Dataset Information

SILAKin: A novel high throughput SILAC-based quantitative mass spectrometry assay to identify the substratome of kinases secreted by pathogens

ABSTRACT: We developed SILAkin, a novel and easy method to identify substrates that is applicable to most kinases. It combines phosphatase treatment, pulse heating, in vitro kinase assay and SILAC (Stable Isotope Labeling with Amino acids in Cell culture)-based quantitative mass spectrometry (MS). SILAkin was developed using the Leishmania casein kinase 1 (L-CK1.2) as experimental model. Leishmania, an intracellular parasite causing Leishmaniasis, releases L-CK1.2 in its host cell. Applying this novel assay allowed us to gain unprecedented insight into host-pathogen interactions through the identification of 225 host substrates phosphorylated by L-CK1.2. The substratome was validated experimentally by L-CK1.2 and human CK1δ, demonstrating the efficiency of SILAkin to identify new substrates and revealing novel regulatory pathways. Finally, SILAkin was instrumental in highlighting host pathways potentially regulated by L-CK1.2 in Leishmania-infected host cells.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Macrophage

DISEASE(S): Leishmaniasis

SUBMITTER: Valentin SABATET

LAB HEAD: Damarys Loew

PROVIDER: PXD026220 | Pride | 2022-02-17

REPOSITORIES: Pride

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Publications

Identification of the Host Substratome of Leishmania-Secreted Casein Kinase 1 Using a SILAC-Based Quantitative Mass Spectrometry Assay.

Smirlis Despina D Dingli Florent F Sabatet Valentin V Roth Aileen A Knippschild Uwe U Loew Damarys D Späth Gerald F GF Rachidi Najma N

Frontiers in cell and developmental biology 20220103

Leishmaniasis is a severe public health problem, caused by the protozoan Leishmania. This parasite has two developmental forms, extracellular promastigote in the insect vector and intracellular amastigote in the mammalian host where it resides inside the phagolysosome of macrophages. Little is known about the virulence factors that regulate host-pathogen interactions and particularly host signalling subversion. All the proteomes of Leishmania extracellular vesicles identified the p ...[more]

PMID: 35047509

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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: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.

Project description:Leishmania (L) are intracellular protozoan parasites which are able to survive and replicate within the harsh and potentially hostile phago-lysosomal environment of mammalian mononuclear phagocytes. A complex interplay then takes place between the macrophage (MM-NM-&) striving to eliminate the pathogen and the parasite struggling for its own survival. To investigate, at the transcriptional level, this host-parasite conflict in the context of monocyte-derived human MM-NM-&s (MDM) infection by L. major metacyclic promastigotes, the quantitative technique of serial analysis of gene expression (SAGE) was used. After extracting mRNA from resting human MM-NM-&s, Leishmania-infected human MM-NM-&s and L. major parasites, three SAGE libraries were constructed and sequenced generating up to 28,173; 57,514 and 33,906 tags respectively (corresponding to 12,946; 23,442 and 9,530 unique tags). Using computational data analysis and direct comparison to 394,059 publicly available experimental human tags, the parasite and the host cell transcriptomes were then simultaneously characterized from the mixed cellular extract, allowing to confidently discriminate host from parasite transcripts. This procedure led us to reliably assign 3,814 tags to MM-NM-&sM-bM-^@M-^Y and 3,666 tags to L. major parasitesM-bM-^@M-^Y transcripts. We focused on those, showing significant changes in their expression that are likely to be relevant to the pathogenesis of parasite infection: (i) human MM-NM-&s genes, belonging to key immune response proteins (i.e. IFNM-NM-3 pathway, S100 and chemokine families) and (ii) a group of Leishmania genes showing a preferential expression at the intra-cellular developing stage of the parasite. Dual SAGE transcriptome analysis provided a useful, powerful and accurate approach to discriminate between genes of human or parasitic origin in Leishmania-infected human MM-NM-&s. The findings presented in this work suggest that the Leishmania parasite is modulating key transcripts in the human MM-NM-&s that may be beneficial for its establishment and survival and provided an overview of gene expression at two developmental stages of the parasite, namely metacyclic promastigotes and intracellular amastigotes, indicating a broad difference between their transcriptomic profiles. Finally, our reported set of expressed genes could deserve future rounds of data mining and gene annotation. Keywords: Leishmania major, Human macrophages, in vitro, infection, transcriptome, SAGE Human monocyte derived macrophages (MDM) from four healthy donors were infected in vitro for 24 hours with metacyclic Leishmania major parasites (ratio 1:5) and the pool was used to construct SAGE library. Non infected MDM from the same donors and from metacyclic Leishmania major parasites were used to construct the two controls' SAGE libraries.

			Action	DRS
	C5730FD-3.msf	Msf
	C5730FD.raw	Raw
	C5731FD-3.msf	Msf
	C5731FD.raw	Raw
	C5732FD-3.msf	Msf

Dataset Information

SILAKin: A novel high throughput SILAC-based quantitative mass spectrometry assay to identify the substratome of kinases secreted by pathogens

Dataset's files

Publications

Identification of the Host Substratome of <i>Leishmania</i>-Secreted Casein Kinase 1 Using a SILAC-Based Quantitative Mass Spectrometry Assay.

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