Dataset Information

Comprehensive proteomic analysis of autophagosomes derived from Leishmania infected macrophages

ABSTRACT: Autophagy has been implicated as a host defense mechanism against intracellular pathogens. However, certain intracellular pathogens such as Leishmania can manipulate the host’s autophagy to promote their survival. Recently, our findings regarding the regulation of autophagy by Leishmania donovani indicate that this pathogen induces non-classical autophagy in infected macrophages, independent of mammalian target of rapamycin complex 1 regulation. This occurs in the background of enhanced mTOR activity, which suggests the fine-tuning of autophagy to optimally promote parasite survival and may involve the sequestration or modulation of specific autophagosome-associated proteins. To investigate how Leishmania potentially manipulates the composition of host-cell autophagosomes, we undertook a quantitative proteomic study of the human monocytic cell line THP-1 following infection with L. donovani. We used stable isotope labeling by amino acid in cell culture and liquid chromatography-tandem mass spectrometry to compare expression profiles between autophagosomes isolated from THP-1 cells infected with L. donovani or treated with known autophagy inducers. Select proteomics results were validated by immunoblotting. In this study, we showed that L. donovani modulates the composition of macrophage autophagosomes during infection when compared to autophagosomes induced by either rapamycin (selective autophagy) or starvation (non-selective autophagy). Among 1787 proteins detected in Leishmania-induced autophagosomes, 146 were significantly modulated compared to the proteome of rapamycin-induced autophagosomes while 57 were significantly modulated compared to starvation-induced autophagosomes. Strikingly, 23 Leishmania proteins were also detected in the proteome of Leishmania-induced autophagosomes. Together, our data provide the first comprehensive insight into the proteome dynamics of host autophagosomes in response to Leishmania infection and demonstrate the complex relations between the host and pathogen at the molecular level.

INSTRUMENT(S): Bruker Daltonics instrument model

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Jenny Moon

LAB HEAD: Leonard J. Foster

PROVIDER: PXD037057 | Pride | 2023-05-10

REPOSITORIES: Pride

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			Action	DRS
	Eleanor_1-1-300ng_7-31-2021_D5_2719.d.1.dar	Other
	Eleanor_1-2-300ng_7-31-2021_D6_2720.d.1.dar	Other
	Eleanor_1-3-300ng_7-31-2021_D7_2721.d.1.dar	Other
	Eleanor_1-4-300ng_7-31-2021_D8_2722.d.1.dar	Other
	Eleanor_1-5-300ng_7-31-2021_D9_2723.d.1.dar	Other

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Publications

Comprehensive proteomic analysis of autophagosomes derived from Leishmania-infected macrophages.

Nandan Devki D Chen Eleanor E Chang Fabian F Moon Kyung-Mee KM Foster Leonard J LJ Reiner Neil N

PloS one 20230407 4

Recently, autophagy has been implicated as a host defense mechanism against intracellular pathogens. On the other hand, certain intracellular pathogens such as Leishmania can manipulate the host's autophagy to promote their survival. Our recent findings regarding the regulation of autophagy by Leishmania donovani indicate that this pathogen induces non-classical autophagy in infected macrophages, independent of regulation by the mammalian target of rapamycin complex 1. This suggests the fine-tun ...[more]

PMID: 37027358

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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.

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

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