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

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.


Introduction
Infection with intracellular pathogens belonging to the genus Leishmania results in a broad spectrum of clinical manifestations, mainly divided into three distinct clinical syndromes: visceral, cutaneous, and mucocutaneous leishmaniasis (1).Leishmania donovani, the focus of this study, causes visceral leishmaniasis, which can be life-threatening if left untreated (2).Leishmania has a digenetic life cycle, alternating between the non-motile amastigote form in the vertebrate host and the motile promastigote form in the invertebrate vector, the phlebotomine sandfly (3).The infection is initiated when an infected female sandfly injects metacyclic promastigotes into a mammalian host during a regular blood meal.Parasites released are rapidly taken up by phagocytic cells, including macrophages and neutrophils (4).The macrophages are the final resident cells which support Leishmania growth and proliferation, establishing chronic infection in humans.After internalization, promastigotes settle in phagolysosomes of macrophages, where they differentiate into amastigote forms, which divide many times and ultimately rupture macrophages and infect other surrounding macrophages (1).
Leishmaniasis is responsible for severe morbidity and mortality in parts of the tropics, subtropics and southern Europe.Leishmaniasis is classified as a neglected tropical disease.Unfortunately, leishmaniasis is rising due to increased global travel and a lack of effective therapeutic and prophylactic methods.Available drugs are expensive, have severe toxicity, and are circumvented by emerging parasite resistance (5).Although tremendous effort has been made to develop Leishmania vaccines, only some have been commercially approved for canine leishmaniasis (6), while none have yet been licensed for human leishmaniasis (5).Expanding the knowledge on the biological interaction between the parasite and the host is crucial for developing a comprehensive strategy to fight the disease and improve leishmaniasis treatment.The pathogenesis of Leishmania involves the alteration of the macrophage phenotype, where Leishmania causes deactivation of the host by interfering with host-cell gene expression and signal transduction (7).One potential mechanism of interest of Leishmania pathogenesis is targeting host small non-coding RNAs (sncRNAs), which are involved in post-transcriptional gene expression regulation, known as RNA interference (RNAi).
It is becoming increasingly clear that non-coding RNAs (ncRNAs), which do not encode proteins but account for the majority of the human genome, play important roles in many biological processes and diseases (8)(9)(10)(11).Among them are small ncRNAs, such as microRNAs (miRNAs), Piwi-interacting RNAs, and tRNA-derived RNA fragments (tRFs), which have been shown to play a critical role in cancer, stress response, metabolic abnormalities and viral infections (12)(13)(14)(15)(16). Out of all species of ncRNAs, sncRNAs represent the core of the regulatory mechanisms of gene expression in eukaryotic cells.The mechanisms of action of most sncRNAs require a group of effector proteins as part of an RNA-induced silencing complex (RISC).These effector proteins are necessary for the stabilization, transport and regulatory activity of ncRNAs on their target RNA transcripts.RNA-binding proteins Argonautes (Agos) are members of the Argonaute family that bind sncRNAs that act as guide RNA and direct them to specific RNA transcripts for silencing.In fact, these highly conserved proteins are central components of the RISC in RNAi (17,18).There are eight Ago family members in humans consisting of four ubiquitously expressed Ago1-4 of the Ago clade and four of the PIWI clade (PIWIL1-4), which are mainly restricted to the germline (19).Deep sequencing of human Ago-associated sncRNAs revealed Ago association with RNA fragments of diverse origin, such as miRNAs, small nucleolar RNAs, vault RNAs, and tRFs, to carry out their gene silencing function (20).
Ago proteins are transiently held in open conformation by heat shock proteins (HSPs) to allow loading of sncRNA, resulting in the closed conformation Ago-sncRNA complex, also known as the mature RISC.The mature RISC further interacts with the scaffolding protein trinucleotide repeat containing 6 (TNRC6)/ glycine-tryptophan repeat protein (GW182), which preferentially binds to mature RISCs over guide-free Ago proteins (21) and facilitates downstream silencing processes, possibly by recruiting other RNA binding proteins (RBPs) (22).Finally, the RISC interacts with and represses expression of messenger RNA (mRNA) targets whose sequences are partially or fully complementary to those of the sncRNAs in the RISC.
Small ncRNAs (sncRNAs), such as microRNAs (miRNAs) and tRNA-derived RNA fragments (tRFs), have been clearly demonstrated to play an important role in microbial infections (12)(13)(14)23).Our recent study showed that Leishmania infection causes genome-wide attenuation of macrophage miRNAs by decreasing miRNAs gene transcription.miRNA downregulation was linked to the upregulation of macrophage transcription factor c-Myc. c-Myc silencing reversed the Leishmania-mediated miRNA suppression and also reduced the intracellular survival of Leishmania (24).We have recently discovered that sncRNAs like tRFs and ribosomal RNA-derived RNA fragments (rRFs) are highly enriched in exosomes secreted by L. donovani and L. braziliensis (25), and these secretory exosomes have been shown to modulate innate and adaptive immune responses through predominantly immunosuppressive effects, such as promoting IL-10 production and inhibiting TNF-alpha in human monocytes and monocyte-derived dendritic cells (26).Overall, the emerging role of sncRNAs (such as miRNAs) from both host and pathogen (such as tRFs) during Leishmania infection represents a novel virulence paradigm that invites further examination.Thus, it was of interest to investigate the possibility that Leishmania targets host macrophage Ago(s) to regulate host gene expression to facilitate its survival.
In this study, we show that L. donovani selectively increases the abundance of host macrophage Ago1.Strikingly, the enhanced level of Ago1 in infected cells correlated with an increased level of Ago1 containing-complexes isolated using a recently published biochemical isolation of Argonaute protein complexes by "Ago proteins Affinity Purification by Peptide" (Ago-APP).Deliberate silencing of host Ago1 significantly attenuated intracellular survival of Leishmania in human macrophages, demonstrating that Ago1 is essential for Leishmania pathogenesis.SILAC (stable isotope labeling using amino acids in cell culture)-based quantitative proteomic analysis revealed that the expression of several previously reported Leishmania pathogenesis-related proteins was dependent on the level of macrophage Ago1, suggesting the role of Ago1 in Leishmania pathogenesis.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.

Parasite culture and in vitro infection
Leishmania donovani Sudan strain 2S was provided by Dr. Kwang Poo Chang (Rockefeller University, NY, USA), and continuously cultured in the lab.Virulence was maintained by regular passages via Syrian Golden hamster from which fresh amastigotes were purified and transformed in vitro into promastigotes (24).Promastigotes were cultured in M199 medium (MilliporeSigma) supplemented with 10% heatinactivated fetal bovine serum, 10 mM HEPES, 10 µg/ml folic acid (MilliporeSigma), 3 µg/ml hemin (MilliporeSigma), 2 mM L-Glutamine, 100 U/ml penicillin/streptomycin and 100 mM adenosine (MilliporeSigma) at 26°C.The parasites were subcultured every three days for a maximum of 25-30 passages.For Leishmania infections, stationary phase prom astigote s we re used at an MOI of 20:1 unless otherwise stated.

Determination of infection rate
To determine the infection rate, Leishmania-infected cells were washed with warm PBS, then fixed with 2% paraformaldehyde in PBS for 20 min on ice, and mounted on glass slides using VECTASHIELD Antifade Mounting Medium with DAPI, which stains macrophage and parasite nuclei.Cells were viewed under fluorescence using the Axioplan II (Carl Zeiss Inc.) microscope.For each treatment, at least 10 images were taken at 40x magnification using the AxioCam MRm Camera and the AxioVision software Version 4.8.2 (Carl Zeiss Inc.).At least 100 cells were counted for each condition to determine the average number of parasites per macrophage and the percentage of macrophages infected.

Parasite rescue and transformation assay
PMA differentiated THP-1 cells (dTHP-1) were infected with stationary-phase L. donovani promastigotes at an MOI of 20:1 for 24 h.After the duration of infection, cells were thoroughly washed with HBSS to remove non-internalized Leishmania.Controlled lysing of infected cells was performed using 0.01% SDS as previously described (24, 27) to release intracellular amastigotes.The rescued Leishmania amastigotes were transformed to promastigotes in M199 media by incubating at 26°C for 72 h.Their growth was evaluated after 72 h of incubation by counting transformed promastigotes stained with trypan blue solution (0.4% w/v in PBS) on a hemocytometer.

THP-1 cell culture in SILAC media
For SILAC-based labeling, RPMI 1640 media without Lglutamine, L-arginine and L-lysine (Caisson labs) was supplemented with 10% heat-inactivated dialyzed fetal bovine serum (Gibco), 2 mM L-glutamine, 10 mM HEPES and 100 U/ml penicillin/streptomycin.The RPMI medium was then split into three: the "light" RPMI was supplemented with normal isotopic abundance arginine (21 mg/l) and lysine (36.5 mg/l), while the "medium" RPMI was supplemented with 13 C 6 -arginine (20.25 mg/l) and 2 H 4 -lysine (37.5 mg/l), and "heavy" RPMI was supplemented with 13 C 6 15 N 4arginine (22.25 mg/l) and 13 C 6 N 2 -lysine (38.5 mg/l).THP-1 cells were spun down, one third of which were resuspended in the respective SILAC media, incubated at 37°C and 5% CO 2 , and passaged for a minimum of five times prior to use.Cells were differentiated using 10 ng/ml of PMA for 16-18 h and then washed with HBSS and rested for 24 h in fresh respective SILAC media prior to infection.

siRNA-mediated knockdown of Ago1 in THP-1 cells
Three siRNAs targeting human Ago1 (SR308857), as well as non-specific control (scrambled) siRNA (SR30004), were purchased from OriGene.Cells were transfected for 52 h with non-specific or Ago1 siRNA (50 pmol/well) using HiPerFect transfection reagent (Qiagen) according to the manufacturer's instructions.We have previously used HiPerFect transfection reagent (Qiagen) to silence the expression of various proteins in THP-1 cells (28) and in human monocyte-derived macrophages (24) using siRNAs from OriGene, with transfection efficiency between 70-80%, as measured by fluorescent-labeled transfection control siRNA (OriGene).After the duration of transfection, THP-1 cells were differentiated with PMA for further use.

Expression and purification of recombinant proteins
For the expression and purification of required recombinant proteins we used a previously published protocol (29).Briefly, GST (glutathione S-transferase) and GST-T6B plasmid constructs were transformed in DH5 alpha E. coli and then expressed in BL21.Subsequently, 2 L isopropyl b-D-1-thiogalactopyranosideinduced culture (OD 600 = 0.6) was grown overnight at 18°C.The cells were harvested by centrifugation at 4,400 × g, 30 min at 4°C and the resulting pellet was then resuspended in resuspension buffer (PBS containing 1 mM PMSF and 1 mM DTT) supplemented with 1 mg/ml lysozyme.After sonication for 4 × 30 sec, the lysate was cleared by centrifugation at 40,000 × g, 40 min at 4°C.Then the supernatant was incubated with 3 ml washed Glutathione-Agarose beads (MilliporeSigma) at 4°C for 3 h.Thereafter, the GST/GST-T6B-coupled beads were loaded onto a column and washed with 10 column volumes (cv) of resuspension buffer.GST/GST-T6B was eluted with 4 cv of elution buffer (PBS supplemented with 20 mM Tris-HCl, pH 8.0, and 10 mM reduced glutathione), pooled and then concentrated with Amicon Ultra-15 centrifugal filter units.The concentrated fractions were loaded onto a desalting chromatography column (Zeba ™ Spin Desalting Columns) that was equilibrated in PBS with 5% (v/v) glycerol.Desalted fractions were adjusted to ∼2 mg/ml proteins, aliquoted and stored at −80°C.The purity of recombinant proteins was assessed by staining with Coomassie brilliant blue.

Preparation of cytosolic fraction
For the preparation of the cytosolic fraction, we followed a published protocol (30).Briefly, dTHP-1 cells were extensively washed and lysed in hypotonic lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl 2 , 0.3% NP-40, 1 mM NaF, 1 mM Na 3 VO 4 ) supplemented with 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 1 mM PMSF, and passed ten times through a 22-gauge needle on ice to disrupt cells.Then disrupted cells were centrifuged at 2,300 × g for 20 min at 4°C.The supernatant was kept as the cytoplasmic fraction.The purity of cytoplasmic fractions was assessed using antibodies against specific markers for the cytoplasm (GAPDH) and nuclear fraction (Lamin A/C) in a Western blot assay.These cytoplasmic fractions were used for the purification of Agos and associated proteins using GST-T6B affinity beads.

GST-T6B affinity chromatography
To prepare the affinity beads, 50 µl of Glutathione-Agarose beads were incubated with approximately 100 µg of GST/GST-T6B recombinant proteins for 3 h at 4°C with end-over-end rotation.Non-bound peptides were removed by washing the beads three times with excess of ice-cold PBS.In order to remove non-specific proteins which might have interacted with GST alone or Glutathione-Agarose beads, the cell lysates of interest prepared in NET buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 10% glycerol, 1 mM NaF; supplemented with 0.5 mM DTT and 1 mM PMSF) were pre-incubated with 50 µl of Glutathione-Agarose beads loaded with 100 µg GST for 1 h at 4°C with end-overend rotation.Non-bound materials were then incubated with the GST-T6B affinity beads for 3 h, at 4°C to capture interacting proteins.Beads were spun down, washed three times with NET buffer to remove non-bound material, and once with PreScission cleavage buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT).The affinity beads were eluted by PreScission enzyme (MilliporeSigma) cleavage at 4°C or by addition of 2x Laemmli sample loading buffer and incubation at 95°C for 7 min.

Liquid chromatography-tandem mass spectrometry and protein identification
For mass spectroscopy analysis of siRNA-mediated Ago1 downregulated macrophages, equal amounts of proteins from each treatment were mixed, reduced, alkylated (31) and run on 10% SDS-PAGE.Proteins in the entire lane were digested in gel with MS-grade trypsin (Promega) (32) and resulting peptides were cleaned on C-18 STop And Go Extraction (STAGE) tips (33) using 40% (v/v) acetonitrile in 0.1% (v/v) formic acid as the elution buffer.Peptides were analysed on NanoDrop One (Thermo Scientific -A205, scopes) to load approximately 200 ng of peptides on Orbitrap Exploris 480 coupled to easy nLC 1200 (Thermo Scientific) with ionopticks' Aurora series 25 cm x 75 µm C18 1.6 µm analytical column heated to 40°C.Peptides were separated on a 62 minute gradient with a LC flow rate of 250 nl/min.Buffer A consisted of 2% (v/v) acetonitrile and 0.1% (v/v) formic acid; buffer B consisted of 80% (v/v) acetonitrile and 0.1% (v/v) formic acid.Spray voltage was set to 1,900 V and ion transfer tube temperature to 290°C.FAIMS was not enabled with data acquisition.Data dependent mode was set for 20 scans and the orbitrap resolution was set to 120,000 for full MS and 15,000 for fragment MS, with AGC target set to 100% at 20 ms for full MS and 50% for fragment MS.Intensity threshold was set at 8,000, isolation window at 2 m/z, with normalized collision energy at 28%.Dynamic exclusion was enabled to exclude after 1 time for 45 s.
Proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD037042.The reviewer account for the PRIDE data has the username reviewer_pxd037042@ebi.ac.uk and password o1Lv0bxR.

Bioinformatic analysis of LC-MS/MS data
All downstream analysis was done using R software (http:// www.r-project.org).Of the total protein groups (hereafter referred to simply as 'proteins') detected by LC-MS/MS, we removed those that were commonly occurring contaminants, identified by peptides found to be part of a protein derived from the reversed part of the decoy database, or non-human, yielding a filtered high-confidence dataset of human proteins.Proteins were further filtered to only keep those with a minimum of two unique peptides per protein identification and which were identified in at least two of the three replicates.For all analyses, log 2 transformed normalized ratios (fold changes) were used.
The one-sample T-test was performed to statistically compare log 2 transformed normalized ratios to the hypothetical value of 0 (representing no change).A p-value of less than 0.05 was considered statistically significant.No cut-off was set as the minimum fold change for a protein to be considered modulated.Instead, where applicable, the significance of each protein modulation was ranked by distance from the origin measured along axes (Manhattan distance), when represented in a volcano plot of -log 10 (p-value) vs. log 2 (fold change).The higher the Manhattan score, the more significant the modulation of the protein was considered.For proteins that were both Leishmaniamodulated and Ago1-dependent, the sum of their Manhattan scores was used to rank their combined significance.
We compared our dataset with the Pathways Commons collection of databases (https://www.pathwaycommons.org/),which collects biological pathway and interaction data from various partner databases-including, but not limited to, BioGRID, SwissProt, NCI Pathway Interaction Database: Pathway, and Innate DB.
For Gene Ontology (GO) annotation, Uniprot Princeton GO Mapper (https://go.princeton.edu/cgi-bin/GOTermMapper) was used with the following parameters: Organism (Annotation) set to "Homo sapiens (GOA @EBI + Ensembl)" and Ontology set to "Generic slim".The proteins were annotated for each of three biological aspects: Biological process ("process"), Molecular function ("function"), and Cellular component ("component").We omitted GO terms with 3 or fewer protein members or that were very broad and encompassed many subcategories ("Organelle", with 68/71 proteins).Conversely, we combined similar highly specific GO terms ("Catalytic activity acting on a protein", "Catalytic activity acting on RNA", "activity acting on DNA") into their corresponding umbrella term ("Catalytic activity").Finally, for sets of two nearly identical GO terms, we retained only one ("Extracellular space" but not "Extracellular region", and "Extracellular matrix" rather than "External encapsulating structure").

The host Ago1 is upregulated during
Leishmania infection and downregulation of Ago1 attenuates Leishmania survival Argonautes 1 and 2 are the most abundant in mammalian cells and have been extensively studied in various biological contexts such as regulation of the biogenesis of small RNA, mRNA translation, small RNA-guided host defense against exogenous nucleic acids, genome editing and even disease progression (17).Thus, we investigated the modulation of host macrophage Ago1 and Ago2 in response to L. donovani infection in PMAdifferentiated THP-1 cells (dTHP-1).These cells have been extensively used as a model system to study human leishmaniasis (35-37).To examine Ago1 and Ago2 levels, dTHP-1 cells were infected with stationary-phase L. donovani promastigotes at MOI 20:1 for 24 h and 48 h.Ago1 and Ago2 levels were monitored by Western blot assay using their respective antibodies.As shown in Figures 1A, B, the expression level of Ago1 was selectively and strongly upregulated, while the Ago2 level remained unaltered in infected cells, both at 24 and 48 h post-infection.We checked the specificity of commercial Ago1 antibody from Cell signaling using whole cell lysate of dTHP-1 cells (positive control) and whole cell lysate of Leishmania promastigotes in a Western blot assay.Anti-Ago1 showed no detectable reactivity in the whole cell lysate of Leishmania promastigotes (data not shown), thus rejecting any potential contribution of Leishmania Ago1 in the observed upregulation of host Ago1 in infected cells.Furthermore, to rule out the possibility that Leishmania-induced Ago1 is due to the general phagocytosis of external particles, we incubated dTHP-1 cells with latex beads at different MOIs (5:1, 10:1 and 20:1) for 24 h.Western blot analysis of the bead-treated cells for Ago1 level revealed no significant induction of Ago1 compared to nontreated control cells (Figure S1).This strongly suggests that the increase in Ago1 abundance following Leishmania infection is not mediated by general phagocytotic processes.
Given that Ago1 binds a variety of sncRNAs, the central component of RISC in the RNA silencing pathway, it seemed likely that Ago1 upregulation would have pleiotropic and important effects on the macrophage phenotype.In addition, it seemed probable that this upregulation of Ago1 itself, independent of any impact on the RNAi pathway, would have consequences for macrophage biology.Thus, to address the biological relevance of Leishmania-mediated selective increase in host Ago1 level, we investigated the possibility that Ago1 may confer a pathogen survival advantage.We therefore assessed Leishmania intracellular survival in host macrophages treated with Ago1 siRNAs.To this end, we downregulated host Ago1 using three unique Ago1-targeting siRNAs.A non-specific scrambled siRNA was used as a negative control.Ago1-knockdown cells and control cells were infected with L. donovani for 24 h.At the end of the experiment, the infected cells were lysed for parasite rescue, as described in "Materials and methods."The results presented in Figure 1C show that reduced Ago1 level correlated with decreased survival of Leishmania.In parallel, the downregulation of Ago1 was confirmed by immunoblotting using Ago1 antibody.The efficiency of siRNA-mediated Ago1 knockdown is shown in Figure 1D.All three unique siRNAs were able to downregulate Ago1.The transfection of siRNAs 2 and 3 resulted in nearly complete downregulation of Ago1.Together, these results show that host Ago1 is essential for the intracellular survival of Leishmania.
Results presented thus far show enhanced levels of Ago1 in infected cells.Deliberate downregulation of Ago1 resulted in decreased survival of Leishmania inside host cells.Given that Argonaute proteins participate in the RNA silencing pathway as the central component of RISC (17,38), it was important to investigate the possibility that an enhanced level of Ago1 in infected cells also results in a higher level of Ago1-containing mature RISC complexes required for RNAi.Thus, we isolated Ago protein complexes from non-infected control and Leishmania-infected cells.

Preparation of GST-TNRC6 affinity beads and validation for its interaction with Argonautes
We used a recently published procedure, "Ago protein Affinity Purification by Peptides," to isolate Ago-containing complexes from control and infected cells (29).This affinity column-based approach allows for the simultaneous isolation of all Argonautes and associated proteins from a variety of species and cell lines.This robust pull-down assay involves the (GST)-T6B peptide, a part of the TNRC6/GW182 protein family that efficiently interacts with all Ago proteins (29).
To prepare recombinant GST-T6B peptide and control affinity beads, purified GST-T6B proteins and GST alone were loaded respectively on Glutathione-Agarose beads as described in "Materials and methods."The purity of concentrated and desalted recombinant proteins is shown in Figure S2A.Without antibodies against the T6B peptide, expected molecular mass and reactivity to anti-GST antibodies were used to correctly identify recombinant proteins (Figure S2B).To test that recombinant GST-T6B peptide is active and successfully isolates Ago proteins from macrophages, whole cell lysate was incubated with GST alone and GST-T6B affinity beads separately.Bound proteins were released by heating in the Laemmli sample buffer.The presence of Agos in the bound materials was investigated by immunoblotting using appropriate antibodies.As expected, Ago1 and Ago2 were detected in material eluted from GST-T6B beads but absent from material eluted from GST alone, thus validating the specificity and effectiveness of peptide affinity beads in the isolation of Ago proteins (Figures S2C, D).

Ago protein-containing complexes isolated from Leishmania-infected cells are enriched in Ago1
As described above, it was of interest to investigate whether this increase in Ago1 correlates with an increase in Ago1 being part of RISC, which is responsible for the RNAi process for gene regulation.
It is well established that most Ago complexes are restricted to the cytoplasm (22, 39) to participate in canonical RNAi.Thus, we used cytoplasmic fractions to isolate Ago protein-containing complexes using GST-T6B affinity beads for further study.GAPDH was used as the cytoplasm marker, and Lamin A/C was used as the nuclear marker.The results presented in Figure 2A show cytoplasmic fraction nearly devoid of nuclear content.Bound complexes from GST alone and GST-T6B affinity beads were released by heating in Laemmli sample buffer.The presence of Agos in the bound materials was investigated by immunoblotting using appropriate antibodies.As expected and shown in Figure 2B, Ago1 and Ago2 are enriched in cytoplasm and nearly completely absent in the nuclear fraction.After confirmation of the enrichment of Agos in the cytoplasm, cytoplasmic and nuclear fractions from control and infected cells were separately incubated with GST-T6B affinity beads to isolate Ago-containing complexes.As shown in Figure 2C, the amount of Ago1 was strikingly enriched in complexes isolated from cytoplasmic fractions of Leishmaniainfected cells compared to non-infected cells.This finding of Ago1 enrichment in RISC isolated from infected cells positively correlates with the abundance of Ago1 in Leishmania-infected cells.Taken together, results obtained suggest that Leishmania infection promotes Ago1-containing mature complexes, which are essential for RNAi.
The results thus far show that Ago complexes are enriched in Ago1-containing complexes, and host macrophage Ago1 is critical for optimal intracellular survival of Leishmania.Together these findings prompted us to further study the role of Ago1 in the pathogenesis of Leishmania infection.To this end, we used a comprehensive, unbiased SILAC-based quantitative proteomic analysis of Leishmania-infected cells in an Ago1-deficient condition.

SILAC-based quantitative proteomic analysis of Leishmania-infected macrophages in normal and Ago1-deficient condition
Our approach to comprehensively identify and characterize host macrophage proteins modulated by Leishmania and their dependence on Ago1 involves the gene silencing of host cell Ago1 by siRNAs unique to human Ago1, followed by SILAC-based quantitative LC-MS/MS analysis (outlined in Figure S3A).For SILAC-based quantitative analysis, THP-1 cells were cultured in corresponding SILAC media as shown in Figure S3A.The "light" (L) cell population was transfected with control siRNA (noninfected).The "medium" (M) cell population was transfected with control siRNA and infected with L. donovani.The "heavy" (H) cell Analysis of cytoplasmic and nuclear fractions from non-infected and infected macrophages for the presence of Ago1.population was transfected with Ago1-specific siRNAs and infected with L. donovani.Due to the restricted availability of SILAC isotopes, we elected to pool two unique siRNAs (#2 and #3, as shown above in Figure 1D).As expected and shown in Figure S3B, pooling of unique siRNAs was effective in downregulating host Ago1, and the use of control siRNA did not prevent Leishmaniamediated upregulation of Ago1.After 24 h incubation with Leishmania, cells were lysed, and equal amounts of proteins from "light," "medium" and "heavy" populations were mixed in a 1:1:1 ratio.Proteins were subsequently identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described in "Materials and methods".

Global proteomic quantitation-based identification of Leishmania-modulated host proteins in infected macrophages
In order to identify Leishmania-modulated proteins which are dependent on Ago1 and which may have relevance to Leishmania pathogenesis, we first identified host macrophage proteins that were differentially expressed after Leishmania infection.As shown in Figure S4A, we filtered the initial 3379 proteins identified through LC-MS/MS to retain only high-confidence human proteins, resulting in 2151 proteins.Of these, we considered only those with ratios detected in at least two out of three replicates.We performed one-sample T-tests comparing the log 2 (M/L) ratios to zero, which represents no change in response to Leishmania infection.Of the total 1778 proteins identified, 331 were significantly modulated by Leishmania (Table S1).212 of the differentially expressed proteins were downregulated, while 119 proteins were upregulated in response to Leishmania infection (Figure 3A).For the ease of readers, Leishmania-modulated proteins were also represented in a volcano plot, where the -log 10 (p-value) was plotted against the log 2 (M/L) ratio (Figure 3B).
For in-depth analysis in terms of their biological relevance, we considered the top 30 host proteins ranked by Manhattan distance (as described in "Materials and methods") that were significantly modulated by Leishmania (Table 1).Interestingly, 14/30 have been shown to be implicated in Leishmania infection-related studies, such as Ferritin heavy chain (FTH1), Cystatin-A, Apoptosis-associated specklike protein containing a CARD (PYCARD), CD44 antigen (CD44) and Heterogeneous nuclear ribonucleoprotein U (HNRNPU).Together, our proteomic analysis of differentially expressed proteins in Leishmania-infected host cells exhibits significant overlap with those that have been previously shown to be implicated in Leishmania infection, thus providing added confidence to our quantitative proteomic analysis.

Global identification of Leishmaniamodulated Ago1-dependent host proteins in infected macrophages
Next, we sought to determine which of the differentially expressed proteins in Leishmania-infected macrophages are dependent on host Ago1 by combining two sets of proteins as shown in Figure S4B: (i) proteins that are upregulated in response to Leishmania infection (log 2 M/L > 0), and whose levels revert back closer to non-infected control levels, i.e. are recovered, (log 2 H/M < 0) in response to Ago1 knockdown, and (ii) proteins that are downregulated by Leishmania infection (log 2 M/L < 0) and which

A B
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 Ago1knockdown 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.4A, S4B.The overall importance of these protein modulations and recoveries were ranked by combining the Manhattan scores of Leishmania modulation and Ago1 modulation (as described in "Materials and methods") and listed in descending order in Table S2.The proteins were also classified by GO terms in three categories: biological process, cellular component, and molecular function (Figures 4B-D).
It was of interest to find out how many of the Ago1-dependent Leishmania-modulated proteins have relevance to Leishmania pathogenesis.For this, we searched the literature relevant to Leishmania-macrophage interactions, mainly using PubMed.Of the 71 Leishmania-modulated Ago1-dependent proteins, 20 have previously been reported in Leishmania infection-related studies.
Together, the quantitative proteomics analysis of Leishmaniamodulated proteins in Ago1-deficient host cells reveals that a significant number of Ago1-dependent proteins have potential implications in Leishmania pathogenesis based on previously published studies.How many of the remaining Leishmaniamodulated Ago1-dependent host proteins have a role to play in Leishmania-infection related activities remains to be investigated.

Discussion
It is well established that Leishmania parasites have evolved to acquire the ability to alter the cell biology of their host cells to favor their survival and pathogenesis.Since sncRNAs are involved in post-transcriptional regulation of gene expression via RNAi, these biomolecules can play critical roles in regulating host-pathogen interactions.In this context, several recent studies have clearly shown that sncRNAs play a crucial role in microbial infections (12)(13)(14)23) including protozoan parasite infections such as Leishmania infection (163-165).There is growing evidence that sncRNAs are involved in the outcome of infections.Dysregulation of host miRNA has been associated with attenuated immune response and thus pathogen survival.On the other hand, sncRNAs can be employed by the host to counter parasite survival.Extracellular vesicles seem to facilitate this communication (164).The current study was initiated based on our hypothesis that Leishmania regulates the host cell RNAi process to promote its survival in infected host cells.Since Argonautes are central components of the RNAi pathway, we began by investigating whether Leishmania regulates the two most abundant host cell Argonautes-Ago1 and Ago2-to its advantage.We found that Leishmania infection selectively enhanced macrophage Ago1 expression.Strikingly, enhanced level of Ago1 in infected cells correlated with higher levels of Ago1-containing complexes, suggesting preference for Ago1 to regulate RNAi-mediated host genes regulation.Our finding of upregulated expression of Ago1 and enrichment of Ago1 complexes in Leishmania-infected host cells prompted us to assess the potential involvement of Ago1 in Leishmania survival.For this study, we knocked down Ago1 using specific siRNAs followed by parasite rescue assay.Interestingly, downregulation of host macrophage Ago1 resulted in significant reduction of Leishmania survival in infected cells.Together, these results strongly demonstrate the involvement of Ago1 in promoting Leishmania survival in infected cells.At present, we do not know the mechanism of Leishmania-mediated upregulation of Ago1 in infected cells.This upregulation can occur at any point in the transcription-translation process.This includes an increase in the rate of transcription, stability of mRNA, rate of translation and stability of protein.
Upregulation of Ago1 during intracellular infection is not unique to Leishmania infection, and changes in host RISC composition have also been reported in other infections.For example, increased expressions of Ago1 and Dicer1 were detected upon slow bee paralysis virus (SBPV) infection in bumblebees and linked to 17 differentially expressed miRNAs upon infection (166).Also, depletion of Ago1 and Dicer1 in the mosquito Anopheles gambiae during Plasmodium berghei infection led to a two-fold increase in the number of oocysts (167).In another study, Ago1 and/or 3 were required for optimal resistance to flu virus (168).It has recently been shown in Epstein-Barr virus (EBV)-infected mammalian cells that sncRNAs other than miRNAs were selectively loaded onto Ago1, but not Ago2 (169).Indeed, differences in the affinity of sncRNAs for Argonaute members have been observed in both lower organisms and mammals.In Drosophila, it has been shown that perfectly matched sncRNAs duplexes are loaded onto Ago2, whereas non-perfectly matched sncRNAs sorted to Ago1 (170,171).Similarly, in the RNAi pathway of Caenorhabditis elegans, sncRNA duplexes that perfectly match their target mRNA are preferentially incorporated into Argonaute RNAi defective-1 (RDE-1) and non-perfectly matched sncRNA duplexes are loaded onto Argonaute family members ALG-1 or ALG-2 (172, 173).In contrast, sncRNA-mediated selectivity of Ago proteins in mammals is not well understood.However, there are reports suggesting the existence of Ago selectivity for certain distinct sncRNAs.For example, RNA-sequencing of Ago1-, Ago2-and Ago3-associated miRNAs revealed that some have a bias toward particular Ago proteins (20).Together, these findings strongly suggest the    To further investigate the role of Ago1 in Leishmania infection, we used SILAC-based quantitative LC-MS/MS proteomics analysis of L. donovani-infected Ago1-deficient cells, which revealed differentially expressed proteins in response to Leishmania infection that are dependent on Ago1.As expected, many of the modulated proteins that we identified have been previously shown to be implicated in Leishmania infection-related processes.For example, strongly modulated proteins Ferritin heavy chain (FTH1) and Cystatin A (CSTA) were upregulated and downregulated respectively, in agreement with previous studies (40,41,174).Apoptosis-associated speck-like protein containing a CARD (PYCARD, also known as ASC), which was downregulated upon Leishmania infection in our study, has previously been reported in multiple studies covering a range of Leishmania species and host cell types to be differentially expressed upon Leishmania infection (42-44).
From the validated set of 331 Leishmania-modulated proteins, a subset of 71 Ago1-dependent proteins was identified.Of these, 20 were found in previous studies relevant to leishmaniasis.Of interest, among the Leishmania-upregulated Ago1-dependent proteins, Cathepsin G (CTSG), has been shown to be significantly enriched at the late chronic phase of visceral leishmaniasis in mice (59).Another study reported a remarkable upregulation of CTSG in murine infection with L. donovani (60).Among the proteins downregulated by infection and recovered by Ago1-knockdown, Cathepsin L (CTSL) has been shown to play a crucial role for a Th1-type immune response during L. major infection by processing of soluble Leishmania antigen (SLA) for presentation on MHC class II molecules (51).Further, treatment of L. major-infected mice with CLIK148, a specific inhibitor of CTSL, exacerbated the disease by enhancing an SLA-specific Th2-type response (51).
In addition, it was of interest to analyze the Leishmania-modulated Ago1-dependent proteins in relation to other intracellular pathogen infections.Interestingly, 53 out of the 71 proteins have been shown to be related to the pathogenesis of other intracellular pathogens (Tables 2, 3).For example, host glyoxylate reductase/ hydroxypyruvate reductase and mitochondrial very long-chain specific acyl-CoA dehydrogenase are both suppressed by the protozoan pathogen Trypanosoma cruzi (67).Protein promyelocytic leukemia protein/tripeptide motif protein (PML/TRIM19) has been reported to show activity against several viruses (80).Lassa virus and lymphocytic choriomeningitis virus replicate to higher levels in PMLdeficient cells (81), whereas human foamy virus replication efficiency was found to be decreased upon overexpression of PML (82).Another protein of interest is calreticulin, which is known to be released onto the cell surface as an eat-me signal as a result of infection, such as in Mycobacterium tuberculosis and cytomegalovirus infections (90,92).Furthermore, deliberate downregulation of calreticulin significantly increased the intracellular survival of M. tuberculosis (92).
Interestingly, based on the Pathways Commons collection of databases, 31 out of 71 Leishmania-modulated Ago1-knockdownrecovered proteins have reported interaction with Ago1, providing additional confidence in our Ago1-dependent proteins.We also note that 346 out of 417 proteins that were differentially expressed upon Ago1-knockdown were not modulated upon Leishmaniainfection.Nevertheless, the data provided can act as a valuable resource for data mining by other investigators interested in Ago1 functions and its role in the RNAi pathway (Figure S5 and Table S1).We note that our data is restricted to proteomic analysis.It will be of interest to complement these findings with relevant biological assays and may be considered for future studies.
Given that Ago1 knockdown results in broad effects on the host cell proteomic profile, our results generate an attractive hypothesis that Leishmania-mediated upregulation of Ago1 is a strategy to regulate host cell gene expression in its favour.Since Ago1 interacts directly with sncRNAs, it is probable that Leishmania skews host RNAi by selectively uploading sncRNAs onto Ago1, including its own sncRNAs (Figure 5).This model also has support of the following published findings: [1] Leishmania exosomes delivered to the host cytosol contain Leishmania sncRNAs (25); [2] Leishmania infection results in a broad-based downregulation of host macrophage miRNA expression, thus freeing host Agos for exogenous sncRNA loading (24); [3] Ago1 does not require full complementarity of sncRNA to target mRNA, making it favorable for the loading of pathogen sncRNAs (170,171); [4] Unloaded Ago proteins are not stable and are degraded by the proteasome degradation pathway (175,176) and thus, increased Ago1 abundance is presumably complemented with increased loading of sncRNAs onto Ago1 for its increased stability (176).Together, this model adds to increasing evidence of cross-kingdom RNAi in which sncRNAs are transported bi-directionally between the host and their pathogens as a host defense mechanism or strategy of pathogens to regulate host cell biology in their favor (177).
To our knowledge, this is the first report showing the role of host macrophage Ago1 in the pathogenesis of an intracellular parasitic infection.The prior understanding of the role of RNA interference mechanisms in infection is mainly based on plants and insects.These findings represent a framework for further study of the role of RNAi in Leishmania pathogenesis in humans.A deep understanding of crosskingdom RNAi in context of Leishmania infection could provide novel therapeutic strategies for managing and treating leishmaniasis and may have implications for other intracellular pathogens.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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FIGURE 1Ago1 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.
(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 noninfected (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.
(A) Pie chart of the log 2 (fold change) distribution of Leishmania-modulated proteins.M/L normalized ratios were log 2 transformed and averaged for at least 2 replicates.P-values < 0.05 from one-sample T-tests were considered significant.Proteins with a negative log 2 (fold change) were downregulated in Leishmania-infected cells compared to non-infected cells, whereas proteins with a positive log 2 (fold change) were upregulated in Leishmaniainfected cells compared to non-infected cells.(B) Volcano plot comparing protein expression levels in Leishmania-infected cells with those in noninfected 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 -log 10 (p-value) = 1.301.The vertical dashed line represents log 2 (M/L) = 0. L, Light (Scrambled siRNA-treated, Non-infected); M, Medium (Scrambled siRNA-treated, Ld-infected).

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FIGURE 4 Proteomic analysis of Ago1-dependent Leishmania-modulated macrophage proteins.(A) Scatter plot of log 2 transformed M/L ratios (y-axis) vs. log 2 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 log 2 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).