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. 2023 Nov 30:14:1287539.
doi: 10.3389/fimmu.2023.1287539. eCollection 2023.

Leishmania infection upregulates and engages host macrophage Argonaute 1, and system-wide proteomics reveals Argonaute 1-dependent host response

Atieh Moradimotlagh #  1 , Stella Chen #  1 , Sara Koohbor  1 , Kyung-Mee Moon  2 , Leonard J Foster  2 , Neil Reiner  1 , Devki Nandan  1
Affiliations

Leishmania infection upregulates and engages host macrophage Argonaute 1, and system-wide proteomics reveals Argonaute 1-dependent host response

Atieh Moradimotlagh et al. Front Immunol. .

Abstract

Leishmania donovani, an intracellular protozoan parasite, is the causative agent of visceral leishmaniasis, the most severe form of leishmaniasis in humans. It is becoming increasingly clear that several intracellular pathogens target host cell RNA interference (RNAi) pathways to promote their survival. Complexes of Argonaute proteins with small RNAs are core components of the RNAi. In this study, we investigated the potential role of host macrophage Argonautes in Leishmania pathogenesis. Using Western blot analysis of Leishmania donovani-infected macrophages, we show here that Leishmania infection selectively increased the abundance of host Argonaute 1 (Ago1). This increased abundance of Ago1 in infected cells also resulted in higher levels of Ago1 in active Ago-complexes, suggesting the preferred use of Ago1 in RNAi in Leishmania-infected cells. This analysis used a short trinucleotide repeat containing 6 (TNRC6)/glycine-tryptophan repeat protein (GW182) protein-derived peptide fused to Glutathione S-transferase as an affinity matrix to capture mature Ago-small RNAs complexes from the cytosol of non-infected and Leishmania-infected cells. Furthermore, Ago1 silencing significantly reduced intracellular survival of Leishmania, demonstrating that Ago1 is essential for Leishmania pathogenesis. To investigate the role of host Ago1 in Leishmania pathogenesis, a quantitative whole proteome approach was employed, which showed that expression of several previously reported Leishmania pathogenesis-related proteins was dependent on the level of macrophage Ago1. Together, these findings identify Ago1 as the preferred Argonaute of RNAi machinery in infected cells and a novel and essential virulence factor by proxy that promotes Leishmania survival.

Keywords: Leishmania; RNAi; host-pathogen interactions; parasitic infection; proteomics.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
Ago1 is upregulated in infected macrophages. (A) dTHP-1 cells were infected with L. donovani (Ld) or not for 24 h and 48 h, whole cell lyates were from non-infected (NI) and Ld-infected macrophages were analysed by Western blotting with antibodies specific for Ago1, Ago2 and Actin (loading control). (B) Densitometric analysis was used to measure Ago1 and Ago2, normalized to Actin. The histogram shown is the mean ± S.D. of the densitometric analyses of three independent experiments. (C) Ago1 knockdown attenuates Leishmania survival. THP-1 cells were treated with control scrambled siRNAs or three different Ago1-specific siRNAs for 52 h Control and Ago1-knockdown cells were differentiated and subsequently incubated with Leishmania for 24 h At the end of the experiment, a parasite rescue assay as described in “Materials and methods” was performed. Shown is the mean ± S.D. of the densitometric analyses of three independent experiments (*p-value < 0.05, **p-value < 0.01, ***p-value < 0.001). (D) In parallel, the cells treated with siRNAs and differentiated for parasite rescue assay were analyzed by Western blotting for Ago1 and Actin levels.
Figure 2
Figure 2
Analysis of cytoplasmic and nuclear fractions from non-infected and infected macrophages for the presence of Ago1. (A) dTHP-1 cells were subjected to cell fractionation for cytoplasmic and nuclear fractions as described in “Materials and methods” and the purity of fractions was assessed using indicated antibodies in a Western blot assay. (B) The cytoplasmic and nuclear extracts from dTHP-1 cells were incubated separately with GST-T6B affinity beads and bound proteins were subjected to Western blotting using indicated antibodies for the presence of Ago1 and Ago2. (C) Immunoblot comparing levels of Ago1 in bound materials eluted from GST-T6B affinity beads incubated with cytosolic or nuclear lysates of non-infected (NI) and Leishmania-infected (Ld) dTHP-1 cells. In parallel, aliquots from the cytoplasmic fraction from NI and Leishmania-infected cells were tested for GAPDH and Lamin A/C level. The data shown is one representative of three independent experiments.
Figure 3
Figure 3
Quantitative proteomic analysis of non-infected and Leishmania-infected macrophages. THP-1 cells were cultured in SILAC media as described in “Materials and methods”. Labeled THP-1 cells were treated with an equal mix of two Ago1 siRNAs or with control scrambled siRNA. Control and Ago1-knockdown cells were differentiated and incubated with Leishmania for 24 h. Subsequently, control and infected cells were washed extensively and mass spectrometry was performed on a mix of three lysates allowing for a quantitative comparison between non-infected, infected cells and infected in Ago1 knockdown condition (three independent experiments). Shown is the comparison between non-infected (L) and Leishmania-infected (M) cells. (A) Pie chart of the log2(fold change) distribution of Leishmania-modulated proteins. M/L normalized ratios were log2 transformed and averaged for at least 2 replicates. P-values < 0.05 from one-sample T-tests were considered significant. Proteins with a negative log2(fold change) were downregulated in Leishmania-infected cells compared to non-infected cells, whereas proteins with a positive log2(fold change) were upregulated in Leishmania-infected cells compared to non-infected cells. (B) Volcano plot comparing protein expression levels in Leishmania-infected cells with those in non-infected cells, both treated with control siRNA. Proteins significantly upregulated by Leishmania infection are shown as green dots, and those that were downregulated, as red dots. The horizontal dashed line marks the p-value cut off of 0.05 or -log10(p-value) = 1.301. The vertical dashed line represents log2(M/L) = 0. L, Light (Scrambled siRNA-treated, Non-infected); M, Medium (Scrambled siRNA-treated, Ld-infected).
Figure 4
Figure 4
Proteomic analysis of Ago1-dependent Leishmania-modulated macrophage proteins. (A) Scatter plot of log2 transformed M/L ratios (y-axis) vs. log2 transformed H/M ratios (x-axis) of proteins. Proteins that were upregulated by Leishmania infection and recovered to any degree following Ago1 knockdown are shown in green. Proteins that were downregulated by Leishmania-infection and recovered to any degree following Ago1 knockdown are shown in red. The Ago1-dependent Leishmania-modulated proteins were also classified by GO terms in three categories: (B) biological process, (C) cellular component, and (D) molecular function. P-values < 0.05 from one-sample T-tests were considered significant for both log2 transformed M/L and H/M fold changes. L, Light (Scrambled siRNA-transfected, Non-infected); M, Medium (Scrambled siRNA-transfected, Ld-infected); H, Heavy (Ago1 siRNA-transfected, Ld-infected).
Figure 5
Figure 5
Hypothetical model of Leishmania-macrophage cross-kingdom RNAi. In canonical RNAi (left panel), endogenous sncRNAs interact with Ago1-4 proteins and then assembles with TNRC6 and other proteins to form the RISC. The RISC then regulates gene expression of the cell at post-transcriptional level by targeting the 3’UTR of mRNAs. In Leishmania-infected cells (right panel), Leishmania induces broad-based downregulation of host miRNAs and upregulation of Ago1 via an unknown mechanism, possibly by secretory factors. Leishmania exosomes containing ncRNAs secreted by amastigotes in the phagolysosomes of either the host cell or infected bystander cells are delivered to the cytosol. For RNAi, the delivered Leishmania exosomal sncRNAs (L-sncRNAs) are preferentially loaded onto host Ago1, which does not require full complementarity to target the mRNA. Created with BioRender.com.
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The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The funding for this research was provided by the Natural Sciences and Engineering Research Council of Canada (RGPIN-2018-04991) and the Canadian Institute of Health Research (PJT-162191) awarded to NR, and Genome British Columbia (264PRO) awarded to LF. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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