms associated with the pathogenicity of Leishmania infection is still limited. RNA interference—collectively RNA silencing pathways—participates in the regulation of various biological processes in most eukaryotic cells. Complexes of Argonaute proteins with small RNAs are core components of the RNA interference system and play a key role in silencing gene expression. It is becoming increasingly clear that several intracellular pathogens target host cell RNA interference pathways to promote their survival. In this study, we investigated the potential role of host macrophage Argonautes in Leishmania pathogenesis. Western blot analysis showed that protein abundance of infected macrophage Argonaute 1 (Ago1) was selectively and significantly higher than that of non-infected control at 24 h post-infection, suggesting that Ago1 plays a role in pathogenicity. In fact, siRNA-mediated downregulation of Ago1 enhanced Leishmania clearance from infected host cells, linking macrophage Ago1 to Leishmania virulence. To investigate the mechanisms of host Ago1 in Leishmania pathogenesis, a stable isotope labeling by amino acids in cell culture (SILAC)-based whole proteome approach was employed, which showed that expression of several previously reported Leishmania pathogenesis-related proteins were dependent on the level of macrophage Ago1. Moreover, the proteomic-based detailed biochemical analysis showed that Leishmania modulated host RNA-induced silencing complex (RISC) composition during infection, strongly suggesting macrophage RISC targeting. Strikingly, Leishmania proteins were detected as part of host RISC in infected cells. Together, our results demonstrate that Leishmania targets host RNA interference machinery to promote its survival inside the host macrophage.

Dataset Information

Proteomic map of the unsequenced kala-azar vector Phlebotomus papatasi

Publications

A proteomic map of the unsequenced kala-azar vector Phlebotomus papatasi using cell line.

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