Medicine, Faculty of
Medicine, Department of
DSpace
UBCV
Moradimotlagh, Atieh
Master of Science - MSc
University of British Columbia
Leishmania is a protozoan parasite that causes a wide range of diseases collectively known as leishmaniasis, which differ in symptoms and severity throughout various clinical forms, including cutaneous, mucocutaneous, and visceral. It is well established that Leishmania exploits the cell biology of host macrophages for successful infection and persistence inside the host. Identification and characterization of Leishmania interactions with host cellular proteins/pathways and the functional consequences of these interactions can pave the way to develop novel anti-leishmanial strategies targeted at these interactions. To date, our understanding of the molecular mechanisms associated with the pathogenicity of Leishmania infection is still limited. RNA silencing pathways—collectively referred to as RNA interference—participates in the regulation of various biological processes in most eukaryotic cells. 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 and found Leishmania macrophage Argonaute 1 (Ago1) was selectively and significantly more abundant than that of non-infected control, 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, we employed whole proteome approach which showed that expression of several previously reported Leishmania pathogenesis-related proteins were dependent on the level of macrophage Ago1. Moreover, 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 identify novel macrophage-Leishmania-RNAi related interactions that could potentially be exploited for anti-leishmanial and therapeutic purposes.
Leishmania donovani upregulates host macrophage Argonaute 1 to persist inside infected host; and quantitative proteomic analysis of RNA-Induced Silencing Complex isolated from infected host by Atieh Moradimotlagh BSc, University of Tehran, 2014 MSc, University of Tehran, 2018 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Experimental Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2023 © Atieh Moradimotlagh, 2023 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Leishmania donovani upregulates host macrophage Argonaute 1 to persist inside infected host; and quantitative proteomic analysis of RNA-Induced Silencing Complex isolated from infected host submitted by Atieh Moradimotlagh in partial fulfilment of the requirements for the degree of Master of Science in Experimental Medicine Examining Committee: Dr. Neil Reiner, Professor, Faculty of Medicine, UBC Supervisor Dr. Laura Sly, Professor, Faculty of Medicine, UBC Supervisory Committee Member Dr. Vince Duronio, Professor, Faculty of Medicine, UBC Supervisory Committee Member Dr. Robert McMaster, Professor, Faculty of Medicine, UBC Additional Examiner iii Abstract Leishmania is a protozoan parasite that causes a wide range of diseases collectively known as leishmaniasis, which differ in symptoms and severity throughout various clinical forms, including cutaneous, mucocutaneous, and visceral. It is well established that Leishmania exploits the cell biology of host macrophages for successful infection and persistence inside the host. Identification and characterization of Leishmania interactions with host cellular proteins/pathways and the functional consequences of these interactions can pave the way to develop novel anti-leishmanial strategies targeted at these interactions. To date, our understanding of the molecular mechanisms associated with the pathogenicity of Leishmania infection is still limited. RNA silencing pathways—collectively referred to as RNA interference—participates in the regulation of various biological processes in most eukaryotic cells. 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 and found Leishmania macrophage Argonaute 1 (Ago1) was selectively and significantly more abundant than that of non-infected control, 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, we employed whole proteome approach which showed that expression of several previously reported Leishmania pathogenesis-related proteins were dependent on the level of macrophage Ago1. Moreover, 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 iv results identify novel macrophage-Leishmania-RNAi related interactions that could potentially be exploited for anti-leishmanial and therapeutic purposes. v Lay Summary Leishmaniasis affects approximately 12 million people worldwide. Currently, there are no approved vaccines to prevent leishmaniases, and existing treatments have limited effectiveness with significant side effects. Leishmaniasis is caused by a protozoan parasite belonging to the genus Leishmania. The bite of an infected sandfly is responsible for transmitting Leishmania. Once under the skin, Leishmania are encountered by phagocytes including macrophages responsible for destroying the intruders. Ironically, macrophages are the host for Leishmania which exploits the cell biology of macrophages for successful infection and persistence inside the host. Here, I found two mechanisms Leishmania seems to use to promote its survival inside infected macrophages: (1) Leishmania upregulates host macrophage Argonaute1 protein. Downregulation of host Argonaute 1 resulted in the reduced survival of Leishmania, thus linking Argonaute 1 to Leishmania virulence and (2) Leishmania alters host RNA-Induced Silencing Complex protein composition during infection, suggesting targeting of host RNA-mediated interference. vi Preface This thesis is based on the experimental data presented and discussed in chapter 3 and chapter 4. None of the text of the thesis is taken directly from previously published or collaborative articles. Results presented in chapter 3 and chapter 4 are based on collaborative work performed in the laboratories of Dr. Neil Reiner (Department of Medicine, UBC) and Dr. Leonard Foster (Department of Biochemistry, UBC). I principally designed the project and the experiments, performed the experiments, interpreted the data, and wrote the manuscript in progress based on chapter 3 and chapter 4. Dr. Devki Nandan helped me extensively during the initial part of this study. Dr. Leonard Foster helped with the proteomics analyses, and Jenny Moon in Dr. Foster lab conducted the mass spectrometry experiments and data generation. Dr. Neil Reiner was the supervisory author on this project and participated in conceptual design and manuscript revision and editing. This project was initiated by Dr. Devki Nandan. He designed and performed the initial experiments regarding modulation of Argonaute proteins in Leishmania-infected cells (Figure 3-1). I then took over the project and designed the subsequent experiments and interpreted the data. vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ................................................................................................................................ v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ................................................................................................................................. x List of Figures ............................................................................................................................... xi List of Abbreviations ................................................................................................................. xiii Acknowledgements .................................................................................................................... xvi 1. Chapter 1: Introduction ...................................................................................................... 18 1.1. Leishmaniasis ................................................................................................................. 18 1.1.1. History of the disease ................................................................................................ 18 1.1.2. Epidemiology .............................................................................................................. 3 1.1.3. Life cycle and transmission ........................................................................................ 6 1.1.4. Treatment and vaccination .......................................................................................... 8 1.1.5. Intracellular fate and survival mechanisms of Leishmania inside the macrophage. . 10 1.1.5.1. Attenuation of microbicidal ROS, NO and superoxide release ....................... 11 1.1.5.2. Inhibition of host cell apoptosis by Leishmania .............................................. 11 1.1.5.3. Th1 and Th2 polarization ................................................................................ 12 viii 1.1.5.4. Regulation of host cell signaling pathways. .................................................... 12 1.2. Small non-coding RNAs modulate host-pathogen interactions ..................................... 15 1.3. RNA-Induced Silencing Complex protein components ................................................. 18 1.3.1. Argonaute proteins and their potential role in pathogenesis ..................................... 19 1.4. Thesis rationale and objectives ...................................................................................... 23 2. Chapter 2: Materials and Methods .................................................................................... 25 2.1. THP-1 cell culture and differentiation ........................................................................... 25 2.2. Parasite culture and in vitro infection ............................................................................ 25 2.3. Determination of intracellular parasite burden............................................................... 26 2.4. Parasite rescue and transformation assay ....................................................................... 26 2.5. Western blotting ............................................................................................................. 27 2.6. THP-1 cell culture in SILAC media ............................................................................... 27 2.7. siRNA-mediated knockdown of Ago1 in THP-1 cells................................................... 28 2.8. Expression and purification of recombinant proteins .................................................... 28 2.9. Cell fractionation for nuclear and cytoplasmic preparations from THP-1 cells ............ 29 2.10. GST-T6B affinity chromatography ............................................................................ 30 2.11. Leishmania cell lysate preparation to isolate GST-T6B interacting proteins ............. 30 2.12. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and protein identification.............................................................................................................................. 31 2.1.3. Bioinformatics analysis of LC-MS/MS data ................................................................... 33 ix 3. Chapter 3: To study the potential role of macrophage Argonaute 1 in promoting a pro-leishmania phenotype ................................................................................................................. 34 3.1 Introduction and rationale .............................................................................................. 34 3.2 Results ............................................................................................................................ 35 3.2.2. Using SILAC-based quantitative proteomic approach to study the impact of Ago1 downregulation via siRNA on Leishmania-infected macrophages ........................................... 38 3.2.3. Identification of host proteins modulated by Leishmania in infected macrophages ....... 40 3.2.3 Leishmania-modulated Ago1-dependent host proteins identified in infected macrophages ............................................................................................................................. 47 3. 3 Discussion .......................................................................................................................... 61 4. Chapter 4. To investigate whether Leishmania modulates host RNA-Induced Silencing Complex composition to its advantage ...................................................................................... 65 4.1 Introduction and rationale ................................................................................................... 65 4.1. Results ............................................................................................................................ 66 4.2.1 Isolation and validation of macrophage Argonaute complexes ................................. 66 4.1.2 Ago proteins are primarily found in macrophage cytoplasm ..................................... 68 4.1.3 Proteomic analysis reveals Leishmania-mediated alterations in the protein composition of Argonaute-containing complexes ..................................................................... 71 5. Chapter 5: Concluding remarks and working hypothesis ............................................... 78 5.1 Scientific significance for the field and future perspectives .......................................... 80 Bibliography ................................................................................................................................ 82 x List of Tables Table 1-1. Leishmania species, clinical manifestation, and geographical locations. ..................... 2 Table 3-1. Top 30 L. donovani infection-modulated proteins ...................................................... 44 Table 3-2. Ago1-dependent Leishmania-modulated host proteins ............................................... 49 Table 3-3. Ago1-dependent Leishmania-modulated host proteins with known pathogenic relevance ....................................................................................................................................... 55 Table 4-1. List of Leishmania proteins that interact with TNRC6, along with descriptions of the corresponding genes...................................................................................................................... 75 xi List of Figures Figure 1-1. Endemicity of visceral and cutaneous leishmaniasis in 2021 ...................................... 5 Figure 1-2. The diagram of Leishmania life cycle. ......................................................................... 7 Figure 1-3. Canonical pathway of microRNA biogenesis and gene silencing mechanism. ......... 16 Figure 1-4. A model for loading of small RNAs onto Ago proteins. ........................................... 19 Figure 1-5. Structure and domain organization of Ago 1–4 proteins. .......................................... 21 Figure 3-1. Ago1 is preferentially upregulated by L. donovani infection in the host macrophage, and its knockdown diminishes parasite survival ........................................................................... 37 Figure 3-2. Ago1 was knocked down in macrophages using siRNA to carry out a comparative proteomics analysis using SILAC approach. ................................................................................ 39 Figure 3-3. Differentially expressed host proteins following Leishmania infection. ................... 42 Figure 3-4. Ago1-dependent Leishmania-modulated host proteins. ............................................. 48 Figure 3-5. Distribution of Ago1-knockdown-affected proteins in the presence of Leishmania infection. ....................................................................................................................................... 60 Figure 4-1. Expression and purification of recombinant proteins. ............................................... 67 Figure 4-2. Ago-complex isolation via GST-T6B affinity beads. ................................................ 68 Figure 4-3. Investigating Ago proteins presence in cytoplasmic and ER-free nuclear fractions of non-infected and Leishmania-infected macrophages. ................................................................... 70 Figure 4-4. Comparative proteomics of TNRC6 interactor proteins from cytosolic fraction of non-infected and Leishmania-infected macrophages ........................................................................... 74 Figure 4-5. Leishmania proteins that interact with TNRC6 in the cytosol of infected macrophages........................................................................................................................................................ 76 xii Figure 5-1. The hypothetical model represents how Leishmania modifies the host RISC in favor of its pathogenicity. ....................................................................................................................... 79 xiii List of Abbreviations Ago Argonaute Agos Argonaute proteins Ago1 Argonaute 1 Ago2 Argonaute 2 CL Cutaneous Leishmaniasis CR Complement Receptor CV Column Volume Cyto Cytoplasmic fraction DAPI 4',6-diamidino-2-phenylindole DCL Diffuse Cutaneous Leishmaniasis DMSO Dimethyl Sulfoxide DNA deoxyribonucleic acid dTHP-1 PMA-differentiated THP-1 EDTA Ethylenediaminetetraacetic Acid ER Endoplasmic Reticulum FBS Fetal Bovine Serum HBSS Hanks' balanced salt solution HGP Human Genome Project HSP Heat Shock Protein iNOS Inducible Nitric Oxide Synthase IPTG isopropyl β-D-1-thiogalactopyranoside LCM Localized Cutaneous Leishmaniasis xiv LC-MS/MS Liquid Chromatography- tandem Mass Spectrometry Ld Leishmania donovani lncRNA Long non-coding RNA LPG Lipophosphoglycan MCL Mucocutaneous Leishmaniasis miRNA micro-RNA MOI Multiplicity of Infection mRNA Messenger RNA MS Mass Spectrometry ncRNA Non-coding RNAs NETs Neutrophil extracellular traps NO Nitric Oxide Nuc Nuclear Fraction PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction piRNA piwi-interacting RNA PKC Protein kinase C PKDL Post-kala-azar dermal leishmaniasis PMA phorbol 12-myristate 13-acetate PTP Protein tyrosine phosphatase RISC RNA-Induced Silencing Complex RNA Ribonucleic Acid RNAi RNA interference xv RNI Reactive Nitrogen Intermediate RNS Reactive Nitrogen Species ROI Reactive Oxygen Intermediate ROS Reactive Oxygen Species rRNA Ribosomal RNA SD Standard Deviation SDS Sodium dodecyl sulfate SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis SILAC Stable Isotope Labeling by Amino Acids in Cell Culture siRNA Small (or short) interfering RNA sncRNAs Small non-coding RNA snoRNA Small nucleolar RNA snRNA Small nuclear RNA SSP. Subspecies TBS Tris-buffered saline tiRNA Translation-interfering tRNA tRF Transfer-RNA derived RNA fragment VL Visceral Leishmaniasis WCL Whole Cell Lysate xvi Acknowledgements To commence, I would like to express my gratitude towards Dr. Neil Reiner for providing me with the opportunity to delve into the field of host-pathogen interactions and conduct research in his laboratory. His guidance and support have been indispensable over the years, and I am truly thankful for his mentorship. Additionally, I extend my sincere appreciation to Dr. Devki Nandan for his ongoing assistance with conducting experiments and sharing his knowledge and support throughout the process. I also wish to acknowledge Dr. Laura Sly and Dr. Vince Duronio, my committee members, for their valuable feedback and suggestions, which have greatly helped refine my work. Moreover, I am grateful to Dr. Alice Mui and Dr. Leonard Foster for their expertise and assistance with experimental methods, which have been an invaluable asset during my research. I would like to extend my appreciation to former and current members of Dr. Reiner's lab for their assistance with experiments and insights. Special thanks to Stella Chen, Harsimran Kaur Khara, and Carolina Torturella Rath for their helpful contributions and meaningful conversations, both in and out of the lab. Their friendship has been a great inspiration to me. I am also thankful to the students who have assisted me with my project, particularly Sara, Eleanor, and Fabian. I am deeply grateful for the unwavering love, support, and encouragement that my family and friends have provided me throughout my journey. Their constant presence in my life has made it easier for me to navigate through the joys and challenges of the past years. I extend special thanks to my parents, siblings, and their families, as well as my beloved nephews and niece, xvii Amirali, Sajad, Paria, and Ali, for their support. Additionally, I want to express my heartfelt appreciation to my dear friends who have been an integral part of my life. Their unwavering loyalty and genuine friendship have been the driving force behind my success, and I cannot thank them enough. I feel incredibly blessed to have such incredible people in my life, and I will always cherish the memories we have created together. xviii 1. Chapter 1: Introduction 1.1. Leishmaniasis Leishmaniasis is a tropical and subtropical zoonotic disease caused by an intracellular parasite transmitted to humans by the bite of an infected sandfly, mainly Phlebotomus (in the Old World) and Lutzomyia (in the New World); exceptionally, transmission has also been reported as a laboratory accident (1,2). Leishmania which causes Leishmaniasis, is a vector-borne, obligate intracellular, protozoan parasite which belongs to the phylum Sarcomastigophora, class Zoomastigophorea, order Kinetoplastida, and family Trypanosomatidae, and causes complex disease in human by multiple subspecies with diverse clinical manifestations, referred to cutaneous (CL), mucocutaneous (MCL), and visceral leishmaniasis (VL) (3). The Leishmania species has unique biological and ecological characteristics, a mammalian reservoir, and a sandfly vector (Table1-1). 1.1.1. History of the disease In 1901, parallel discoveries by William Leishman and Charles Donovan led to the identification of the leishmaniasis causative parasite. They independently visualized microscopic single-celled parasites (ovoid bodies later called Leishman-Donovan bodies) living within the in spleen of patients who were suffering from fever, anemia, and splenomegaly (4). Afterwards, the transmission of the Leishmania parasites through the bite of female phlebotomine sandflies was well established in 1942 (5). Leishmania donovani was the first identified Leishmania species taking its name from William Leishman and Charles Donovan in order to honor their academic endeavors (6). Since the initial diagnosis via microscopical identification, medical technological 2 advancement has transformed diagnosis into PCR-based DNA sequencing capable of identifying specific Leishmania species. Table 1-1. Leishmania species, clinical manifestation, and geographical locations. Adapted from Steverding et al (7). Subgenus Species Old/New World Clinical disease Distribution Leishmania L. aethiopica OW LCL, DCL East Africa (Ethiopia, Kenya) L. amazonensis NW LCL, DCL, MCL South America (Brazil, Venezuela, Bolivia) L. donovani OW VL, PKDL Central Africa, South Asia, Middle East, India, China L. infantum (syn. L. chagasi) OW, NW VL, CL Mediterranean countries (North Africa and Europe), Southeast Europe, Middle East, Central Asia, North, Central and South America (Mexico, Venezuela, Brazil, Bolivia) L. major OW CL North and Central Africa, Middle East, Central Asia L. mexicana (syn. L. pifanoi) NW LCL, DCL USA, Ecuador, Venezuela, Peru L. tropica OW LCL, VL North and Central Africa, Middle East, Central Asia, India L. venezuelensis NW LCL Northern South America, Venezuela L. waltoni NW DCL Dominican Republic Viannia L. braziliensis NW LCL, MCL Western Amazon Basin, South America (Guatemala, Venezuela, Brazil, Bolivia, Peru) L. guyanensis NW LCL, MCL Northern South America (French Guinea, Suriname, Brazil, Bolivia) L. lainsoni NW LCL Brazil, Bolivia, Peru L. lindenbergi NW LCL Brazil L. naiffi NW LCL Brazil, French Guinea L. panamensis NW LCL, MCL Central and South America (Panama, Columbia, Venezuela, Brazil) L. peruviana NW LCL, MCL Peru, Bolivia L. shawi NW LCL Brazil Mundinia L. martiniquensis NW, OW LCL, VL Martinique, Thailand 3 1.1.2. Epidemiology Leishmaniasis is a disease with a worldwide distribution; it is found in about 89 countries (8). Even though its effects are primarily seen in developing or undeveloped nations, the Leishmania parasite has spread to Europe and the United States due to traveling to and immigration from these regions. Asia, Africa, the Mediterranean, and the Middle East all have endemic cases of Old World leishmaniasis. The four common species that cause Old World leishmaniasis are Leishmania tropica, Leishmania major, Leishmania aethiopica, and Leishmania donovani. On the other hand, Leishmania mexicana, Leishmania braziliensis, and Leishmania guyanensis are the three species that cause New World leishmaniasis. In total, 90% of leishmaniasis reports come from six Old World nations (Afghanistan, Algeria, Arabian Peninsula, Syria, Sudan, and Iran) and two New World nations (Brazil and Peru) (9). 350 million individuals worldwide are at risk of getting the illness, and there are between 12 and 15 million affected people. An estimated 1.5 to 2 million new cases and 70,000 fatalities are reported annually (10,11). There are three main forms of the disease. Visceral leishmaniasis (VL), also known as kala-azar, is fatal if left untreated in over 95% of cases. It is distinguished by irregular fever attacks, weight loss, spleen and liver enlargement, and anemia. Brazil, east Africa, and India account for the majority of instances. An estimated 50,000 to 90,000 new cases of VL occur worldwide annually, with only 25–45% reported to WHO. On the other hand, the most prevalent type of leishmaniasis, classified as cutaneous leishmaniasis (CL), results in skin lesions, primarily ulcers, on exposed body parts. These may cause severe disability or social stigma and leave permanent scars. The Americas, the Mediterranean basin, the Middle East, and central Asia account for about 95% of all CL cases. A total of 600,000 to 1 million new cases are thought to arise annually throughout the world, although only about 200,000 are reported to WHO. In the third form, which 4 is known as mucocutaneous leishmaniasis (MCL), the nose, mouth, and throat's mucous membranes may be destroyed either partially or completely. Brazil, Ethiopia, Peru, Bolivia (the Plurinational State of), and Brazil account for more than 90% of cases of mucocutaneous leishmaniasis. To track the patterns in incidence and the number of imported cases, the WHO Global leishmaniasis Program has begun reporting the number of new endemic cases separately since 2013. The WHO Global leishmaniasis Program received data for 2021 from 51 VL-endemic countries (64%) and 53 CL-endemic countries (59%) as of November 2022. Eight countries, including Brazil, Eritrea, Ethiopia, India, Kenya, Somalia, South Sudan, and Sudan, reported around 89% of all cases of VL worldwide. Afghanistan, Algeria, Brazil, Colombia, Iran (Islamic Republic of), Iraq, Pakistan, Peru, and the Syrian Arab Republic are among the nine countries that reported more than 5000 CL cases in 2021. These nine countries collectively account for 88% of the global recorded CL incidence. 54 cases of visceral leishmaniasis and 340 cases of cutaneous leishmaniasis were reported in 2021 (Figure 1-1). 5 Figure 1-1. Endemicity of visceral and cutaneous leishmaniasis in 2021 Adapted from WHO (https://www.who.int/data/gho/map-gallery-search-results) 6 1.1.3. Life cycle and transmission Leishmania’s complex life cycle alternates between vertebrate and invertebrate hosts and has two distinct developmental stages: extracellular promastigotes and intracellular amastigotes. Promastigotes are the motile, flagellated forms of Leishmania species. They are present inside the sandfly and develop through several morphologically distinct phases of differentiation before becoming the infectious, non-dividing promastigotes known as "metacyclic" promastigotes that are transmitted when a sandfly bites a person. Amastigotes are intracellular parasites that thrive in a variety of mammalian cells, including specialized phagocytes like macrophages. Amastigotes lack an exteriorized flagellum (Figure 1-2). The promastigote morphology in the sandfly and the amastigote morphology in the mammalian host are Leishmania’s two major distinct cell morphologies. However, the fundamental cellular architecture, which is characterized by cross-linked sub-pellicular corset microtubules, is constant between the two types of Leishmania cell morphologies. Cell division depends on the insertion and elongation of microtubules into the preexisting array, which is maintained throughout the cell cycle. The nucleus and other single-copy organelles, including the mitochondrion and the Golgi apparatus, are located within the cell. The kinetoplast, a collection of concatenated mitochondrial DNA, is located anterior to the nucleus and is directly related to the basal body from which the flagellum extends (12–14). At the base of the flagellum is an invagination of the cell membrane forming a vase-like structure called the flagellar pocket, which is important in these parasites as it is the only site of endocytosis and exocytosis and is hence a critical interface between the parasite and its host environment (15). Interestingly, it has been established that sandfly saliva enhances Leishmania infection and immunization with a single salivary protein or exposure to uninfected bites was shown to result in a protective immune response against leishmaniasis (16). 7 Figure 1-2. The diagram of Leishmania life cycle. Leishmania life cycle consists of the following steps: A macrophage bearing Leishmania amastigotes is consumed by a sandfly after it takes a blood meal from an infected mammalian host. The amastigotes change into procyclic promastigotes once they are inside the sandfly midgut. Procyclic promastigotes subsequently transform into nectomonad promastigotes, which exit the peritrophic matrix, connect to the midgut's microvilli, and then go to the thoracic midgut and stomodeal valve, where they differentiate into leptomonad promastigotes. The leptomonad promastigotes separate into two different forms: haptomonad promastigotes, which adhere to the stomodeal valve, and metacyclic promastigotes, which are the mammalian infective form that are transmitted when the sandfly consumes blood again (17). The sandfly regurgitates metacyclic promastigotes during blood feeding along with different salivary and immunomodulatory parasite-derived proteophosphoglycans. After that, one of several potential cell types present in the immediate vicinity phagocytizes the metacyclic promastigotes. Metacyclic promastigotes develop into aflagellate amastigotes after settling inside the cell. Amastigotes replicate inside host cells, and when there are too many of them, the host cells burst, allowing phagocytes in the area to get reinfected. When another sandfly consumes infected phagocytes along with the blood meal, the transmission cycle continues(18). Adapted from Sunter et al (17). 8 1.1.4. Treatment and vaccination Depending on the efficacy of the host immune system, the type of Leishmania affecting the host, as well as how the parasite spreads, there are several treatment options. Host factors such as genetics or immune response, treatment related factors such as dosage, duration, and completion of the therapy, and parasite related factors such as species intrinsic sensitivity or lack of resistance to the medication are crucial determinants regarding to the disease treatment. The Leishmania parasite's long incubation period makes it difficult to identify and apply early treatment. The lack of efficient human vaccines has driven the disease to a crucial stage (19). Some of the often-used medications in standard therapy include pentavalent antimonials, sodium stibogluconate and N-methylglucamine, liposomal amphotericin B, miltefosine, and paramycin (20,21). Conventional amphotericin B has a difficult application process and negative side effects, making liposomal amphotericin B, which is also an antifungal medication, a preferable option in treating CL and VL with less toxicity. Pentavalent antimonials are still utilized in some underdeveloped or developing countries which are not able to afford liposomal amphotericin B therapy. Although pentavalent antimonials are toxic to the kidneys and liver, they are still effective enough for treatment (22). On the other side, the therapy is frequently limited by increased parasite resistance to drug in use (23). In some endemic areas, novel drug combinations have been investigated to reduce treatment toxicity and cost while also halting the development of parasite resistance. It was discovered that nitroquinolines had leishmanicidal properties. Dermaseptin, Andropin, and Cecropin are a few antimicrobial peptides that have been discovered to be beneficial against CL. An oral medication called edelfosine has far more action than miltefosine. Additionally, substances extracted from plants have been found to have antileishmanial action. For instance, the treatment 9 of VL with the polyphenolic flavonoid quercetin showed antileishmanial action (24). Moreover, Azadirachta indica, Eclipta alba, and Piper longum are three plant species that also demonstrated significant antileishmanial activity (25–27). Another cutting-edge strategy is macrophage targeted drug delivery which affects Leishmania parasites that use the macrophages as their mode of infection. Liposomes, microspheres, nanoparticles, and carbon nanotubes are some of the several drug carriers being researched to target macrophages as getting into them is complicated (28). Macrophage-specific expressed receptors can be exploited for active drug delivery to the infected cell (29). Regarding disease prevention, significant obstacles still exist in developing an effective human vaccine that will permanently eradicate leishmaniasis. Different vaccinations are, however, now being examined in mouse models. One of them stimulates the host immune system by using parasites that are "dead but metabolically active". Compared to mice infected with live parasites, mice treated with "dead but metabolically active" L. infantum chagasi did not exhibit organomegaly or parasite presence six months after infection, yet still triggered a protective host immune response against the parasite that is comparable to the reaction triggered by live Leishmania (30). Taken together, there is no approved vaccine against leishmaniasis, and available drugs are costly and have severe side effects. Truly, novel approaches and paradigms are needed to advance drug and vaccine development. 10 1.1.5. Intracellular fate and survival mechanisms of Leishmania inside the macrophage. Following Leishmania entry to the mammalian host via infected sandfly bite, the parasite can infect multiple cell types, including macrophages, neutrophils, and dendritic cells (DCs). However, mononuclear phagocytes, macrophages and DCs are the most central to Leishmania replication and long-term survival (31). In order to enable promastigotes to reach macrophages without invoking the host innate antimicrobial defenses, neutrophils may serve as "Trojan horses" (32). Leishmania ssp. contain an assortment of invasive/evasive and pathoantigenic factors, known as virulence factors, that play crucial roles in infecting macrophages and lead to an effective intracellular parasitism in the parasitophorous vacuoles (PV). Virulence factors allow the parasite to pre-adapt to the mammalian host's elevated temperature and lowered pH inside the macrophages, which are necessary for the parasite's initial establishment at the bite site and for the invasion of macrophages (33). Some of the most studied virulence factors include cysteine proteinases (CPs), such as GP63 metalloprotease and lipophosphoglycan (LPG) molecules found on the surface of the parasite (34,35). CPs are more expressed in the amastigotes form of Leishmania, and they are considered to be a major molecular factor related to Leishmania virulence (33). Serine proteinases (SPs) have also been shown to act as virulence factors during Leishmania infection (34). The main effector cells in charge of eliminating the Leishmania parasites are macrophages. Leishmania are ideally internalized inside phagosomes, which develop into phagolysosomes following to the fusion to lysosomes, and the parasites are digested inside phagolysosomes (36). As discussed above macrophages act as the primary host cells for Leishmania. In fact, macrophages play a paradoxical role. They participate in immunomodulation and antileishmanial 11 activity; on the other hand, they act as a replication niche during early phase of infection, and also support long-term chronic survival of Leishmania (37). Leishmania survives and multiplies within the mammalian macrophage in amastigote form. This form is an obligatory intracellular form of the parasite that resides in parasitophorous vacuoles (PV). The pH of PV ranges from 4.7-5.2 and shares features of late endosomes/lysosomes. In addition, after host macrophage activation, Leishmania is vulnerable to being eliminated by reactive oxygen intermediates (ROI) or reactive nitrogen intermediates (RNI). It is known that ROI and RNI can access the lumen of PV (38). In spite of a highly adverse condition, Leishmania survives, raising many questions regarding survival mechanisms of the pathogen inside the macrophage. During its evolutionary process, Leishmania ssp. have developed smart strategies to not only escape from host defense but also to manipulate them to support their own survival and multiplication. Below we review examples of macrophage cellular functions and properties that are affected by Leishmania. 1.1.5.1. Attenuation of microbicidal ROS, NO and superoxide release Leishmania amastigotes suppress the formation of Reactive Oxygen Species (ROS) within the phagolysosomes by promoting heme degradation (39) and preventing NADPH oxidase from assembling at the phagosome membrane (40). Thus, they interfere with either the production or action of host-derived ROI. The synthesis of Nitric oxide (NO) by the inducible isoform of NO synthase is the most important mechanism known in the resolution of Leishmania ssp. in murine models (41). Additionally, hand, host cells infected with Leishmania have attenuated ability to induce NO production in response to IFN-gamma (42,43). 1.1.5.2. Inhibition of host cell apoptosis by Leishmania It is known that Leishmania inhibits host cell apoptosis to keep the cell alive longer (44). Programmed cell death-1 (PD-1) downregulation (45), induction of cytokine signaling 12 (SOCS) protein suppressors (46), and regulation of the glycogen synthase kinase 3β (GSK-3β)/β-catenin/Forkhead box protein O1 (FOXO-1) axis (47), are different mechanisms described for apoptosis inhibition by Leishmania. 1.1.5.3. Th1 and Th2 polarization The activation state of macrophages depends on optimal concentrations of stimulatory and inhibitory cytokines. The Th1 immune response plays a critical role in protecting the host against primary infection (48,49). A protective immune response against Cutaneous leishmaniasis, due to L. major, L. mexicana, or L. amazonensis, as well as Visceral leishmaniasis caused by L. donovani or L. infantum, relies on pro-inflammatory T cell profile (50). CD4+ Th1 cells induce macrophage-driven proinflammatory response which secrete TNF-α, IL-1β, IL-6, IL-12, IL-18, and IL-23 cytokines, induce ROS generation, and enhance phagocytosis (51,52); together, they are very effective in generating protective immunity against Leishmania (53). On the other hand, cytokines such as TGF-beta, IL-10 and IL-4 inhibit Th1 response. Moreover, these cytokines are involved in deactivation of macrophages (54). Leishmania has acquired the ability to upregulate IL-10 in infected macrophages (55). Leishmania also increases the production of TGF-beta in infected cells (54,56). In addition to upregulating the production of immunosuppressive cytokines, Leishmania infect macrophages without inducing the production of proinflammatory cytokines. 1.1.5.4. Regulation of host cell signaling pathways. Several independent findings suggest Leishmania ssp. have evolved mechanisms to cause deactivation of macrophage through effects on host cell signaling. Leishmania infection induces an imbalance of protein kinase levels (inhibited) relative to protein phosphatases activities (activated) in macrophages, resulting in a dramatic hypophosphorylation of macrophage proteins and inhibition of several macrophage functions, including phagocytosis and cytokine production, 13 which are essential for the host immune response against Leishmania (57). In L. donovani-infected macrophages, the host cell protein tyrosine phosphatase (PTP) SHP-1 exhibits increased activity, which is correlated with reduced response to pro-inflammatory cytokines, decrease in nitric oxide production, and prolonged survival of intracellular parasites (58). This increase in SHP-1 activity is associated with impaired IFN-γ -triggered Janus kinase 2 (JAK2) activation (59). A defect in JAK/signal transducers and activators of transcription (STAT) signaling explain the unresponsiveness of infected cells to IFN-γ. A more recent article suggests that the activation of SHP-1 prevents IFN-γ dependent nitric oxide production also through inactivation of extracellular signal-regulated kinase 1/2 (ERK1/2) and prevention of nuclear translocation of nuclear factor-κB (NF-κB) and activator protein 1 (AP-1) (60). Other PTPs, such as PTP1B and TCPTP, as well as the mitogen-activated protein kinase (MAPK)-directed phosphatases MKP1 and MPK3, have been identified as being engaged in the pathogenesis of Leishmania (61,62). Taken together, these findings provide clear evidence that Leishmania attenuates host cell signaling by the activation of host phosphatases. Some of the earliest studies in the field demonstrated that Leishmania attenuated oxidative burst by inhibiting protein kinase C (PKC) activity, which seemingly involves Leishmania surface molecule lipophosphoglycan (LPG) (63). Leishmania also attenuates PKC-dependent depolymerization of periphagosomal F-actin, which is necessary for phagosome maturation (64,65). Recently, PI3K/Akt pathway has been demonstrated to have a role in Leishmania pathogenesis. According to Ruhland et al, L. major, L. pifanoi, and L. amazonensis infection of RAW264.7 macrophages results in a prompt and pronounced Akt phosphorylation that inhibits host cell apoptosis (66). Moreover, L. amazonensis' activation of PI3K results in the inhibition of 14 IL-12 production (67), whereas L. donovani's IL-10 production induction is mediated by PI3K-dependent inactivation of GSK-3β (68). Modulation of these signaling pathways induced by Leishmania in the infected macrophage results in the inhibition of the induction of inflammatory mediators (TNF-α, IL-12), the induction of IL-10 production, the prevention of host cell apoptosis, the impairment of oxidative burst, the suppression of iNOS expression and nitric oxide production, and the downregulation of various genes, such as major histocompatibility complex (MHC)-I and II, required for mounting the adaptive immunity (57). Thus, over the years, we have learned that Leishmania ssp. employ multiple evasion mechanisms to survive inside the hostile environment of the host macrophage. The molecular and functional characterization of Leishmania virulence factors on cell signaling components are likely to produce new insight into pathogenesis. 15 1.2. Small non-coding RNAs modulate host-pathogen interactions Recently, it is becoming clear that host defense mechanisms and microbial strategies are regulated through small non-coding RNA-induced interference in eukaryotes. These small non-coding RNAs are generated through DICER or DICER like endonucleases and loaded onto Ago leading to gene silencing either by degrading complementary mRNA or blocking translation (Figure 1-3). Non-coding RNAs are classified as small non-coding RNAs (sncRNAs) for those that are shorter than 200 nucleotides, and long non-coding RNAs (lncRNAs) for those that are longer (69). Small non-coding RNAs include microRNAs (miRNAs), tRNA-derived fragments (tRFs), small nucleolar RNAs (snorRNAs), small nuclear RNAs (snRNAs), small interfering (siRNAs) and germline-restricted piwi-interacting RNAs (piRNAs) while lncRNAs include circular RNAs (cirRNAs) and antisense, intronic, intergenic and overlapping bidirectional transcripts (70,71). It has been recognized that sncRNAs are crucial for many biological functions and diseases (72–75). For instance, it has been extensively proven that microRNAs (miRNAs) and transfer-RNA derived RNA fragment (tRFs) play a crucial role in microbial infections, cancer, the stress response, and metabolic diseases (76–79). Conversely, in pathogens, these sncRNAs play active role in developing offensive strategies and pathogenesis (80–82). These sncRNAs have attracted considerable attention of researchers in the area of host-pathogen interactions as they are thought to be involved in the cross-kingdom host-microbe interactions (83). These cross-kingdom phenomena include induction of gene silencing between non-related species. Cross-kingdom RNAi involves two organisms, where one acts as donor and initiates gene silencing and the other is tilted as a receptor. For example, as a defense mechanism, host RNAi may mediate the pathogen silencing (84,85). Contrary to this, pathogens also mount offensive strategy involving production of sncRNAs, which move into host genome to initiate host gene silencing (86,87). 16 Figure 1-3. Canonical pathway of microRNA biogenesis and gene silencing mechanism. The miRNA gene is transcribed to generate a primary microRNA (pri-miRNA) precursor molecule that undergoes nuclear cleavage to form a precursor microRNA (pre-miRNA). The pre-miRNA is cleaved in the cytoplasm to create a microRNA duplex. The duplex unwinds and the mature miRNA assembles into the RISC. Depending on the degree of complementarity between the miRNA and the target mRNA, the miRNA drives gene silencing via mRNA cleavage or via translation repression. Adapted from Winter et al (88). 17 Recently, we made a major advance when we identified a novel, exosome-based protein secretion pathway used by Leishmania. We found that Leishmania secrete exosomes into the extracellular space and that these 30–100 nm vesicles derived from endocytic compartments are capable of immune-modulation leading to a pro-parasitic phenotype (89–91). Of additional interest, our most recent data show that exosomes released from L. donovani and Leishmania braziliensis are enriched in a conserved subset of small non-coding RNAs (sncRNAs), with particular enrichment of fragments derived from rRNAs and tRNAs which are efficiently delivered to host macrophages (92). The finding that Leishmania exosomes carry a select and biased repertoire of sncRNA cargo that is conserved across old and new world Leishmania species is of special interest. This is particularly so since—as discussed above—progress in the identification and functional analysis of sncRNAs—including pathogen sncRNAs—has begun to reveal previously unappreciated roles for these molecules in pathogen cell regulation. While such a role for parasite-derived L-sncRNAs is of considerable interest in its own right, our most recent data suggest that host sncRNAs may also be linked to the pathobiology of leishmaniasis. We recently found that human monocyte-derived macrophages infected with Leishmania show a striking, broad-based down regulation of host cell miRNA expression (93). Together, these findings provide a basis for the premise that the biology of sncRNAs—both host and pathogen—may contribute to Leishmania pathogenesis. Leishmania sncRNAs (L-sncRNAs) may act as novel parasite effectors by competing with endogenous host sncRNAs to hijack the host RNAi pathway, leading to altered cell phenotype. 18 1.3. RNA-Induced Silencing Complex protein components The RISC is a multiprotein complex that includes several proteins, such as Agos, GW182/TNRC6 proteins, TRBP, and Hsp90 (Figure 1-4). Ago2 is a crucial contributor to the gene silencing function of RISC due to its cleavage activity. TRBP is a dsRNA binding protein and processes pre-microRNAs into mature miRNAs in miRNA biogenesis (94,95). Hsc70/Hsp90 chaperone machinery mediates ATP-dependent RISC loading of small RNA duplexes (95). The interaction between Ago and GW182 family members is essential for the formation of the minimal RISC. GW182/TNRC6 proteins are present in high molecular weight RISC (HMW-RISC) containing Ago2-bound miRNAs upon mitogenic stimulation (96). The Ago-binding domain of GW182 is essential for the interaction between GW182 and Ago proteins. One GW182 protein may stabilize and bind cooperatively to multiple Ago proteins on adjacent miRNA target sites. Comprehensive identification of nuclear and cytoplasmic localization signals shows that GW182 belongs to an evolutionarily conserved protein family restricted to metazoans, and it can shuttle between the nucleus and cytoplasm via the NLS and NES in the central region (97). The GW182 protein family consists of two conserved domains, N-terminal and C-terminal domains. The N-terminal domain contains a GW-rich region, which is responsible for binding to miRNA-associated factors such as DDX6 and RCK/p54 and the C-terminal domain contains a UBA (ubiquitin-associated) domain, a Q-rich region, and an RRM (RNA recognition motif) domain (96). 19 A B Figure 1-4. A model for loading small RNAs onto Ago proteins. (A) Dicer binds dsRNAs/pre-miRNAs and cleaves both strands using its RNase III domains and generates a shorter dsRNA (~21 nucleotides). The dsRNA then separates from Dicer and reassociates with it at a different position. Meanwhile, TRBP senses the asymmetry and positions the RNA in an orientation, allowing correct strand selection and loading. Afterwards, the dsRNA is transferred to a bound Ago protein that is kept in an open conformation by HSP90. Cleavage-competent Ago proteins cleave the passenger strand (black arrow) and facilitates strand displacement. (B) Unloaded Ago are held in open conformation by HSP90 dimer binding along with co-chaperones. After small RNA binding, the passenger strand is removed, HSP90 hydrolyses ATP, and the Ago protein loaded with the single-stranded RNA transitions into a closed conformation and HSP90 and potential co-chaperones leave the complex. Adapted from Meister et al (98). 1.3.1. Argonaute proteins and their potential role in pathogenesis As described earlier, through the cytoplasmic RNAi, double strand RNA is processed by the endonuclease Dicer and loaded onto an Ago proteins. After selecting the guide strand, the newly formed RISC is equipped to silence target genes by one of several mechanisms. Ago proteins are key players in all small-RNA-guided gene-silencing processes that have been identified so far. These proteins are present in a wide range of organisms, including eukaryotes, bacteria, and archaea. Although eukaryotic and prokaryotic Ago proteins have very low sequence homology, their overall architecture and functions are highly conserved. Gene expression 20 regulation through small RNA and thus Ago proteins are critical for many biological processes, including cell proliferation, differentiation, and homeostasis (98–103). Based on sequence similarities, the mammalian Ago protein family has been separated into the Ago and Piwi subfamilies. In human, the Ago subfamily consists of hsAgo1–4 (also known as elF2C1-4) which are expressed in many tissues (104). Ago1-4 are highly conserved with a sequence identity of about 85% and structurally similar, with few non-conserved amino acids in their functional domains. Ago2 has been known to have mRNA slicing activity (105,106), while Ago3 was recently discovered to have slicing activity only when loaded with certain miRNAs, and the slicing activity was dependent on the pairing of the post seed region of the guide RNA as well as on the 5ˊ and 3ˊ flanking regions of the target RNA (107). Ago proteins have four highly conserved domains, which are crucial for their function. These domains include N-terminal domain, the PIWI/Ago/Zwille (PAZ) domain, the Middle (MID) domain, and the P-element-induced whimpy test (PIWI) domain (Figure 1-5). The N- terminal domain drives duplex unwinding during RISC assembly, and it is strictly required for this step, but not for precedent duplex loading or subsequent target cleavage. The PAZ and MID domains, recognize single-stranded 3ˊ and 5ˊ ends of siRNA/miRNA respectively; and the PIWI domain contains a slicer active site leading to target cleavage. The overall structure of Ago proteins is characterized by a two-lobed scaffold, with one lobe composed of the N-PAZ domains and the other consisting of the MID-PIWI domains. These two lobes are connected by Linker 1 (L1) and Linker 2 (L2) and undergo structural rearrangements upon binding to small RNA molecules (107–110). 21 Figure 1-5. Structure and domain organization of Ago 1–4 proteins. The Ago proteins share a similar domain organization. The two N-terminal motifs labeled I and II and the residues of the catalytic tetrad in the PIWI domain D, E, D, H, R, and G are indicated. Also labeled are cluster 2 (CL2) in Ago1 and Ago4, the Ago3-specific insertion (3SI) and the Ago4-specific insertion (4SI). Abbreviation: N: N-terminus; L1: linker domain 1; PAZ: PIWI/Ago/Zwille domain; L2: linker domain 2; MID: MID domain; PIWI: P-element-induced whimpy testes domain; X: Ago1x additional 33 aa; D: aspartate; E: glutamate; H: histidine; R: arginine; G: glycine. Adapted from Müller et al (102). Besides cytoplasmic RNAi, it has been recently discovered that Ago2 and RNAi factors Dicer, TRBP, and TRNC6A/GW182 are present in the human nucleus and associate together in multiprotein complexes. Consequently, small RNAs can silence nuclear RNA and guide site-specific cleavage of the targeted RNA (111,112). Ago1 is also found in nucleus, and it has been reported to interact with RNA Polymerase Ⅱ directly and act as a transcriptional regulator in gene expression. RNA-mediated gene repression/activation can be mediated by different mechanisms inducing histone modifications and change of chromatin conformation, blocking the binding of repressors, and promoting the binding of activators like transcription enhancers. Ago1 also functions in constitutive and alternative splicing (111,113–116). Research has shown that many sncRNAs require Ago proteins to be loaded onto RISC to guide the Ago proteins to their mRNA targets. By using deep-sequencing techniques to analyze 22 human Ago-associated small RNAs, it was discovered that Ago proteins associate with RNA fragments of various origins apart from miRNAs, including small nucleolar RNAs, vault RNAs, and tRFs, to carry out their gene silencing function (117). It is therefore likely that sncRNAs compete to load onto Ago proteins so they can conduct their function in post-transcriptional gene regulation via RNA silencing pathways. Interestingly, researchers have indicated that in Epstein-Barr virus (EBV)-infected cells, novel sncRNAs—distinct from miRNAs—are specifically loaded onto mammalian Ago1 rather than Ago2. This observation was made by comparing deep-sequenced RNAs obtained from immunoprecipitations of Ago1 and Ago2 (118). This strongly suggests the existence of distinct Ago sorting mechanisms that direct discrete small ncRNA onto specific Ago-containing RISCs. A similar Ago sorting mechanism that directs distinct sncRNA duplexes onto specific Ago-containing RISCs has been shown in Drosophila. In fact, It was revealed that Drosophila small RNAs are sorted between Ago1 and Ago2 according to their duplex structure and the identity of their first nucleotide (119). Considering that Leishmania exosomes are rich in specific sncRNAs, such as tRFs and rRNA fragments, and are transported into the cytosol of infected macrophages (92), it is reasonable to speculate that Leishmania may employ a specific host macrophage Ago protein to load their sncRNAs. Consequently, the Ago-specific Leishmania sncRNAs may play a vital role in regulating host macrophage biology to its advantage. 23 1.4. Thesis rationale and objectives Our long-term goals have been to identify and characterize mechanisms involved in Leishmania-macrophage interactions that contribute to chronic infection. A previous study from our lab regarding Leishmania exosomes analysis unveiled the presence of Leishmania HSP70 and small non-coding RNAs (especially tRFs and rRNAs) as cargo, which could be delivered to the host macrophage via exosome-mediated delivery (91,92). Moreover, the genome-wide downregulation of host miRNAs at the level of gene transcription following Leishmania infection was discovered and linked to the induced upregulation of host transcription factor c-Myc by Leishmania. In addition, the abundance of c-Myc was correlated to parasite survival (93). Besides ncRNAs as a key player in RNAi, Ago proteins are the other major component of the RISC. It is well established that Ago1, unlike Ago2, does not require full complementarity between mRNA, and that ncRNA to inhibit the target mRNA and ncRNAs loading to Ago1 is not random, but has a preferred pattern (118). All in all, it was of interest to study the role of host RNAi in Leishmania pathogenesis. The first aim was to study the potential role of macrophage Ago1 in promoting a pro-leishmania phenotype. This aim was based on a previous finding in the laboratory that Leishmania infection selectively increased the abundance of Ago1 in host cells, suggested a link between host Ago1 and Leishmania virulence (Nandan et al unpublished data). The studies described here were designed to investigate the mechanisms of involvement of host Ago1 in Leishmania pathogenesis, starting with the question of whether deliberate downregulation of Ago1 would result in enhanced clearance of Leishmania from infected host cells. After obtaining positive results, we focused on the whole proteome level to investigate how Leishmania-induced changes in macrophage protein expression are affected by the level of host Ago1. The technique of choice was quantitative 24 proteome profiling combining SILAC and LC-MS/MS. SILAC (Stable Isotope Labeling with Amino Acids in Cell Culture) is a method used for protein expression analysis by mass spectrometry (LC-MS/MS). It involves in vivo metabolic incorporation of "heavy" 13C- or 15N-labeled amino acids into proteins, followed by LC-MS/MS analysis for accelerated comprehensive identification, characterization, and quantitation of proteins. The second aim was to investigate whether Leishmania modulates host RISC composition to its advantage. This aim was designed after confirming that host Ago1 plays a role in the survival of Leishmania in infected macrophages. Although the level of host Ago2 does not seem to be modulated in Leishmania-infected cells, we cannot rule out the possibility that Leishmania induces changes in the composition of Ago2 complexes and its associated proteins qualitatively and/or quantitatively. In addition, it is also possible that Leishmania alters the composition of Ago1, Ago3 and/or Ago4 complexes and their associated proteins to its advantage. Thus, we investigated the possibility of Leishmania hijacking host Ago complexes in general. This investigation could be important to further understand the role of Ago proteins in Leishmania pathogenesis. To determine the possibility that Leishmania hijacks host Ago complexes, a recently published procedure "Ago protein Affinity Purification by Peptides" or "Ago-APP" was used (120) followed by SILAC-based quantitative proteomic analysis. This affinity column-based procedure allows for the simultaneous isolation of all Agos and associated proteins from a variety of species and cell lines. 25 2. Chapter 2: Materials and Methods 2.1. THP-1 cell culture and differentiation THP-1 cells (human leukemia monocytic cells) were obtained from ATCC (TIB-202TM), and maintained at 37°C, 5% CO2 in RMP1 1640 media (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 10 mm HEPES (MilliporeSigma), 100 U/ml penicillin/streptomycin (MilliporeSigma) and 2 mM L-glutamine (MilliporeSigma). Cells in suspension were routinely passaged every 2–3 days to maintain a density between 0.3 x 106 and 0.8 x 106 cells/ml. For differentiation, THP-1 cells were treated with 10 ng/ml Phorbol 12-myristate 13-acetate (PMA) overnight (16–18 h). Adhered dTHP-1 cells were washed with Hanks' balanced salt solution (HBSS) (MilliporeSigma) and given fresh media without PMA. The cells were rested for 24 hr prior to further treatment. 2.2. Parasite culture and in vitro infection Leishmania donovani Sudan strain 2S was provided by Dr Kwang Poo Chang (Rockefeller University, NY, USA), continuously cultured in the lab, and virulence was maintained by regular passages via Syrian Golden hamster from which fresh amastigotes were purified and transformed in vitro into promastigotes (93). Promastigotes were cultured in M199 medium (MilliporeSigma) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco), 10 mM HEPES (Stemcell), 10 μg/ml folic acid (MilliporeSigma), 6 μg/ml hemin (MilliporeSigma), 2 mM L-Glutamine (Stemcell), 100 U/ml penicillin/streptomycin (MilliporeSigma) 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, day 5 stationary L. donovani promastigotes were used at an MOI of 20:1 unless otherwise stated. 26 2.3. Determination of intracellular parasite burden To determine the infection rate and burden, dTHP-1 cells grown on coverslips were infected with Leishmania donovani for 24 h. Thereafter, infected cells were washed with warm PBS to remove unbound parasites, 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. ImageJ software was used to analyze the captures microscopic photos of the infected cells. 2.4. Parasite rescue and transformation assay dTHP-1 cells 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 (93,121) to release intracellular amastigotes. The rescued Leishmania amastigotes were transformed to promastigotes in M199 media by incubating at 26°C for 48-72 h. Their growth was evaluated by counting transformed promastigotes stained with trypan blue solution (0.4% w/v in PBS) on a hemocytometer. Counts were performed in triplicate for each treatment group. 27 2.5. Western blotting dTHP-1 cells were washed three times with HBSS and resuspended in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM NaF, 1 mM Na3VO4, 1 mM EDTA), supplemented with 1 mM PMSF, 5 μg/ml aprotinin and 5 μg/ml leupeptin. Whole cell lysates were heated at 95C in equal volumes of 4x Laemmli sample loading buffer for 7 min. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad) using a semi-dry transfer apparatus. Transferred proteins were probed with the appropriate antibodies, according to the manufacturer’s instructions: Anti-Ago1 (Cell Signaling 5053), anti-Ago2 (Abcam 2897), anti-GST (Santa Cruz sc-138), anti-GAPDH (Abcam G041), anti-Lamin A/C (Cell Signaling 2032), anti-Calreticulin (Cell Signaling 12238), anti-Calnexin (Cell Signaling 2679), anti-HSC 70 (HSP70) (SantaCruz SC-7298), and anti-Actin (SantaCruz SC-47778). Reactive protein signals were captured on Blue X-ray film (Carestream) using SuperSignal West Pico Plus or Femto Chemiluminescence Substrate (Thermo). Densitometric analysis was performed in ImageJ, and results were normalized to Actin. 2.6. THP-1 cell culture in SILAC media For SILAC-based labeling, RPMI 1640 media without L-glutamine, L-arginine and L-lysine (Caisson labs) was supplemented with 10% heat-inactivated dialyzed fetal bovine serum (Gibco), 2mM 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 13C6-arginine (20.25 mg/l) and 2H4-lysine (37.5 mg/l), and “heavy” RPMI was supplemented with 13C615N4-28 arginine (22.25 mg/l) and 13C615N2-arginine (38.5 mg/l). The prepared media were filtered using Stericup Quick Release-VP Sterile Vacuum Filtration System (MilliporeSigma S2VPU02RE). Fresh THP-1 cells (fewer than 2 passages) were spun down, one third of which were resuspended in the respective SILAC media. THP-1 cells were incubated at 37°C, 5% CO2 and passaged for a minimum of 5 cycles prior to use. Cells were differentiated using 10 ng/ml of PMA for 16-18 hr and then washed three times with HBSS and rested for 24 hr in fresh supplemented SILAC media prior to infection. 2.7. siRNA-mediated knockdown of Ago1 in THP-1 cells Three oligoribonucleotides targeting human Ago1, as well as non-specific control (scrambled) siRNA, were purchased from OriGene (SR308857). THP-1 cells were transfected with either non-specific or Ago1 siRNA (50 pmol/well) using HiPerFect transfection reagent (Qiagen) according to the manufacturer’s instructions. After 52 hr of transfection, the cells were differentiated with 10 ng/ml PMA and infected with L. donovani (MOI 20:1) for 24 hr before preparation of whole cell lysates for Western blotting or controlled lysis for parasite rescue. 2.8. Expression and purification of recombinant proteins For the expression and purification of required recombinant proteins we used a previously published protocol with minor modifications (120). Briefly, GST and GST-T6B plasmid constructs were transformed in DH5 alpha E. coli and then expressed in BL21. Subsequently, 2 L of isopropyl β-D-1-thio-galactopyranoside (IPTG)–induced culture (OD600 = 0.6) was grown overnight at 18°C. The cells were harvested by centrifugation at 4,400 × g for 30 min at 4°C and the resulting pellet was then resuspended in resuspension buffer (PBS containing 1 mM PMSF and 29 1 mM DTT) supplemented with 1 mg/ml lysozyme. After sonication for 4 cycles of 30 sec (Microson ultrasonic cell disruptor/Heat Systems Ultrasonics inc.), 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 (UFC901096). The concentrated fractions were loaded onto a desalting chromatography column (Zeba™ Spin Desalting Columns) that was equilibrated in PBS with 5% (vol/vol) glycerol. Desalted fractions were adjusted to ∼2 mg/ml proteins, aliquoted and stored at −80°C. 2.9. Cell fractionation for nuclear and cytoplasmic preparations from THP-1 cells For the preparation of cytosolic- and endoplasmic reticulum (ER)- free nuclear fractions, we followed a published protocol (111). 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 MgCl2, 0.3% NP-40, 1 mM NaF, 1 mM Na3VO4) 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 cells were centrifuged at 2,300 × g for 20 min at 4°C. The supernatant was kept as the cytoplasmic fraction and the nuclear pellet was washed with PBS, and then heated at 95C in 4x Laemmli loading buffer for Western blotting. The purity of nuclear and cytoplasmic fractions was assessed using antibodies against specific markers for the cytoplasm and nucleus. Potential ER contamination in the nuclear fraction was assessed using antibodies against Calnexin (Cell Signaling #2679) and 30 Calreticulin (Cell Signaling #12238). These ER-free nuclear and cytoplasmic fractions were used for the purification of Ago proteins and associated proteins using GST-T6B affinity beads. 2.10. GST-T6B affinity chromatography To prepare the affinity beads, 50 μl of Glutathione-Agarose were incubated with approximately 100 μg of GST/GST-T6B recombinant proteins for 3 hr 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 l of Glutathione-Agarose beads loaded with 100 µg GST for 1 hr at 4°C with end-over-end rotation. Non-bound materials were then incubated with the GST-T6B affinity beads for 3 hr, 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. 2.11. Leishmania cell lysate preparation to isolate GST-T6B interacting proteins 2×109 stationary-phase Leishmania were collected by centrifugation at 4°C. The pellet was washed twice with HBSS and once with TBS (10 mM Tris-HCl, 0.15M NaCl). Then the pellet was frozen and thawed twice using liquid nitrogen to lyse Leishmania. Subsequently, the pellet was resuspended in 1 ml ice-cold NET lysis buffer (50 mM Tris pH7.5, 150 mM NaCl, 5 mM 31 EDTA, 0.5% NP-40, 10% glycerol, 1mM NaF) supplemented with 1 mM DTT, cOmplete-EDTA-free protease inhibitor cocktail (Roche), 1 mM PMSF, and 5 µg/ml each of aprotinin, leupeptin and pepstatin A (MilliporeSigma), and incubated on ice for 20 min, followed by centrifugation at 23,000 × g, 20 min, 4°C. The supernatant was incubated with GST-T6B affinity beads to isolate interacting proteins as described above in the previous section (2.10). 2.12. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and protein identification For mass spectroscopy analysis of siRNA-mediated Ago1-downregulated macrophages, equal amounts of proteins from each treatment were mixed, reduced, alkylated (122) and run on 10% resolving SDS-PAGE. Proteins in the entire lane were digested in gel with MS-grade trypsin (Promega) (123) and resulting peptides were cleaned on C-18 STop And Go Extraction (STAGE) tips (124) 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 32 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. Acquired data were searched on MaxQuant version 2.1.0.0 (125) against Uniprot’s human sequences (UP000005640), Leishmania donovani BPK282A1, and common contaminant sequences. SILAC labels of medium arginine (13C6), medium lysine (D4), heavy arginine (13C6, 15N4), and heavy lysine (13C6, 15N2) were set for quantitation enabling iBAQ, requantify, and match-between-runs options. The data were filtered for 1% false discovery at protein, peptide and PSM levels. For GST-T6B interacting proteins, samples were pooled, and processed as described above for mass spectroscopy analysis of whole cell lysates. To increase confidence, resulting peptides were run on two different instruments: Bruker Daltonik’s TimsTof Pro and Impact II Qtof, coupled to either to nanoElute (Bruker Daltonik) or easy nLC 1200 (Thermo Scientific) nanoflow liquid chromatography system. Both instruments used Ionopticks’s Aurora series 25 cm x 75 μm C18 1.6 μm analytical column heated to 50°C with either 120 min or 90 min separation of either 750 ng or 600 ng peptides determined by NanoDrop One (Thermo Scientific – A205, scopes). Impact II Qtof analysis used acquisition settings as Kerr et al. described (126). Equivalent settings were used on TimsTof Pro analysis while TIMS mode was disabled. Duplicate MS runs were combined as fractions and searched on MaxQuant version 1.6.17.0 (125) against sequences and parameters as described above. Both proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (127) 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. 33 2.1.3. Bioinformatics 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-MC/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. For the Ago1-knockdown experiments, 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, log2 transformed normalized ratios (fold changes) were used. The one-sample T-test was performed to statistically compare log2 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 -log10(p-value) vs. log2(fold change). The higher the Manhattan score, the more significant the modulation of the protein was considered. For proteins that were both Leishmania-modulated and Ago1-dependent, the sum of their Manhattan scores was used to rank their combined significance. Regarding host TNRC6-interacting proteins, the ones which were identified in at least two of the three replicates were reported. UniProt was used to find biological and molecular functions. The bubble plots to categorize proteins by their function were created on Lucidchart App. 34 3. Chapter 3: To study the potential role of macrophage Argonaute 1 in promoting a pro-leishmania phenotype 3.1 Introduction and rationale During the last decade, progress in the identification and functional analysis of sncRNAs—including pathogen sncRNAs—has begun to reveal their role in host-pathogen interactions. In one older study, sncRNAs from the plant fungal pathogen Botrytis cinera were shown to act as effectors to block the immune systems of Arabidopsis and tomato (86). This involved hijacking the host RNA interference (RNAi) machinery by binding of the pathogen sncRNAs to plant Ago, leading to silencing of host defense genes. In another elegant example, human adenovirus was shown to block the host RNAi pathway by saturating the RISC with virus-associated (VA) sncRNAs (128). These fascinating examples of cross-kingdom RNAi make it clear that pathogen sncRNAs represent a novel virulence paradigm. They also beg the question as to how widespread this phenomenon is in nature and whether these novel effectors are also operative in major human eukaryotic pathogens such as Leishmania. sncRNAs have been described in a number of human protozoan pathogens such as Toxoplasma gondii, Giardia lamblia, Trichomonas vaginalis, Entamoeba histolytica, Trypanosoma brucei, Trypanosoma cruzi and L. braziliensis (129–134). Of special interest, a homogeneous population of tRNA-derived small RNAs (tsRNAs) was described in T. cruzi (135), and these were found to be secreted within extracellular vesicles (136). Although the exact function and mechanism of action of T. cruzi tsRNAs is still unknown, there have been some indications that they may play a role in disease pathogenesis (136,137). It is well established that sncRNAs need to be loaded onto Ago proteins to guide the Ago proteins to their cognate mRNA for gene silencing. It is also known that Ago proteins associate 35 with RNA fragments of various origins apart from miRNAs, including tRFs, to carry out their gene silencing function (117). It is therefore likely that sncRNAs compete to load onto Ago proteins for their function via RNA silencing pathways. Interestingly, in Epstein-Barr virus (EBV)-infected cells, novel sncRNAs, distinct from miRNAs, are selectively loaded onto mammalian Ago1(118), indicating the existence of distinct Ago sorting mechanisms that direct discrete small ncRNA onto specific Ago-containing RISCs. A similar distinct Ago sorting mechanism has been shown in Drosophila. (119). Leishmania exosomes are rich in specific sncRNAs, such as tRFs and rRNA fragments, and are transported into the cytosol of infected host (92), suggesting that Leishmania may employ a specific host macrophage Ago protein to load their sncRNAs and Ago1 could be the preferred choice. Thus, Leishmania sncRNAs may play a vital role in regulating host macrophage biology to promote its survival. 3.2 Results 3.2.1 Leishmania selectively upregulates host macrophage Ago1, and downregulation of Ago1 attenuates parasite survival to survive inside the host. Ago1 and 2 are the most abundant Agos in mammalian cells and have been extensively studied in a variety of biological contexts (101). Therefore, we were interested to know if L. donovani infection affected host macrophage Ago1 and Ago2 protein expression in PMA differentiated THP-1 cells (dTHP-1). PMA (phorbol 12-myristate 13-acetate)-activated THP-1 cells, a human monocytic cell line, have been widely used as a model system to study the interaction between Leishmania parasites and host cells (138–140). PMA treatment induces differentiation of THP-1 cells into a macrophage-like phenotype, which is the primary host cell for Leishmania parasites. 36 Accordingly, in the present study, dTHP-1 cells were infected for 24 hr with stationary-phase L. donovani promastigotes using three different MOIs to see whether Leishmania affects the protein levels of Ago1 and Ago2. Western blotting was used to measure the levels of Ago1 and Ago2 using the corresponding antibodies. As shown in Figure 3-1 A and B, Leishmania selectively increased the abundance of host Ago1 without affecting the level of Ago2 post infection. This interesting observation prompted us to study the potential role of host Ago1 in the pathogenesis of Leishmania infection. We next investigated whether host Ago1 plays a role in promoting the survival of Leishmania inside the infected host. To address this, we downregulated host Ago1 using three unique Ago1-targeting siRNAs. A non-specific scrambled siRNA was used as a negative control (Figure 3-1 C). Ago1-knockdown cells and control cells were infected with Leishmania for 24 hr. 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 3-1 D show that reduced Ago1 level correlated with decreased survival of Leishmania. In parallel, downregulation of Ago1 was confirmed by immunoblotting using Ago1 antibody. The efficiency of siRNA-mediated Ago1 knockdown is shown in Figure 3-1 C. In fact, treatment of siRNAs 2 and 3 resulted in nearly complete downregulation of Ago1. Taken together, these results indicate that host Ago1 is critical for optimal intracellular survival of Leishmania. 37 A B C D Figure 3-1. Ago1 is preferentially upregulated by L. donovani infection in the host macrophage, and its knockdown diminishes parasite survival (A) L. donovani (Ld) promastigotes were delivered into dTHP-1 cells at the indicated MOIs. Following a 24 hr Leishmania incubation period, cells were washed with HBSS, and whole cell lysates were prepared. Actin- (internal control) and Ago1-, Ago2-specific antibodies were used to perform an immunoblot analysis. NI denotes for non-infected. (B) The mean and standard deviation of the densitometric assessments of three different experiments are represented by the histogram. (C) In parallel, knock down of Ago1 protein was verified in cells treated with siRNAs. (D) Three distinct Ago1-specific siRNAs were transfected into THP-1 cells in addition to a control scrambled siRNA. After using PMA to differentiate control and Ago1-knockdown cells, they were treated with L. donovani promastigotes for 24 hr. Subsequently, infected cells were washed, internalized parasites were released by lysing cells using a low concentration of SDS, and then transferred to the transformation media as explained within "Materials and Methods." The densitometric analyses of three different experiments are represented by their mean and standard deviation (* p-value< 0.05, ** p-value< 0.01, *** p-value< 0.001). 38 3.2.2. Using SILAC-based quantitative proteomic approach to study the impact of Ago1 downregulation via siRNA on Leishmania-infected macrophages The above results presented in Figure 3-1 prompted us to study in depth 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. Our strategy consists of silencing the gene for dTHP-1 Ago1 with siRNAs specific to human Ago1 to comprehensively identify host macrophage proteins affected by Leishmania and their dependence on Ago1. Due to the restricted availability of SILAC isotopes, we elected to pool two unique siRNAs (#2 and #3 as shown above in Figure 3-1 C). As expected, and shown in Figure 3-2 B, pooling of unique siRNAs was effective in downregulating host Ago1 and the use of control siRNA did not prevent Leishmania-mediated upregulation of Ago1. For SILAC-based quantitative analysis, THP-1 cells were cultured in corresponding SILAC media as described in “Material and Methods” and shown in Figure 3-2 A. The “light” (L) cell population was transfected with control siRNA (non-infected). The “medium” (M) cell population was transfected with control siRNA and infected with L. donovani. The “heavy” (H) cell population was transfected with Ago1-specific siRNAs and infected with L. donovani. After 24 hr incubation, 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). 39 A B Figure 3-2. Ago1 was knocked down in macrophages using siRNA to carry out a comparative proteomics analysis using SILAC approach. In SILAC-RPMI media with isotopically labelled arginine and lysine, THP-1 cells were cultured. To guarantee high labelling, cells were cultured for at least five passages in the appropriate SILAC media. (A) Schematic representation of the SILAC quantitative proteomic approach. (B) To transfect labelled THP-1 cells, two human Ago1 siRNAs were mixed equally, or the scrambled siRNA was used for control group. PMA was used to differentiate control and Ago1-knockdown cells, and both cell groups were then treated with L. donovani for 24 hr. After washing, whole cell lysates of control and infected cells were obtained for SILAC-based proteomic analysis. In addition, samples were examined using immunoblotting to demonstrate that Ago1 siRNAs reduced Ago1 protein levels while Leishmania-mediated Ago1 induction is preserved in the presence of scrambled siRNA. The fold changes are compared to non-infected, scrambled siRNA-transfected cells. The densitometric analyses of three separate experiments' means and standard deviations are displayed (** p value 0.01). 40 3.2.3. Identification of host proteins modulated by Leishmania in infected macrophages We initially selected host macrophage proteins that were differentially expressed after Leishmania infection in order to shed light on Leishmania-modulated proteins that are dependent on host Ago1, and which may be relevant to Leishmania pathogenesis. As can be observed in Figure 3-3 A, we kept only the 2151 high-confidence human proteins from the initial 3379 proteins identified by LC-MS/MS. Only those modulated proteins that had ratios found in at least two out of three replicates were taken into consideration. We used one-sample T-tests to compare the log2(M/L) ratios to zero, which implies a response to Leishmania infection that has not changed. Leishmania significantly modulated 331 out of the total 1778 proteins. In response to Leishmania infection, 119 proteins were elevated and 212 of the differentially expressed proteins were downregulated (Figure 3-3 B). Leishmania-modulated proteins were also depicted in a volcano plot, where the log2(M/L) ratio and -log10(p-value) were plotted against each other (Figure 3-3 C). We considered the top 30 host proteins listed by Manhattan distance (as mentioned in "Materials and Methods") that were significantly altered by Leishmania for in-depth study in terms of their biological relevance (Table 3-1). It is interesting to note that 14/30 have been previously implicated in leishmaniasis related studies. For instance, the highest ranked modulated protein, Ferritin heavy chain (FTH1) is a protein that is involved in the transport and sequestration of iron within cells. In previous studies, it has been shown to be upregulated at the transcript level by L. amazonensis. This suggests that Leishmania infection may influence the regulation of iron metabolism within host cells, potentially to facilitate its own survival and proliferation. The upregulation of FTH1 in response to L. amazonensis infection may reflect a host cell response to try to limit the availability of iron to the parasite, which is known to require iron for growth and 41 replication. Cystatin-A (CSTA), the most significantly Leishmania-downregulated protein, is a natural cysteine protease inhibitor that plays a role in completely curing murine visceral leishmaniasis when used in synergy with IFN-γ. This is believed to be due to CSTA's ability to promote the synthesis of proinflammatory cytokines and nitric oxide, which can help fight the infection. However, further research is needed to fully understand the mechanisms behind CSTA's potential therapeutic effects in leishmaniasis (141). Thus, it is expected that Leishmania would downregulate Cystatin A if its overexpression put its survival in danger. Our study of the proteins that were affected by Leishmania infection showed a significant overlap with previous research that has identified proteins involved in Leishmania infection. This overlap gives us more confidence in the accuracy of our proteomic data. 42 Figure 3-3. Differentially expressed host proteins following Leishmania infection. 43 (A) Schematic diagram showing the data processing steps required to identify host proteins that are altered by Leishmania infection (B) Pie chart representing the distribution of Leishmania-modulated proteins transformed to log2(fold change). M/L normalized ratios underwent log2 transformation and were averaged over a minimum of two replicates. One-sample T-test P-values < 0.05 were regarded as significant. Positive log2 (fold change) proteins were increased in Leishmania-infected cells relative to non-infected cells, whereas negative log2 (fold change) proteins were downregulated in Leishmania-infected cells. (C) A volcano plot comparing the levels of protein expression in Leishmania-infected and non-infected cells that were both transfected with control scrambled siRNA. Green dots indicate proteins that were highly elevated by Leishmania infection, whereas red dots indicate proteins that were downregulated. Based on Manhattan distance, the top 30 modulated proteins are tagged. 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 transfected, Non-infected), M: Medium (Scrambled siRNA transfected, Ld-infected).44 Table 3-1. Top 30 L. donovani infection-modulated proteins Protein names Gene names Uniprot ID Average log2(M/L) P-value log2(M/L) Manhattan distance Uniprot function Known roles related to Leishmania Ferritin heavy chain;Ferritin heavy chain, N-terminally processed;Ferritin FTH1 P02794 0.703 1E-04 4.714 Stores iron in a soluble, non-toxic, readily available form. Important for iron homeostasis. L. amazonensis infection causes iron accumulation and ferritin upregulation in macrophages (142) Endoplasmin HSP90B1 P14625 -0.205 2E-04 3.821 Molecular chaperone that functions in the processing and transport of secreted proteins N/A Cystatin-A;Cystatin-A, N-terminally processed CSTA P01040 -1.737 0.008 3.818 An intracellular thiol proteinase inhibitor. Has an important role in desmosome-mediated cell-cell adhesion in the lower levels of the epidermis. Cystatin cured experimental visceral leishmaniasis by switching the differentiation of Th2 cells to Th1 type, as well as upregulating NO (143); cystatin and IFN-γ inhibited the growth of amastigotes in macrophages (141) Apoptosis-associated speck-like protein containing a CARD PYCARD Q9ULZ3 -0.228 3E-04 3.780 Functions as key mediator in apoptosis and inflammation. L. major infection downregulates PYCARD (144); PYCARD transcript upregulation observed in L. infantum-infected dog glomeruli and tubules (145); PYCARD downregulated in L. amazonensis-infected macrophages, but upregulated in L. major-infected cells (146) CD44 antigen CD44 P16070 0.534 7E-04 3.663 Cell-surface receptor that plays a role in cell-cell interactions, cell adhesion and migration, helping them to sense and respond to changes in the tissue microenvironment Leishmania-encoded migration inhibitory factor (LmMIF) upregulated both CD74 and its associated coreceptor, CD44 (147) Heterogeneous nuclear ribonucleoprotein U HNRNPU Q00839 -0.228 5E-04 3.519 DNA- and RNA-binding protein involved in several cellular processes such as nuclear chromatin organization, telomere-length regulation, transcription, mRNA alternative splicing and stability, Xist-mediated transcriptional silencing and mitotic cell progression HNRNPU was upregulated at the mRNA level by L. donovani amastigotes, while its levels were unchanged by L. donovani promastigotes (148); An increased abundance of proteins involved in RNA splicing (heterogeneous nuclear ribonucleoproteins [hnRNPs]) was observed at 24 hr post-infection with L. donovani (139) Dedicator of cytokinesis protein 10 DOCK10 Q96BY6 0.258 7E-04 3.423 Guanine nucleotide-exchange factor (GEF) that activates CDC42 and RAC1 by exchanging bound GDP for free GTP N/A Thymidine phosphorylase TYMP P19971 -0.834 0.003 3.415 Catalyzes the reversible phosphorolysis of thymidine. L. major (unlike L. mexicana) appears to metabolically incorporate 5F-uridine, probably through a uridine or thymidine phosphorylase (149); The major pathway of thymidine metabolism in L. donovani was cleavage of the deoxyriboside linkage to form thymine, probably via the action of a thymidine phosphorylase (150) Protein PML (TRIM19) PML P29590 -0.205 6E-04 3.410 Functions via its association with PML-nuclear bodies (PML-NBs) in a wide range of important cellular processes, including tumor suppression, transcriptional regulation, apoptosis, DNA damage response, and viral defense mechanisms. N/A 40S ribosomal protein S9 RPS9 P62263 -0.162 6E-04 3.405 Component of the ribosome Upregulated by L. major (151) 40S ribosomal protein S14 RPS14 P62263 -0.388 0.001 3.340 Component of the ribosome N/A Calumenin CALU O43852 -0.378 0.001 3.270 Involved in regulation of vitamin K-dependent carboxylation of multiple N-terminal glutamate residues. Seems to inhibit gamma-carboxylase GGCX. N/A 45 Protein names Gene names Uniprot ID Average log2(M/L) P-value log2(M/L) Manhattan distance Uniprot function Known roles related to Leishmania E3 ubiquitin-protein ligase CBL CBL P22681 0.101 9E-04 3.168 Adapter protein that functions as a negative regulator of many signaling pathways that are triggered by activation of cell surface receptors. Acts as an E3 ubiquitin-protein ligase, which accepts ubiquitin from specific E2 ubiquitin-conjugating enzymes, and then transfers it to substrates promoting their degradation by the proteasome. N/A Calreticulin CALR P27797 -0.212 0.001 3.164 Calcium-binding chaperone that promotes folding, oligomeric assembly and quality control in the endoplasmic reticulum (ER). N/A Actin-related protein 2/3 complex subunit 4 ARPC4;ARPC4-TTLL3 P59998 -0.162 0.001 3.148 Actin-binding component of the Arp2/3 complex, a multiprotein complex that mediates actin polymerization upon stimulation by nucleation-promoting factor (NPF). N/A Ubiquitin-like modifier-activating enzyme ATG7 ATG7 O95352 0.231 0.001 3.143 E1-like activating enzyme involved in the 2 ubiquitin-like systems required for cytoplasm to vacuole transport (Cvt) and autophagy. N/A Protein disulfide-isomerase A4 PDIA4 P13667 -0.129 0.001 3.119 Catalyzes the rearrangement of -S-S- bonds in proteins. Upregulated by L. amazonensis and L. major (152) Costars family protein ABRACL ABRACL Q9P1F3 0.099 0.001 3.085 Plays a role in the regulation of actin filament-based process. Upregulated in the splenic interstitial fluid of malnourished BALB/c mice infected with L. infantum (153) Annexin A6;Annexin ANXA6 P08133 -0.324 0.002 3.018 May associate with CD21. May regulate the release of Ca2+ from intracellular stores. N/A TIP41-like protein TIPRL O75663 0.215 0.002 3.000 May be an allosteric regulator of serine/threonine-protein phosphatase 2A (PP2A). N/A Cathepsin L1;Cathepsin L1 heavy chain;Cathepsin L1 light chain CTSL P07711 -0.559 0.004 2.985 Thiol protease important for the overall degradation of proteins in lysosomes. Plays a critical for normal cellular functions such as general protein turnover, antigen processing and bone remodeling. Plays a role in the processing of soluble Leishmania antigen (SLA) in L. major (154) Heterogeneous nuclear ribonucleoprotein M HNRNPM P52272 -0.083 0.001 2.959 Pre-mRNA binding protein in vivo, binds avidly to poly(G) and poly(U) RNA homopolymers in vitro. Involved in splicing. Host nucleoplasmic HNRNPM increased in abundance in a L. major GP63-dependent manner (155) 40S ribosomal protein SA RPSA P08865 -0.348 0.003 2.941 Required for the assembly and/or stability of the 40S ribosomal subunit. Required for the processing of the 20S rRNA-precursor to mature 18S rRNA in a late step of the maturation of 40S ribosomal subunits. Also functions as a cell surface receptor for laminin. N/A 60 kDa heat shock protein, mitochondrial HSPD1 P10809 -0.168 0.002 2.924 Chaperonin implicated in mitochondrial protein import and macromolecular assembly. Together with Hsp10, facilitates the correct folding of imported proteins. May also prevent misfolding and promote the refolding and proper assembly of unfolded polypeptides generated under stress conditions in the mitochondrial matrix Upregulated in L. amazonensis infection (156) 46 Protein names Gene names Uniprot ID Average log2(M/L) P-value log2(M/L) Manhattan distance Uniprot function Known roles related to Leishmania Cytoplasmic dynein 1 heavy chain 1 DYNC1H1 Q14204 0.074 0.002 2.879 Cytoplasmic dynein 1 acts as a motor for the intracellular retrograde motility of vesicles and organelles along microtubules. N/A Far upstream element-binding protein 1 FUBP1 Q96AE4 -0.273 0.003 2.853 Regulates MYC expression by binding to a single-stranded far-upstream element (FUSE) upstream of the MYC promoter. May act both as activator and repressor of transcription N/A Protein S100-A8;Protein S100-A8, N-terminally processed S100A8 P05109 -0.917 0.012 2.837 S100A8 is a calcium- and zinc-binding protein which plays a prominent role in the regulation of inflammatory processes and immune response. S100A8/S100A9 can act as a biomarker in experimental leishmaniasis for phagocyte activation linked to an effective Th1-response (157); S100A8 transcript was repressed upon L. major infection (158) Epididymal secretory protein E1 NPC2 P61916 -0.334 0.003 2.825 Intracellular cholesterol transporter which acts in concert with NPC1 and plays an important role in the egress of cholesterol from the lysosomal compartment Retinoic acid restores the levels of cellular cholesterol in L. donovani infected macrophages by increasing npc1 and npc2 expressions (159) UDP-N-acetylglucosamine--peptide N-acetylglucosaminyltransferase 110 kDa subunit OGT O15294 0.299 0.003 2.825 Catalyzes the transfer of a single N-acetylglucosamine from UDP-GlcNAc to a serine or threonine residue in cytoplasmic and nuclear proteins resulting in their modification with a beta-linked N-acetylglucosamine (O-GlcNAc) N/A Nucleophosmin NPM1 P06748 -0.089 0.002 2.809 Involved in cellular processes such as ribosome biogenesis, protein chaperoning, histone assembly, cell proliferation, and regulation of tumor suppressors p53/TP53 and ARF. Downregulated in the thymic interstitial fluid of mice infected with L. infantum (160) 47 3.2.3 Leishmania-modulated Ago1-dependent host proteins identified in infected macrophages Since one of our interests was to identify which of the differentially expressed proteins in Leishmania-infected macrophages are dependent on host Ago1, we proceeded by merging two sets of proteins as shown in Figure 3-4 A: (i) proteins that are upregulated in response to Leishmania infection (log2M/L > 0), and whose levels revert back closer to non-infected control levels (log2H/M < 0) in a low-Ago1 condition, and (ii) proteins that are downregulated by Leishmania infection (log2M/L < 0) and which are recovered by Ago1 knockdown (log2H/M > 0), where levels in L. donovani-infected, Ago1 siRNA-transfected cells are represented by H (heavy) and levels in L. donovani-infected, scramble siRNA-transfected cells are represented by M (medium). As a result of statistical analysis, we observed a significant change in the abundance of 97 out of 331 proteins following the knockdown of Ago1 in Leishmania-infected macrophages, as compared to the control infected macrophages with normal levels of Ago1. Out of these 97 proteins, 21 were upregulated and 50 were downregulated which means that the levels of these 71 proteins were recovered by Ago1 knockdown, as demonstrated in Figure 3-4 B. We used the Manhattan scores of Leishmania modulation and Ago1 modulation to rank the significance of the protein modulations and recoveries and presented in a descending manner in Table 3-2. 48 Figure 3-4. Ago1-dependent Leishmania-modulated host proteins. (A) The identification of Ld-modulated host proteins that are dependent on Ago1 (i.e., those that are affected by Ago1 knockdown) is shown schematically. (B) Scatter plot of log2 transformed M/L ratios (y-axis) vs. log2 transformed H/M ratios (x-axis) of proteins. Green proteins are those that, after Ago1 knockdown, recovered to some extent from Leishmania infection-induced upregulation. Red represents proteins that Leishmania infection-induced downregulation and any degree of recovery after Ago1 knockdown. Both log2 transformed M/L and H/M fold changes were considered significant by one-sample T-tests with P-values < 0.05. L: Light (Scrambled siRNA transfected, Non-infected), M: Medium (Scrambled siRNA transfected, Ld-infected), and H: Heavy (Ago1 siRNA transfected, Ld-infected).49 Table 3-2. Ago1-dependent Leishmania-modulated host proteins Protein names Gene names Uniprot ID Avg log2(M/L) P-value log2(M/L) Avg log2(H/M) P-value log2(H/M) M/L Manhattan distance H/M Manhattan distance M/L + H/M Manhattan distance Uniprot Function L. donovani infection-upregulated Ago1-knockdown affected cAMP-dependent protein kinase type II-alpha regulatory subunit PRKAR2A P13861 0.373 0.008 -0.531 0.004 2.467 2.982 5.449 Regulatory subunit of the cAMP-dependent protein kinases involved in cAMP signaling. Type II regulatory chains mediate membrane association by binding to anchoring proteins, including the MAP2 kinase. Dihydropyrimidinase-related protein 2 DPYSL2 Q16555 0.612 0.039 -0.996 0.005 2.021 3.296 5.317 Plays a role in neuronal development and polarity, as well as in axon growth and guidance, neuronal growth cone collapse and cell migration. May play a role in endocytosis. Cathepsin G CTSG P08311 1.211 0.048 -1.248 0.035 2.534 2.705 5.238 Serine protease with trypsin- and chymotrypsin-like specificity ATP-dependent 6-phosphofructokinase, liver type PFKL P17858 0.142 0.004 -0.334 0.006 2.535 2.529 5.064 Catalyzes the phosphorylation of D-fructose 6-phosphate to fructose 1,6-bisphosphate by ATP, the first committing step of glycolysis. Negatively regulates the phagocyte oxidative burst in response to bacterial infection. Septin-9 SEPT9 Q9UHD8 0.242 0.034 -0.204 0.002 1.715 2.821 4.536 Filament-forming cytoskeletal GTPase (By similarity). May play a role in cytokinesis. May play a role in the internalization of 2 intracellular microbial pathogens, Listeria monocytogenes and Shigella flexneri Glyoxylate reductase/hydroxypyruvate reductase GRHPR Q9UBQ7 0.174 0.007 -0.448 0.018 2.299 2.195 4.494 Enzyme with hydroxy-pyruvate reductase, glyoxylate reductase and D-glycerate dehydrogenase enzymatic activities. Prostaglandin G/H synthase 1 PTGS1 P23219 0.625 0.049 -0.554 0.013 1.931 2.442 4.373 Dual cyclooxygenase and peroxidase in the biosynthesis pathway of prostanoids, with a particular role in the inflammatory response. Glia maturation factor gamma GMFG O60234 0.209 0.028 -0.381 0.007 1.755 2.550 4.305 N/A Exosome complex component RRP41 EXOSC4 Q9NPD3 0.297 0.043 -0.699 0.013 1.668 2.573 4.241 Non-catalytic component of the RNA exosome complex which has 3'->5' exoribonuclease activity and participates in a multitude of cellular RNA processing and degradation events. Cytoplasmic aconitate hydratase ACO1 P21399 0.358 0.028 -0.621 0.022 1.903 2.285 4.188 Bifunctional iron sensor that switches between 2 activities depending on iron availability. Gamma-glutamylcyclotransferase GGCT O75223 0.206 0.037 -0.616 0.013 1.637 2.501 4.138 Catalyzes the formation of 5-oxoproline from gamma-glutamyl dipeptides and may play a significant role in glutathione homeostasis. Induces release of cytochrome c from mitochondria with resultant induction of apoptosis. Aconitate hydratase, mitochondrial ACO2 Q99798 0.158 0.022 -0.265 0.014 1.825 2.113 3.938 Catalyzes the isomerization of citrate to isocitrate via cis-aconitate. Phosphatidylinositol-binding clathrin assembly protein PICALM Q13492 0.045 0.010 -0.065 0.016 2.024 1.866 3.890 Cytoplasmic adapter protein that plays a critical role in clathrin-mediated endocytosis which is important in processes such as internalization of cell receptors, synaptic transmission or removal of apoptotic cells. Golgi-associated plant pathogenesis-related protein 1 GLIPR2 Q9H4G4 0.475 0.027 -0.431 0.047 2.038 1.762 3.801 N/A Histidine triad nucleotide-binding protein 2, mitochondrial HINT2 Q9BX68 0.212 0.017 -0.247 0.038 1.988 1.667 3.655 Exhibits adenosine 5'-monophosphoramidase activity. Reticulon-1;Reticulon RTN1 Q16799 0.176 0.046 -0.254 0.018 1.516 1.996 3.512 Inhibits amyloid precursor protein processing, probably by blocking BACE1 activity. 50 Protein names Gene names Uniprot ID Avg log2(M/L) P-value log2(M/L) Avg log2(H/M) P-value log2(H/M) M/L Manhattan distance H/M Manhattan distance M/L + H/M Manhattan distance Uniprot Function NAD(P) transhydrogenase, mitochondrial NNT Q13423 0.098 0.031 -0.327 0.031 1.614 1.831 3.444 Performs transhydrogenation between NADH and NADP, which is coupled to respiration and ATP hydrolysis, and functions as a proton pump across the membrane. Peptidyl-prolyl cis-trans isomerase FKBP8;Peptidyl-prolyl cis-trans isomerase FKBP8 Q14318 0.297 0.022 -0.078 0.049 1.960 1.391 3.351 Constitutively inactive PPiase, which becomes active when bound to calmodulin and calcium. Seems to act as a chaperone for BCL2. Rho GTPase-activating protein 1 ARHGAP1 Q07960 0.132 0.028 -0.124 0.031 1.677 1.634 3.311 GTPase activator for the Rho, Rac and Cdc42 proteins, converting them to the putatively inactive GDP-bound state. Hematopoietic lineage cell-specific protein HCLS1 P14317 0.044 0.040 -0.210 0.031 1.440 1.718 3.157 Substrate of the antigen receptor-coupled tyrosine kinase. Plays a role in antigen receptor signaling for both clonal expansion and deletion in lymphoid cells. Very long-chain specific acyl-CoA dehydrogenase, mitochondrial ACADVL P49748 0.150 0.046 -0.147 0.036 1.490 1.589 3.080 One of the acyl-CoA dehydrogenases that catalyze the first step of mitochondrial fatty acid beta-oxidation, an aerobic process allowing the production of energy from fats L. donovani infection-downregulated Ago1-knockdown affected Thymidine phosphorylase TYMP P19971 -0.834 0.003 1.848 0.000 3.415 5.256 8.671 Catalyzes the reversible phosphorolysis of thymidine. The produced molecules are then utilized as carbon and energy sources or in the rescue of pyrimidine bases for nucleotide synthesis. Protein PML (TRIM19) PML P29590 -0.205 0.001 0.913 0.002 3.410 3.664 7.074 Functions via its association with PML-nuclear bodies in a wide range of cellular processes, including tumor suppression, transcriptional regulation, apoptosis, senescence, DNA damage response, and viral defense mechanisms. Cytosol aminopeptidase LAP3 P28838 -0.131 0.012 0.731 0.000 2.049 4.490 6.540 Cytosolic metallopeptidase that catalyzes the removal of unsubstituted N-terminal hydrophobic amino acids from various peptides. Cathepsin L1;Cathepsin L1 heavy chain;Cathepsin L1 light chain CTSL P07711 -0.559 0.004 0.903 0.010 2.985 2.902 5.887 Thiol protease important for the overall degradation of proteins in lysosomes (Probable). Plays a critical for normal cellular functions such as general protein turnover, antigen processing and bone remodeling. Serpin H1 SERPINH1 P50454 -0.394 0.004 0.570 0.007 2.761 2.739 5.500 Binds specifically to collagen. Could be involved as a chaperone in the biosynthetic pathway of collagen. Protein disulfide-isomerase A6 PDIA6 Q15084 -0.188 0.026 0.214 0.000 1.778 3.611 5.389 May function as a chaperone that inhibits aggregation of misfolded proteins; Negatively regulates the unfolded protein response (UPR) through binding to UPR sensors such as ERN1. Syntenin-1 SDCBP O00560 -0.481 0.005 0.567 0.011 2.805 2.514 5.319 Multifunctional adapter protein involved in diverse array of functions including trafficking of transmembrane proteins, neuro and immunomodulation, exosome biogenesis, and tumorigenesis Calreticulin CALR P27797 -0.212 0.001 0.181 0.012 3.164 2.097 5.261 Calcium-binding chaperone that promotes folding, oligomeric assembly and quality control in the endoplasmic reticulum (ER) via the calreticulin/calnexin cycle. Protein disulfide-isomerase A4 PDIA4 P13667 -0.129 0.001 0.259 0.016 3.119 2.052 5.171 Catalyzes the rearrangement of -S-S- bonds in proteins. Eukaryotic translation initiation factor 3 subunit A EIF3A Q14152 -0.101 0.016 0.305 0.001 1.886 3.272 5.159 RNA-binding component of the eukaryotic translation initiation factor 3 (eIF-3) complex, which is required for several steps in the initiation of protein synthesis. Fructose-bisphosphate aldolase A;Fructose-bisphosphate aldolase ALDOA P04075 -0.160 0.008 0.088 0.002 2.274 2.835 5.109 Catalyzes the reversible conversion of beta-D-fructose 1,6-bisphosphate (FBP) into two triose phosphate and plays a key role in glycolysis and gluconeogenesis. 51 Protein names Gene names Uniprot ID Avg log2(M/L) P-value log2(M/L) Avg log2(H/M) P-value log2(H/M) M/L Manhattan distance H/M Manhattan distance M/L + H/M Manhattan distance Uniprot Function 40S ribosomal protein S4, X isoform RPS4X P62701 -0.223 0.025 0.114 0.001 1.824 3.280 5.104 Component of the ribosome. NAD(P)H dehydrogenase [quinone] 1 NQO1 P15559 -0.429 0.029 0.437 0.003 1.970 3.019 4.988 Flavin-containing quinone reductase that catalyzes two-electron reduction of quinones to hydroquinones using either NADH or NADPH as electron donors. 40S ribosomal protein S3 RPS3 P23396 -0.156 0.007 0.089 0.003 2.298 2.618 4.916 Involved in translation as a component of the 40S small ribosomal subunit. 6-phosphogluconate dehydrogenase, decarboxylating PGD P52209 -0.284 0.044 0.357 0.002 1.639 3.137 4.776 Catalyzes the oxidative decarboxylation of 6-phosphogluconate to ribulose 5-phosphate and CO2, with concomitant reduction of NADP to NADPH. 40S ribosomal protein S13 RPS13 P62277 -0.192 0.004 0.124 0.009 2.557 2.180 4.738 Component of the ribosome. Annexin A6;Annexin ANXA6 P08133 -0.324 0.002 0.218 0.032 3.018 1.714 4.732 May associate with CD21. May regulate the release of Ca2+ from intracellular stores. Glutamine--fructose-6-phosphate aminotransferase [isomerizing] 1 GFPT1 Q06210 -0.144 0.014 0.719 0.011 1.993 2.670 4.664 Controls the flux of glucose into the hexosamine pathway. Most likely involved in regulating the availability of precursors for N- and O-linked glycosylation of proteins. Annexin A2;Annexin;Putative annexin A2-like protein ANXA2;ANXA2P2 P07355 -0.128 0.004 0.240 0.015 2.546 2.063 4.609 Calcium-regulated membrane-binding protein whose affinity for calcium is greatly enhanced by anionic phospholipids. May be involved in heat-stress response. Protein disulfide-isomerase P4HB P07237 -0.160 0.005 0.170 0.011 2.448 2.122 4.569 This multifunctional protein catalyzes the formation, breakage and rearrangement of disulfide bonds. HLA class I histocompatibility antigen, A-2 alpha chain HLA-A P01892 -0.582 0.018 0.607 0.028 2.329 2.167 4.496 Antigen-presenting major histocompatibility complex class I (MHCI) molecule. Proteasome activator complex subunit 2 PSME2 Q9UL46 -0.247 0.005 0.445 0.035 2.512 1.899 4.411 Implicated in immunoproteasome assembly and required for efficient antigen processing. The PA28 activator complex enhances the generation of class I binding peptides by altering the cleavage pattern of the proteasome. 26S proteasome non-ATPase regulatory subunit 7 PSMD7 P51665 -0.154 0.007 0.285 0.016 2.304 2.077 4.381 Component of the 26S proteasome, a multiprotein complex involved in the ATP-dependent degradation of ubiquitinated proteins. Elongation factor 1-alpha 1;Putative elongation factor 1-alpha-like 3;Elongation factor 1-alpha EEF1A1;EEF1A1P5 P68104 -0.381 0.005 0.175 0.032 2.655 1.676 4.331 Promotes the GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes during protein biosynthesis. Plays a role in the positive regulation of IFNG transcription in T-helper 1 cells as part of an IFNG promoter-binding complex with TXK and PARP1 Protein disulfide-isomerase A3 PDIA3 P30101 -0.229 0.034 0.200 0.004 1.693 2.634 4.327 Disulfide isomerase which catalyzes the formation, isomerization, and reduction or oxidation of disulfide bonds. Peptidyl-prolyl cis-trans isomerase B PPIB P23284 -0.226 0.010 0.267 0.017 2.244 2.043 4.287 PPIase that catalyzes the cis-trans isomerization of proline imidic peptide bonds in oligopeptides and may therefore assist protein folding. Heat shock protein HSP 90-beta HSP90AB1 P08238 -0.315 0.006 0.280 0.033 2.514 1.764 4.277 Molecular chaperone that promotes the maturation, structural maintenance and proper regulation of specific target proteins involved for instance in cell cycle control and signal transduction. Peroxiredoxin-1 PRDX1 Q06830 -0.328 0.013 0.400 0.029 2.204 1.942 4.146 Thiol-specific peroxidase that catalyzes the reduction of hydrogen peroxide and organic hydroperoxides to water and alcohols, respectively. Plays a role in cell protection against oxidative stress by detoxifying peroxides and as sensor of hydrogen peroxide-mediated signaling events. Myeloid-derived growth factor MYDGF Q969H8 -0.144 0.027 0.402 0.011 1.712 2.362 4.074 Bone marrow-derived monocyte and paracrine-acting protein that promotes cardiac myocyte survival and adaptive 52 Protein names Gene names Uniprot ID Avg log2(M/L) P-value log2(M/L) Avg log2(H/M) P-value log2(H/M) M/L Manhattan distance H/M Manhattan distance M/L + H/M Manhattan distance Uniprot Function angiogenesis for cardiac protection and/or repair after myocardial infarction (MI). Stimulates endothelial cell proliferation through a MAPK1/3-, STAT3- and CCND1-mediated signaling pathway. Inhibits cardiac myocyte apoptosis in a PI3K/AKT-dependent signaling pathway (By similarity). 60S ribosomal protein L17 RPL17;RPL17-C18orf32 P18621 -0.091 0.028 0.117 0.005 1.652 2.400 4.052 Component of the large ribosomal subunit. Long-chain-fatty-acid--CoA ligase 3 ACSL3 O95573 -0.425 0.033 0.381 0.018 1.908 2.114 4.022 Acyl-CoA synthetases (ACSL) activate long-chain fatty acids for both synthesis of cellular lipids, and degradation via beta-oxidation Pyruvate kinase PKM;Pyruvate kinase PKM P14618 -0.190 0.005 0.122 0.045 2.509 1.464 3.974 Controls the rate of glycolysis. Catalyzes the final rate-limiting step of glycolysis by mediating the transfer of a phosphoryl group from phosphoenolpyruvate (PEP) to ADP, generating ATP Heat shock protein 105 kDa HSPH1 Q92598 -0.239 0.017 0.387 0.028 2.018 1.942 3.960 Acts as a nucleotide-exchange factor (NEF) for chaperone proteins HSPA1A and HSPA1B, promoting the release of ADP from HSPA1A/B thereby triggering client/substrate protein release Translation initiation factor eIF-2B subunit alpha EIF2B1 Q14232 -0.345 0.038 0.379 0.016 1.763 2.182 3.945 Catalyzes the exchange of eukaryotic initiation factor 2-bound GDP for GTP. Elongation factor 2 EEF2 P13639 -0.122 0.040 0.211 0.008 1.524 2.334 3.857 Catalyzes the GTP-dependent ribosomal translocation step during translation elongation. Prelamin-A/C;Lamin-A/C LMNA P02545 -0.083 0.046 0.503 0.012 1.423 2.433 3.857 Components of the nuclear lamina. 40S ribosomal protein S20 RPS20 P60866 -0.199 0.022 0.124 0.015 1.855 1.943 3.798 Component of the ribosome ATP synthase subunit O, mitochondrial ATP5O P48047 -0.097 0.028 0.045 0.010 1.644 2.039 3.683 Mitochondrial membrane ATP synthase (F1F0 ATP synthase or Complex V) produces ATP from ADP in the presence of a proton gradient across the membrane which is generated by electron transport complexes of the respiratory chain. Staphylococcal nuclease domain-containing protein 1 SND1 Q7KZF4 -0.032 0.008 0.127 0.040 2.120 1.526 3.645 Endonuclease that mediates miRNA decay of both protein-free and Ago2-loaded miRNAs 78 kDa glucose-regulated protein HSPA5 P11021 -0.210 0.033 0.272 0.021 1.687 1.948 3.635 Endoplasmic reticulum chaperone that plays a key role in protein folding and quality control in the endoplasmic reticulum lumen Nucleoside diphosphate kinase;Nucleoside diphosphate kinase B NME1-NME2;NME2;NME1 P22392 -0.165 0.042 0.140 0.012 1.545 2.060 3.605 Major role in the synthesis of nucleoside triphosphates other than ATP. Acts as a transcriptional activator of the MYC gene Ribonuclease inhibitor RNH1 P13489 -0.234 0.037 0.160 0.017 1.666 1.917 3.583 Ribonuclease inhibitor which inhibits RNASE1, RNASE2 and ANG. May play a role in redox homeostasis. Aldose reductase AKR1B1 P15121 -0.274 0.034 0.068 0.018 1.746 1.804 3.549 Catalyzes the NADPH-dependent reduction of a wide variety of carbonyl-containing compounds to their corresponding alcohols. 60S ribosomal protein L13 RPL13 P26373 -0.164 0.027 0.175 0.024 1.731 1.803 3.534 Component of the ribosome, a large ribonucleoprotein complex responsible for the synthesis of proteins in the cell 60S ribosomal protein L5 RPL5 P46777 -0.108 0.036 0.221 0.023 1.554 1.851 3.406 Component of the ribosome, a large ribonucleoprotein complex responsible for the synthesis of proteins in the cell. 60S ribosomal protein L10a RPL10A P62906 -0.163 0.034 0.163 0.028 1.637 1.720 3.357 Component of the large ribosomal subunit. 53 Protein names Gene names Uniprot ID Avg log2(M/L) P-value log2(M/L) Avg log2(H/M) P-value log2(H/M) M/L Manhattan distance H/M Manhattan distance M/L + H/M Manhattan distance Uniprot Function T-complex protein 1 subunit theta CCT8 P50990 -0.071 0.022 0.095 0.043 1.723 1.460 3.183 Component of the chaperonin-containing T-complex (TRiC), a molecular chaperone complex that assists the folding of proteins upon ATP hydrolysis Heat shock 70 kDa protein 4 HSPA4 (HSP70) P34932 -0.084 0.039 0.200 0.044 1.497 1.559 3.056 Chaperone-mediated protein complex assembly Elongation factor 1-gamma EEF1G P26641 -0.172 0.043 0.153 0.046 1.534 1.493 3.027 Probably plays a role in anchoring the complex to other cellular components. 60S ribosomal protein L6 RPL6 Q02878 -0.062 0.031 0.150 0.049 1.569 1.457 3.026 Component of the large ribosomal subunit. 54 It was of interest to investigate how many of the Ago1 dependent Leishmania-modulated proteins were relevant to the pathogenesis of Leishmania. To find out, we conducted a literature review for studies related to the interaction between Leishmania and the proteins of interest. Out of the 71 Ago1-dependent proteins that were modulated by Leishmania, 23 were found to have been previously reported in Leishmania infection-related studies (as shown in Table 3-3 and the references therein). Among these 23 proteins, 7 were upregulated by Leishmania (DPYSL2, CTSG, PFKL, SEPT9, PTGS1, ACO1, and ACO2), while 16 were downregulated by Leishmania (CTSL, EIF3A, ALDOA, NQO1, GFPT1, ANXA2, P4HB, HLA-A, HSP90AB1, PRDX1, PKM, HSPH1, LMNA, HSPA5, RPL13 and HSPA4). These discoveries may help clarify how Ago1 influences Leishmania pathogenesis and its underlying mechanisms. Based on previously published findings, our quantitative proteomics analysis of Leishmania-modulated proteins in Ago1-deficient host cells suggests that a considerable number of Ago1-dependent proteins may play a role in Leishmania pathogenesis. 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. How many of the remaining Leishmania-modulated Ago1-dependent host proteins have a role to play in Leishmania-infection related activities remains to be investigated.55 Table 3-3. Ago1-dependent Leishmania-modulated host proteins with known pathogenic relevance Protein names Gene names Uniprot ID Avg log2(M/L) Avg log2(H/M) Ago1 Lit* Roles related to Leishmania Roles related to other pathogens and immunology L. donovani infection-upregulated Ago1-knockdown affected Cathepsin G CTSG P08311 1.211 -1.248 U2AF2 interacts with CTSG, AGO1 and AGO2 Significantly enriched in both vaccinated and non-vaccinated mice at late chronic visceral leishmaniasis phase (161); 33-fold upregulated in murine infection (162) Part of the THBS1 sub-network interactome mobilized in cells by T. cruzi (163); can clear pathogens, regulate inflammation by modifying the chemokines, cytokines, cell surface receptors (164); plays a dichotomous role in modulating levels of MHC Class I molecules (165) ATP-dependent 6-phosphofructokinase, liver type PFKL P17858 0.142 -0.334 N/A Upregulated in BALB/c mouse macrophages upon L. amazonensis infection (166); Transcription was significantly enhanced by L. donovani infection (167) N/A Septin-9 SEPT9 Q9UHD8 0.242 -0.204 RSP17 interacts with SEPT9 and AGO1, AGO2, AGO3 Has residues phosphorylated by leishmania casein kinase 1 (L-CK1.2), which is present in leishmania extracellular vesicles and is essential for parasite survival (168) Septin 9 induces lipid droplets growth by a phosphatidylinositol-5-phosphate and microtubule-dependent mechanism hijacked by HCV (169); Septin-regulated actin dynamics promote Salmonella invasion of host cells (170) Glyoxylate reductase/hydroxypyruvate reductase GRHPR Q9UBQ7 0.174 -0.448 N/A N/A Repressed by protozoan pathogen T. cruzi (171) Prostaglandin G/H synthase 1 PTGS1 P23219 0.625 -0.554 N/A Downregulated more than two-fold in response to L. major infection (172) Overexpression of human prostaglandin G/H synthase-1 and -2 by recombinant vaccinia virus (173) Glia maturation factor gamma GMFG O60234 0.209 -0.381 N/A N/A regulates the intracellular growth of Salmonella via modulation of Ferroportin in murine macrophages (174) Cytoplasmic aconitate hydratase ACO1 P21399 0.358 -0.621 N/A Aco1 is one of the genes related to the response of Leishmania ssp. (175) Mycobacterium tuberculosis-infected Macrophages exhibit increased transcripts for Aco1/Irp1 (176) Aconitate hydratase, mitochondrial ACO2 Q99798 0.158 -0.265 N/A N/A Upregulated in Mycobacterium tuberculosis-infected Macrophages via increased Aco1/Irp1 (176) Phosphatidylinositol-binding clathrin assembly protein PICALM Q13492 0.045 -0.065 N/A N/A Exosomal MicroRNA-155 Inhibits Enterovirus A71 Infection by Targeting PICALM (177) Reticulon-1;Reticulon RTN1 Q16799 0.176 -0.254 N/A N/A Host reticulon proteins play a role in rearranging membranes for positive-strand RNA virus replication (178) NAD(P) transhydrogenase, mitochondrial NNT Q13423 0.098 -0.327 N/A N/A Has vital non-mitochondrial functions in malaria parasite transmission (179) Rho GTPase-activating protein 1 ARHGAP1 Q07960 0.132 -0.124 N/A N/A Host ARHGAP1 identified in Legionella-Containing vacuole within Human Macrophages (180) Hematopoietic lineage cell-specific protein HCLS1 P14317 0.044 -0.210 N/A N/A Upregulated by human cytomegalovirus (181); Of the mitochondrial proteins affected by Mycobacterium tuberculosis infection, HCLS1 is upregulated (182) Very long-chain specific acyl-CoA dehydrogenase, mitochondrial ACADVL P49748 0.150 -0.147 N/A N/A T. cruzi suppresses lipid, fatty acid and steroid metabolism, including related protein ACADVL (171); following low-virulence Porcine reproductive and respiratory syndrome virus infection, ACADVL (FC = 7.26) was upregulated in primary alveolar macrophages (183) L. donovani infection-downregulated Ago1-knockdown affected Thymidine phosphorylase TYMP P19971 -0.834 1.848 N/A N/A Increased in COVID-19 patients in an acuity-dependent manner (184) Protein PML (TRIM19) PML P29590 -0.205 0.913 N/A N/A Has potential activity against several viruses (185); restricts HIV Infection in certain cells types (186); controls Listeria monocytogenes infection (187,188) Cathepsin L1;Cathepsin L1 heavy chain;Cathepsin L1 light chain CTSL P07711 -0.559 0.903 N/A Crucial for a Th1-type immune response during L. major infection, plays a role in the processing of soluble Leishmania antigen (SLA) (154); Treatment with cathepsin Plays a key role in SARS-CoV-2 infection in humans and humanized mice (154); Upregulated in human macrophages upon infection with M. tuberculosis (190); CTSL maturation and activity is impaired in macrophages harboring M. avium and M. tuberculosis (191); CTSL 56 Protein names Gene names Uniprot ID Avg log2(M/L) Avg log2(H/M) Ago1 Lit* Roles related to Leishmania Roles related to other pathogens and immunology L inhibitor potentiates Th2-type immune response in L. major-infected BALB/c mice (189) protects mice from mycoplasmal infection and is essential for airway lymphangiogenesis (192) Calreticulin CALR P27797 -0.212 0.181 CALR interacts with AGO1 N/A Normally functions as a chaperone in the endoplasmic reticulum, but can be released onto the cell surface or secreted, as a result of ER stress, inflammation, or infection (193); modulates the intracellular survival of mycobacteria by regulating ER-stress-mediated apoptosis (194) Protein disulfide-isomerase A4 PDIA4 P13667 -0.129 0.259 N/A N/A The downregulation of PDIA4 expression led to significant reduction of influenza A and B virus replication in cells (195); The structural proteins of coronavirus go through post-translational modifications in the ER, triggering ER stress and inducing the expression of ER chaperones such as PDIA4 (196) Eukaryotic translation initiation factor 3 subunit A EIF3A Q14152 -0.101 0.305 EIF3A interacts with AGO1 Upregulated on a transcriptional level in BMDMs upon infection with L. donovani amastigotes (148) Host eIF3A is essential for HEV translation/replication. Depletion of host translation factors, such as eIF3A, significantly reduced the viral replication (197) Fructose-bisphosphate aldolase A;Fructose-bisphosphate aldolase ALDOA P04075 -0.160 0.088 N/A Upregulated in L. major amastigote-infected bone marrow-derived macrophages (BMDMs) (172,198); Upregulated in BALB/c mouse macrophages upon L. amazonensis infection (166) Inhibition of ALODA blocks biogenesis of ATP and attenuates Japanese encephalitis virus production (199) 40S ribosomal protein S4, X isoform RPS4X P62701 -0.223 0.114 RPS4X interacts with AGO1 N/A In the acute phase of influenza virus infection, there was a decreased expression of host genes involved in translational elongation and protein biosynthesis, such as RPS4X (200) NAD(P)H dehydrogenase [quinone] 1 NQO1 P15559 -0.429 0.437 N/A NRF2, which positively regulates NQO1, was strongly upregulated in infection with Leishmania, whose activation promoted parasite persistence but limited TNF-a production and tissue destruction (201) HIV-1 Rev downregulates Tat expression and viral replication via modulation of NQO1 (202); Dicoumarol, an NQO1 inhibitor, blocks Hepatitis B virus cccDNA transcription by promoting degradation of viral protein HBx (203) 40S ribosomal protein S3 RPS3 P23396 -0.156 0.089 RPS3 interacts with AGO1 N/A RPS3 might be involved in P. aeruginosa pathogenesis (204); RPS3 expression levels may also be important to mouse resistance to the H5N1 influenza virus (205) 6-phosphogluconate dehydrogenase, decarboxylating PGD P52209 -0.284 0.357 N/A N/A Significant up-regulation of the PPP key enzymes in influenza-infected cells, including 6-phosphogluconate dehydrogenase (6PGD), was reported by Janke et al (206) 40S ribosomal protein S13 RPS13 P62277 -0.192 0.124 RPS13 interacts with AGO1 N/A At 1 hpi of Chlamydia infection in epithelial cells, increased chromatin accessibility was associated with a variety of genes involved in the regulation of transcription and translation, including RPS13 (207) Annexin A6;Annexin ANXA6 P08133 -0.324 0.218 N/A N/A Human ANXA6 interacts with influenza A virus (IAV) protein M2 and negatively modulates infection (208); Overexpression of ANXA6 decreased cholesterol levels in the cell membrane, thereby inhibiting IAV replication and propagation. (209); Enterohemorrhagic Escherichia coli effector protein EspF interacts with host protein ANXA6 (210) Glutamine--fructose-6-phosphate aminotransferase [isomerizing] 1 GFPT1 Q06210 -0.144 0.719 N/A L. donovani infection is associated with increased glutamine metabolism, including transient induction of GFPT2 mRNA expression, but note that GPFT1 is not mentioned (211) Adenovirus 5 infection causes increases in transcript levels of hexosamine biosynthesis pathway enzymes, including glutamine fructose-6-phosphate amidotransferase 1 (GFAT1), encoded by the GFPT1 gene (212) Annexin A2;Annexin;Putative annexin A2-like protein ANXA2; ANXA2P2 P07355 -0.128 0.240 ANXA2 interacts with AGO1 In the presence of L. major infection, Two S100 family members were up-regulated (S100A10, S100A11), which interact with annexins A1 and A2, forming a sophisticated Ca2+ sensing system. ANXA2 was downregulated (158) Cytomegalovirus exploits ANXA2 to enhance infection, opposing the protection of cells conferred by A1 or A5 (213); ANXA2 and S100A10 regulate human papillomavirus type 16 entry and intracellular trafficking in human keratinocytes (214); ANXA2 mediates the localization of measles virus matrix protein at the plasma membrane (215) Protein disulfide-isomerase (PDI) P4HB P07237 -0.160 0.170 P4HB interacts with AGO1 Downregulated (0.27 fold change) in L. major-infected human macrophages (158); associates with NADPH oxidase and is required for phagocytosis of L. chagasi promastigotes by macrophages (216) Has many roles in host-pathogen interaction (217); over-expression of PDI increases the fusion of viral membranes, leading to internalization of HIV-1. Cell surface PDI facilitates infection of HeLa cells by mouse polyoma virus. Mediates the entry of cholera toxin (218) 57 Protein names Gene names Uniprot ID Avg log2(M/L) Avg log2(H/M) Ago1 Lit* Roles related to Leishmania Roles related to other pathogens and immunology HLA class I histocompatibility antigen, A-2 alpha chain HLA-A P01892 -0.582 0.607 N/A The total number of HLA class I-restricted self-peptides and source proteins identified from infected THP-1 cells was four-fold lower compared to those identified in non-infected cells, and were heterogeneous and individualized (219) HLA-A cytoplasmic domains are more susceptible to HIV-1 Nef-mediated downregulation than HLA-B cytoplasmic domains. some studies indicate certain HLA-A variants, such as HLA-A*03 and HLA-A*30, are associated with effective antiviral therapy (220) 26S proteasome non-ATPase regulatory subunit 7 PSMD7 P51665 -0.154 0.285 N/A N/A Upon SARS-CoV-2 infection, several proteasomal subunits, including PSMD7, were downregulated (221) Elongation factor 1-alpha 1;Putative elongation factor 1-alpha-like 3;Elongation factor 1-alpha EEF1A1; EEF1A1P5 P68104 -0.381 0.175 EEF1A1 interacts with AGO1 N/A Over expressed eEF1A1 was able to restore HIV-1 reverse transcription efficiency in HEK293T cells with endogenous eEF1A knocked-down, and HIV-1 reverse transcription efficiency correlated with the level of eEF1A1 mRNA (222); eEF1A1 is critical for genome replication of the Paramyxovirus Respiratory Syncytial Virus (223) Protein disulfide-isomerase A3 PDIA3 P30101 -0.229 0.200 PDIA3 interacts with AGO1 N/A The presence of PDIA3 on the cell surface helps different viruses to enter the cells and also helps in replication (224) Peptidyl-prolyl cis-trans isomerase B PPIB P23284 -0.226 0.267 N/A N/A ppiB is essential for proteome homeostasis and virulence in Burkholderia pseudomallei (225) Heat shock protein HSP 90-beta HSP90AB1 P08238 -0.315 0.280 HSP90AB1 interacts with AGO1 Upregulated at the transcriptional level in L. donovani promastigote-infected macrophages (148) HSP90AB1 restores infectivity to HIV with incompletely processed capsid-spacer protein 1 (CA-SP1) (226); Impaired infectivity of ritonavir-resistant HIV is rescued by HSP90AB1 (227) Peroxiredoxin-1 PRDX1 Q06830 -0.328 0.400 PRDX1 interacts with AGO1 Upregulated at the transcriptional level in L. donovani promastigote-infected macrophages (148) Upregulated in response to M. tuberculosis infection(182); PRDX1 contributes to host defenses against M. tuberculosis (228); Cells lacking Prdx-1 showed increased levels of reactive oxygen species formation and a higher level of protein carbonylation in response to Respiratory Syncytial Virus infection (229) 60S ribosomal protein L17 RPL17; RPL17-C18orf32 P18621 -0.091 0.117 RPL17 Interacts with AGO1 N/A N/A Long-chain-fatty-acid--CoA ligase 3 ACSL3 O95573 -0.425 0.381 N/A N/A Required for polio replication (230) Pyruvate kinase PKM;Pyruvate kinase PKM P14618 -0.190 0.122 PKM interacts with AGO1 Significantly upregulated at the transcriptional level upon L. donovani infection in neutrophils (167) Activation of PKM2 in vivo inhibited LPS and Salmonella typhimurium-induced IL-1b production (231) Heat shock protein 105 kDa HSPH1 Q92598 -0.239 0.387 HSPH1 interacts with AGO1 Upregulated in bone marrow-derived macrophages (BMDMs) infected by L. donovani amastigotes (148) Downregulated by Helicobacter pylori (232) Translation initiation factor eIF-2B subunit alpha EIF2B1 Q14232 -0.345 0.379 N/A N/A Sandfly fever Sicilian phlebovirus (SFSV) expresses a protein that strongly binds to eIF2B, in order to evade host PKR-mediated translation inhibition (233) Elongation factor 2 EEF2 P13639 -0.122 0.211 EEF2 interacts with AGO1 N/A eEF2 depletion results in impaired HIV-1 virus production and infectivity (234) Prelamin-A/C;Lamin-A/C LMNA P02545 -0.083 0.503 LMNA interacts with AGO1 Enhances Th1 differentiation and response against L. major (235) Enhances Th1 differentiation and response against vaccinia virus (235) 40S ribosomal protein S20 RPS20 P60866 -0.199 0.124 RPS20 interacts with AGO1 N/A RPS20 ubiquitination is required for poxvirus protein translation (236) ATP synthase subunit O, mitochondrial ATP5O P48047 -0.097 0.045 ATP5O interacts with AGO1 N/A N/A Staphylococcal nuclease domain-containing protein 1 SND1 Q7KZF4 -0.032 0.127 Reported to be a component of RISC (237) N/A SND1 depletion leads to inhibition of Kaposi's sarcoma associated herpesvirus (KSHV) early gene expression (238); SND1 promotes Th1/17 immunity against chlamydial lung infection (239) 78 kDa glucose-regulated protein (heat shock 70kDa protein 5) HSPA5 P11021 -0.210 0.272 HSPA5 interacts with AGO1 Upregulated in macrophages at 24h infection with L. infantum (240) Essential host factor for Ebola virus infection (241); Plays a role in Coxsackievirus A9 infection (242). 58 Protein names Gene names Uniprot ID Avg log2(M/L) Avg log2(H/M) Ago1 Lit* Roles related to Leishmania Roles related to other pathogens and immunology Nucleoside diphosphate kinase;Nucleoside diphosphate kinase B (Ndk) NME1-NME2; NME2; NME1 P22392 -0.165 0.140 NME1 interacts with AGO1 N/A Epstein-Barr virus (EBV), Human Papillomavirus (HPV) and Kaposi’s Sarcoma-associated herpesvirus (KSHV) have been shown to directly interact with human-NME1, resulting in functional suppression of the enzyme (243) Ribonuclease inhibitor RNH1 P13489 -0.234 0.160 N/A N/A Attenuates inflammasome activation and is associated with increased COVID-19 severity (244); Influences HIV-1 reservoirs (245) 60S ribosomal protein L13 RPL13 P26373 -0.164 0.175 RPL13 interacts with AGO1 Among the top 10 downregulated genes in macrophages infected with L. major (246) Participates in the antiviral immune response induced by foot-and-mouth disease virus (FMDV), inhibiting FMDV replication (247); RPL13 is a critical regulator of IRES-driven translation during FMDV infection (248). 60S ribosomal protein L5 RPL5 P46777 -0.108 0.221 RPL5 interacts with AGO1 N/A Potato Spindle Tuber Viroid (PSTVd) modulates its replication through a direct interaction with RPL5, the host splicing regulato (249,250) 60S ribosomal protein L10a RPL10A P62906 -0.163 0.163 RPL10A interacts with AGO1 N/A RPL10a regulates translation from the Hepatitis C virus (HCV) and cricket paralysis virus (CrPV) IRESs (251) T-complex protein 1 subunit theta CCT8 P50990 -0.071 0.095 CCT8 interacts with AGO1 N/A Molecular chaperones that play a role in the folding of actin and tubulin (CCT2-CCT8) were present on Legionella-containing vacuoles (180); The chaperonin CCT8 facilitates spread of tobamovirus infection in plants (252) Heat shock 70 kDa protein 4 (HSP70) HSPA4 P34932 -0.084 0.200 HSPA4 Interacts with AGO1 Hypoxia modulates expression of HSPA4 and reduces Leishmania infection in macrophages (253) Present on the surface of host cells can act as a site of attachment for enterovirus 71 (254); involved in dengue virus entry into host cells (255); can inhibit Influenza A virus replication (256) Elongation factor 1-gamma EEF1G P26641 -0.172 0.153 EEF1G interacts with AGO1 N/A Foot and mouth disease virus (FMDV) may hijack cellular eEF1G to facilitate its own transcription, localization, and translation (257); eEF1G plays a role in the translation of influenza A virus proteins in a strain-specific manner (258); eEF1A and eEF1G subunits are critical reverse transcription complex stability cofactors required for efficient completion of HIV-1 reverse transcription (259) 60S ribosomal protein L6 RPL6 Q02878 -0.062 0.150 RPL6 interacts with AGO1 N/A RPL6 are among the hub genes of the ribosome pathway to be significantly associated with viral acute respiratory infection (ARI) in infants (260); Depleting RPL6 decreases ubiquitin-dependent influenza A virus-encoded peptide presentation by MHC class I (261). *All interaction data are from Pathway Commons Protein-Protein Interactions (https://www.pathwaycommons.org/) 59 After characterizing the Ago1-dependent Leishmania-modulated host proteins, we point out that Leishmania infection had no effect on 346 of the 417 proteins whose expression changed when Ago1 was knocked down. Nevertheless, the supplied data can serve as a useful resource for data mining by additional researchers curious about the roles of Ago1 and its part in the RNAi pathway (Figure 3-5). 60 Figure 3-5. Distribution of Ago1-knockdown-affected proteins in the presence of Leishmania infection. H/M normalized ratios were log2 transformed and averaged for the 2 or 3 replicates. One-sample T-test P values < 0.05 were regarded as significant. Proteins with a negative log2(fold change) were downregulated upon Ago1-knockdown, whereas positive log2(fold change) represents protein upregulation upon Ago1-knockdown in presence of Leishmania infection. 61 3. 3 Discussion It is well established that Leishmania parasites have evolved to be able to alter the cell biology of their host cells to facilitate their survival. In this context, numerous recent studies have firmly demonstrated that the small sncRNAs, such as miRNAs, tRFs, and ribosomal RNA-derived RNA fragments, are essential for microbial infections, cancer, stress response, and metabolic problems (76–79). Moreover, recent work from this laboratory have shown that the exosomes released by L. donovani and L. braziliensis are considerably enriched in sncRNAs like tRFs and ribosomal RNA-derived RNA fragments (92). Additionally, it has been demonstrated that the effects of these secretory exosomes on dendritic cells and monocytes can modify innate and adaptive immune responses (89). Recent findings (92,93) strongly suggest that the biology of sncRNAs from both the host and the pathogen may contribute to Leishmania pathogenesis and call for further study. We began this study presented in this thesis based on our hypothesis that Leishmania modulates the RNAi process in host cells to support its survival and pathogenicity in infected host cells. We started by examining whether Leishmania modulates the two most prevalent host cell Ago proteins—Ago1 and Ago2—to its advantage since Ago proteins are the key elements of the RNAi pathway. In this study, we examined the potential function of Ago proteins during Leishmania infection by comparing the quantitative expression of Ago1 and Ago2, in infected and non-infected cells. We found that Leishmanial infection particularly elevated macrophage Ago1 expression while Ago2 levels did not change. This suggested Leishmania possibly prefers Ago1 over Ago2 for loading of its own sncRNAs to Ago1. The sorting of sncRNAs into Ago1 or Ago2 is regulated by their degree of complementarity to the target mRNA. sncRNAs that are perfectly complementary to the target mRNA are preferentially sorted into Ago2, whereas those with 62 imperfect complementarity are sorted into Ago1. This is because Ago2 has a more stringent requirement for base pairing between the sncRNA and the target mRNA, which ensures specific and efficient cleavage of the target. In contrast, Ago1 can tolerate some mismatches between the sncRNA and the target mRNA, which allows for more flexible regulation of gene expression (119,262). Since Leishmania secretory exosomes are enriched in sncRNAs like tRFs and ribosomal RNA derived RNAs-may not have full complementarity to host mRNAs it is tempting to hypothesize that Leishmania prefers Ago 1 over Ago 2 for loading of its own sncRNAs to regulate host gene expression. After observing a notable increase in Ago1 expression in cells infected with Leishmania, we used siRNAs to knock down Ago1 in order to study the potential role of Ago1 in Leishmania survival. Interestingly, compared to infected cells treated with control scrambled siRNA, siRNA-mediated downregulation of host macrophage Ago1 resulted in a considerable reduction of Leishmania survival in infected cells. These findings taken together clearly imply that Ago1 is involved in facilitating Leishmania survival in infected cells. To the best of our knowledge, upregulation of Ago1 and its seeming role in Leishmania survival are novel findings. However, there have also been reports of changes in the RISC composition of the host in other infections. For example, infected bumblebees with the slow bee paralysis virus (SBPV) showed elevated dicer-1 and Ago1 expression, which was associated with seventeen differentially expressed miRNAs (263). In another study, it has been indicated that mice models need Ago1 and/or Ago3 proteins for optimal resistance to the flu virus, as deleting these proteins increased their vulnerability to infection. This suggests that Ago proteins play a crucial role in the mouse immune system's defense against the flu virus (264). Probably, this is the first finding to describe the part played by the host Ago1 in the pathogenesis of an intracellular parasite infection. 63 Ago1-dependent differentially expressed proteins in response to L. donovani infection were revealed using SILAC-based quantitative LC-MS/MS proteomics investigation of Leishmania-infected Ago1-deficient cells. We compared our data on host proteins altered by Leishmania to previously published host cell proteins affected by Leishmania infection to confirm our methodology. As predicted, it is interesting to note that many of the modulated proteins that we have discovered have been demonstrated to be involved in Leishmania infection-related activities in the prior studies. Numerous of highly modulated proteins in our search, including Ferritin heavy chain (FTH1) and Cystatin A (CSTA), showed up- and down-regulation in conformity with earlier research (41–43) as shown in Table 3-1. We discovered several Leishmania-modulated Ago1-dependent proteins in addition to the proteins mentioned above that have already been connected to Leishmania pathogenesis (Table 3-3). It was interesting to examine these proteins in context of other intracellular pathogen infections. It's interesting to note that 53 out of the 71 Ago1-dependent proteins altered by Leishmania have been attributed to the pathogenesis of other intracellular infections. (Table 3-3). We also compared our data with that of the Pathways Commons database collection (https://www.pathwaycommons.org/), which compiles information on biological pathways and interactions from a number of partner databases, such as BioGRID, SwissProt, NCI Pathway Interaction Database: Pathway, and Innate DB. Surprisingly, 31 of the 71 Leishmania-modulated Ago1-knockdown-recovered proteins interacted with Ago1, bringing credibility to our Ago1-dependent proteins. In some organisms, Ago proteins have also been discovered to act as a transcriptional regulator in gene expression. As a result, it has been demonstrated that nuclear RNAi happens in presence of Ago proteins (111,112,114–116,116). Thus, we investigated the possible presence of 64 Ago proteins in the nuclei of human macrophages. However, using multiple approaches we did not find evidence of the presence of Ago proteins (Ago1 and Ago2) in the nuclei of our model macrophages. However, we can't completely rule out the possibility that our methods may not have been sensitive enough to detect very low amount of Ago proteins. In spite of this, the proteins discovered in this study, which are both Leishmania infection- and/or Ago1-dependent, are promising candidates that consider for additional research into their biological function for a profound understanding of intracellular pathogenesis and the function of Agos in mammalian cells broadly. 65 4. Chapter 4. To investigate whether Leishmania modulates host RNA-Induced Silencing Complex composition to its advantage 4.1 Introduction and rationale On the molecular level, RNAi is executed by a family of ribonucleoprotein complexes called RNA-Induced Silencing Complexes, abbreviated as RISCs. In fact, RISC is a generic term for a family of heterogenous molecular complexes, that can be designed to target almost any gene. The underlying common feature of each complex is a member of the Ago protein family that binds to the small regulatory RNA. Second, in every RISC, the small regulatory RNA functions as a guide that leads RISC to its target through Watson-Crick base pairing with cognate mRNA. Thus, the composition of RISC depends on a particular Ago protein and associated proteins. For functional RISC the minimum requirement is Ago protein, small regulatory RNA and HSP-70 protein. Due to the heterogenous nature of RISC, an exact molecular composition for RISC has never been defined. Results presented in chapter 3 clearly indicate that Leishmania increases the abundance of host Ago1 which plays a role in the survival of Leishmania in infected macrophages. It is tempting to assume that Leishmania is biased towards Ago1 containing RISC. In addition, it is also possible that Leishmania alters the composition of Ago2, Ago3 and/or Ago4 complexes and their associated proteins to its advantage and warrants further investigation. Thus, we investigated the possibility of Leishmania hijacking host Ago complexes in general. For this, a recently published procedure "Ago protein Affinity Purification by Peptides" or "Ago-APP" was used (120). This affinity column-based procedure allows for the simultaneous isolation of all Agos and associated proteins from a variety of species and cell lines. This powerful pull-down assay involves the (GST)-T6B peptide, which is a part of the TNRC/GW182 protein family which efficiently interacts with all Ago proteins (120). In the context of Leishmania pathogenesis, this technique 66 can be used to isolate Ago complexes from Leishmania-infected cells and compare them to Ago complexes from uninfected cells. This comparison can reveal changes in the composition of Ago complexes that may have occurred because of Leishmania hijacking host Ago proteins to its advantage. Thus, the Ago-APP column has the potential to provide important insights into the host RISC composition in response to Leishmania infection. This investigation could be important to further understand the role of Ago proteins in Leishmania pathogenesis and may pave the way for the development of effective therapeutic strategies for the treatment of Leishmaniasis. 4.1. Results 4.2.1 Isolation and validation of macrophage Argonaute complexes To prepare GST-T6B peptide and GST-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 recombinant proteins was evaluated using SDS-PAGE followed by staining with Coomassie Blue R and shown in Figure 4-1 A. Expected molecular mass and reactivity to anti-GST antibodies were used for the identity of recombinant proteins to accurately identify recombinant proteins in the absence of availability of antibodies against the T6B peptide (Figure 4-1 B). 67 Figure 4-1. Expression and purification of recombinant proteins. In E. coli strain BL-21, the expression of the GST-T6B construct and the GST alone construct were induced by IPTG in 2xYT media. Bacteria then were collected, lysed, and sonicated. Centrifugation was used to separate insoluble materials and non-broken bacteria. Supernatant of lysates were then loaded onto Glutathione-Agarose, washed to remove unbound materials, and then released recombinant expressed GST-coupled proteins with reduced glutathione in PBS. Eluted proteins were concentrated and desalted. Protein amounts were measured, aliquoted, and kept at -80 o C. (A) Small aliquots of eluted and concentrated fraction of GST and GST-T6B, and whole cell lysates were examined by running on SDS-PAGE and stained with Coomassie Brilliant Blue R to determine the purity of eluted proteins. (B) Aliquots were also examined simultaneously in an immunoblot assay blot for the presence of anti-GST reactivity. For the validation of affinity chromatography, whole cell lysate from dTHP-1 was incubated with GST alone and GST-T6B affinity beads as illustrated in the flow diagram to confirm that recombinant GST-T6B peptide effectively isolates Ago proteins from macrophages (Figure 4-2 A). After incubation of whole cell lysate with GST alone and GST-T6B, followed by extensive washings, bound materials were released either by PreScission enzyme cleavage or heating in Laemmli sample buffer. The presence of Ago proteins (Ago1 and Ago2) were tested using immunoblotting with appropriate antibodies. As expected, Ago1 and Ago2 were absent from 68 the material eluted from GST alone but were detected in the material eluted from GST-T6B beads, demonstrating the functionality and specificity of GST-T6B beads (Figure 4-2 B, C). Figure 4-2. Ago-complex isolation via GST-T6B affinity beads. (A) Ago-complex affinity purification is shown schematically. Glutathione-Agarose was incubated with around 100 g of GST or GST-T6B purified recombinant proteins for 3 hours at 4°C for each of the GST or GST-T6B affinity beads. The dTHP-1 lysate, which was made in NET buffer with the addition of 0.5 mM DTT and protease inhibitors as mentioned in "Materials and Methods," was then treated with the affinity beads. Through multiple washing, non-bound proteins were taken out and bound proteins on affinity beads were released by the addition of 2x Laemmli sample buffer. (B), (C) By using immunoblotting, the eluted materials were examined for the presence of Ago1 and Ago2. One of three separate experiments' results is represented. 4.1.2 Ago proteins are primarily found in macrophage cytoplasm It was of interest to separate the Ago complexes from the cytoplasm and the nucleus after the GST-T6B peptide affinity column's interaction with Ago proteins from whole cell lysate had been verified. It is reasonably established that most Ago complexes are confined to the cytoplasm to participate in canonical RNAi (98,265). Nevertheless, a few recent data suggest that Ago proteins control transcription and alternative splicing in the nuclei of human cells (148,149). This raises the 69 possibility that Leishmania may alter Ago proteins in the cytoplasm, nucleus, or both. In order to investigate the above possibility, first it was important to find the presence of Ago proteins in the nucleus of our model human macrophages. To do this, we performed subcellular fractionation (Figure 4-3 A), which produced intact nuclei without any presence of endoplasmic reticulum (ER) contamination. Calreticulin, Calnexin, Lamin A/C, and GAPDH antibodies were utilized as markers for the ER lumen, ER membrane, inner nuclear membrane, and cytoplasm, respectively. Lamin A/C was easily and exclusively recognized in the nuclear lysates, as shown in Figure 4-3 B, although Calreticulin, Calnexin, and GAPDH were not present in nuclear fraction. First, a Western blot test was used to check the cytoplasmic and nuclear extracts for the presence of Ago1 and Ago2. Unexpectedly but interestingly, Ago1 and Ago2 could not be detected in the nuclear extract, but both were clearly present in the cytoplasmic fraction (data not shown). The absence of Ago1 and Ago2 in the nuclear fraction could be due to small amounts. In order to enrich the presence of Ago1 and Ago2 in the nucleus, nuclear and cytoplasmic extracts of cells were subjected to GST-T6B affinity purification. As seen in Figure 4-3 C, Ago2 was not detectable in the nuclei of dTHP-1 cells, although Ago1 was barely detectable in the nucleus following prolonged exposure of the blot to the film. In case Leishmania causes the translocation of Ago proteins from the cytoplasm to the nucleus, we also examined dTHP-1 cells that were infected with Leishmania. Once more, nuclear fractions failed to reveal the presence of Ago1 and Ago2 (Figure 4-3 D). Furthermore, we used LC-MS/MS analysis to examine nuclear GST-T6B-interacting Ago complexes. However, this technique was unable to determine whether Ago proteins were present in the nuclear fraction. Taken together, it seems Ago proteins are largely absent in the nucleus of our model host. However, we cannot completely rule out the absence of Ago1 and Ago2 proteins from the nucleus. 70 In the light of these results, we opted to use cytoplasmic fractions from non-infected and Leishmania-infected dTHP-1 cells for further investigation of Agos and associated complexes. Figure 4-3. Investigating Ago proteins presence in cytoplasmic and ER-free nuclear fractions of non-infected and Leishmania-infected macrophages. (A) Schematic diagram showing simplified steps of the cell fractionation (B) As in "Materials and Methods," dTHP-1 cells were fractionated into cytoplasmic and nuclear fractions. Using the designated antibodies, immunoblotting was used to evaluate the purity of the cytoplasmic and the ER-free nuclear fractions. (C) Following that, the cytoplasmic and nuclear extracts of dTHP-1 cells were each incubated separately with GST-T6B affinity beads, and the bound proteins were examined by immunoblotting with Ago1 and Ago2 antibodies. (D) An immunoblot comparing the levels of Ago1 in materials extracted from GST-T6B affinity beads after they were treated with nuclear and cytosolic lysates from dTHP-1 cells that were non-infected with Leishmania (NI) and those that were (Ld). Parallel to this, the levels of GAPDH and Lamin A/C were determined in aliquots from the cytoplasmic or nuclear fraction of NI and Leishmania-infected cells, respectively. The data shown is one representative of three independent experiments. 71 4.1.3 Proteomic analysis reveals Leishmania-mediated alterations in the protein composition of Argonaute-containing complexes In order to determine the composition of RISC in non-infected and Leishmania-infected dTHP-1 cells, again we used SILAC-based quantitative proteomics approach. The main aim of this study was to investigate the possible modulation of cytoplasmic Ago-containing complexes, with focus on their protein components. As described in "Materials and Methods," we performed the GST-T6B affinity pull-down assay followed by SILAC-based quantitative mass spectrometry analysis to comprehensively define the proteomics of Ago-complexes. Leishmania-infected cells were cultured in "Medium" (M) SILAC medium while non-infected cells were maintained in "Light" (L) SILAC medium for analysis. Cells from both Leishmania-infected and control groups that were non-infected were used to prepare cytoplasmic fractions. To identify Ago-complexes and associated proteins, equal amounts of proteins from each fraction were incubated individually with GST-T6B affinity beads. Cytoplasmic fractions were pre-cleared by incubation with GST-alone beads to remove non-specific interactions. PreScission enzyme cleavage released proteins bound to GST-T6B beads. Proteins that have been released were concentrated for LC-MS/MS analysis. With the use of the proteomics analysis, we were able to recognize 160 proteins, including Ago1-3, that were present in GST-T6B associated materials. Even though we were unable to identify Ago4 in our preparations, the presence of the other Ago proteins confirmed that the GST-T6B pull-down assay has the capability to purify complexes that contain Ago proteins. For 53 proteins identified in at least two of three replicates, quantitative ratios contrasting the quantities of protein bound to GST-T6B in Leishmania-infected versus non-infected cells were assessed. The 72 one-sample T-test revealed that Ago-complexes made from non-infected and Leishmania-infected cells differed in the quantity of 17 human proteins (Figure 4-4 A). Amongst these modulated proteins, 11 proteins were downregulated in Ago-complexes obtained from Leishmania-infected cells and 6 proteins were upregulated. The differentially expressed proteins underwent a gene ontology (GO) analysis using the UniProt database. A bubble plot was used to display the data, showing how the macrophage cytosolic T6B-interacting proteins changed as a result of Leishmania infection. "Chaperone," which comprised HSP90B1, GRPEL1, and five proteins from the HSP70 family, was the most common biological process GO term (7/17) (Figure 4-4 B). Furthermore, HSPs, most of which are downregulated, make up the majority of the T6B-interactor proteins that Leishmania significantly modifies (Figure 4-4 C). Following Leishmania infection, it was shown that host proteins involved in RNA interference, protein translation, ATP binding, transferases, oxidases, and host-virus interaction were altered in the RISC (Figure 4-4 B). As expected, some RISC modulated proteins investigated via Ago-App were detected as Leishmania-modulated proteins (described in Chapter 3). HSPA5, PRDX1, EEF1G (presented results in Table 3-2, Table 3-3 and Figure 4-4 A), and UBC are the proteins which were detected in both assays as downregulated proteins. 73 A B 74 C Figure 4-4. Comparative proteomics of TNRC6 interactor proteins from cytosolic fraction of non-infected and Leishmania-infected macrophages Light and medium SILAC media were used to cultivate THP-1 cells. Labeled THP-1 cells were differentiated with PMA and then medium SILAC medium treated cells were infected with L. donovani for 24 hr. Light SILAC medium treated cells considered as non-infected (control). The cytoplasmic fractions from NI and infected cells were next precleared with GST beads alone, followed by an incubation with GST-T6B affinity beads. Mass spectrometry was used to quantitatively compare NI and infected cells by mixing released interacting proteins in a 1:1 ratio (three independent experiments). (A) By using the log2(M/L) ratio, the histogram displays the host proteins that are differentially expressed in cells with Leishmania infection with a p-value <0.2 (B) Modulated proteins with a p-value< 0.2 are shown as a bubble plot according to their molecular and biological functions. (C) The volcano plot represents modified proteins with a p-value < 0.1 threshold of all 53 identified proteins in at least two out of three experiments. * p-value < 0.05. L: Light (Non-infected), M: Medium (Ld-infected). Surprisingly, 10 of the total detected proteins matched to L. donovani (Table 4-1) which were analyzed using the UniProt database and a bubble plot was used to display the data (Figure 4-5 A). We would like to point out that because parasites were not labelled using SILAC medium, the identification of Leishmania proteins as components of Ago-complexes in infected cells is only qualitative. We incubated Leishmania whole cell lysate with GST-T6B affinity beads to rule out the potential that Leishmania proteins could directly interact with GST-T6B, rather than as part of 75 Ago-complexes. In a Western blot assay, bound proteins were examined for the presence of HSP70, the only Leishmania protein for which an antibody is available. There was no HSP70 signal seen in the bound material, as seen in Figure 4-5 B. This strongly suggests that Leishmania HSP70 was separated as a component of Ago-complexes rather than through direct interaction with GST-T6B and implies that it may be integrated into host RISC during Leishmania infection. Table 4-1. List of Leishmania proteins that interact with TNRC6, along with descriptions of the corresponding genes. Protein ID Gene ID Gene description 1 E9BLZ1 13385668 heat shock 70-related protein 1, mitochondrial precursor, putative 2 E9BK18 13387132 heat-shock protein hsp70, putative 3 E9BLG9 13385496 co-chaperone GrpE, putative 4 E9BQG8 13392588 malate dehydrogenase, putative 5 E9BQC4 13392415 small GTP-binding protein Rab18, putative 6 E9BAG2 13389313 hypothetical protein 7 E9BI56 13388877 hypothetical protein 8 E9BD23 13393231 hypothetical protein 9 E9BMZ6 13389645 hypothetical protein 10 E9BBT6 13391409 hypothetical protein 76 A B Figure 4-5. Leishmania proteins that interact with TNRC6 in the cytosol of infected macrophages. (A) Proteins are categorized by their roles and illustrated using a bubble plot. (B) The input material, whole cell lysate of L. donovani, was precleared with GST beads alone and then incubated for three hours with GST-T6B affinity beads. After a couple of washings of the beads to remove non-bound materials, the bound proteins were eluted by adding 2x Laemmli sample buffer and incubating at 95°C for 7 minutes. By immunoblotting, the presence of the HSP70 protein in released proteins was examined. The results shown represents one of three independent experiments. 77 4.3. Discussion After connecting host cell Ago1 to Leishmania pathogenicity, it was of interest to investigate whether Leishmania may affect other host Ago complexes and associated proteins. We used the GST-T6B affinity pull-down assay, which has the ability to isolate all Ago-complexes, for a comprehensive investigation of Ago complexes (120). The interactome of Ago proteins in the cytoplasm of Leishmania-infected and non-infected control host cells were determined using a quantitative SILAC-based LC-MS/MS technique. This allowed for the identification of various RISC components, and the detection of associated factors Ago1, Ago2 and Ago3 in the data validated the LC-MS/MS results. Additionally, 53 high-confidence interactors common between infected and non-infected cells were found in this analysis in at least two out of three data sets. 17 human proteins out of the 53 total interactors had their levels altered in Ago-complexes from Leishmania-infected cells. Interestingly, compared to Ago complexes obtained from non-infected cells, host HSP family members that interact with Ago were primarily downregulated in infected cells. This observation was remarkable because it is well known that the ATP-dependent conformational shift of Ago proteins to an open and active configuration is mediated by the Hsp70/Hsp90 multi-chaperone systems. This procedure makes it possible for Ago proteins to fit the RNA duplex, making it a crucial component of the RISC-loading (266). It is noteworthy that of the 10 Leishmania proteins found in Ago-complexes, there were two putative HSP70s. This suggests that Leishmania HSPs and host HSP70 may compete for binding to Ago-complexes. This hypothesis is further supported by the earlier finding of HSP70s in the secretory exosomes of L. donovani via mass spectrometry analysis (90). 78 5. Chapter 5: Concluding remarks and working hypothesis It is known that unloaded Ago proteins are not stable and are degraded by the proteasome degradation pathway. This suggests that miRNA (or sncRNA) availability plays an important role in maintaining the stability of Ago proteins and in regulating the activity of the RISC (267,268). Thus, it is reasonable to assume that increased Ago1 abundance must be complemented with increased loading of sncRNAs onto Ago1 for its increased stability. Moreover, since non-perfectly matched sncRNA duplexes can be loaded into Ago1, Ago1 seems to be a favorable choice for the loading of pathogen sncRNAs other than miRNAs. Together, our results suggest an attractive hypothesis that Leishmania-mediated upregulation of Ago1 is a strategy to selectively upload sncRNAs, including its own sncRNAs, to regulate host cell gene expression in its favor. Therefore, it is rational to assume that higher sncRNA loading onto Ago1 is necessary to support increasing Ago1 abundance for Ago1 to remain stable. Ago1 appears to be a good option for the loading of pathogen sncRNAs since it can be loaded by sncRNA that are not properly matched. Together, our findings support the intriguing concept that Leishmania uses Ago1 overexpression as a tactic to selectively upload sncRNAs, including some of its own sncRNAs, to influence the gene expression of the host cell. The finding that Leishmania exosomal enriched sncRNAs _mainly tRFs and ribosomal RNA fragments_ are secreted in the cytoplasm of infected cells gives support to this hypothesis. These exosomal sncRNAs do not seem to have perfect match to host mRNAs. In this context, cross-kingdom RNAi describes bidirectional transfer of sncRNAs between a host and a parasite, resulting in the regulation of gene expression in both organisms. Cross-kingdom RNAi has been observed in various host-parasite interactions, and it provides a mechanism of inter-organismal communication that can influence the outcome of the interaction (269). Further supporting this hypothesis is our recent finding that Leishmania brings about a broad-based 79 downregulation of host macrophage miRNA expression (93), thus releasing unloaded host Ago proteins for Leishmania sncRNAs loading. The hypothesis is presented in Figure 5-1. Figure 5-1. The hypothetical model represents how Leishmania modifies the host RISC in favor of its pathogenicity. Leishmania effector molecules such as Ld-HSP70 already loaded with Ld-sncRNAs released from Ld exosomes compete with host sncRNA-HSP70 complex loading onto Ago1, thus regulating host macrophage gene silencing to create a pro-parasitic environment. Shown are the steps of Leishmania-mediated regulation of host RNAi: (1) exosomes secreted by parasites either in the phagolysosomes of the host cell or from infected bystander cells are delivered to the cytosol, where (2) the exosomal cargo, including Ld-HSP70 already loaded with Ld-sncRNAs, are released and (3) loaded, in competition with host RISC components, onto the host Ago1-containing RISC. (4) As part of the canonical mechanism of the RNAi pathway, the loaded RISC recognizes the target mRNA by base pairing in the 3ˊ untranslated region of mRNAs. Ago1 then interacts with TNRC6, (5) recruiting the PAN2–PAN3 and the CCR4–NOT deadenylase complexes which leads to deadenylation of the target mRNA (270,271). (6) The mRNA is decapped by decapping protein 2 (DCP2) and associated cofactors, and subsequently degraded by the XRN1 exonuclease (271). (7) Leishmania upregulates host Ago1 as the preferred Ago for uploading Leishmania exosomal sncRNAs. 80 5.1 Scientific significance for the field and future perspectives These findings open opportunities for further research into the role of RNAi in Leishmania pathogenesis, with possible implications for other intracellular infections. Additionally, the majority of our knowledge of the function of RNAi mechanisms in infection is based on research on plants and insects, therefore our study using the human macrophage-Leishmania as a model system is also a significant contribution. Our investigation is centered on examining the changes in protein composition of the host macrophage RISC during Leishmania infection, and the specific role of Ago proteins, particularly Ago1, in this process. By studying how Leishmania infection alters the composition of the host RISC and the function of Ago1, we aim to gain insights into the molecular mechanisms underlying the host-pathogen interaction. Our investigation provides a basis for more in-depth studies to further elucidate the molecular mechanisms underlying host-pathogen interactions in Leishmania infections. Additionally, the implications of our study may extend beyond Leishmania infections and could potentially inform research on the role of RNAi in other intracellular pathogens. Further research in this area may provide valuable insights into the development of novel therapeutic strategies for combatting intracellular infections. Our study represents a novel and pioneering investigation into the alterations induced by Leishmania in host RNAi complexes. By demonstrating the presence of Leishmania proteins in the host RISC, we provide evidence for a potentially sophisticated strategy employed by Leishmania to hijack the host RNAi machinery. It would be of significant interest to determine the precise molecular mechanism underlying the Leishmania-mediated upregulation of host cell Ago1, such as identifying the specific Leishmania factor responsible for this effect. Nevertheless, our study represents an important starting point for future investigations aimed at understanding the 81 biological consequences of Leishmania-mediated modulations of host RISC composition. With a deepened understanding of cross-kingdom RNAi in the context of Leishmania infection, there may be significant potential for developing novel therapeutic strategies for managing and treating leishmaniasis. It is also worthwhile to investigate loaded sncRNAs onto the host RISC following infection to see if Leishmania ncRNAs will be detected in the host RISC to obtain insights about the molecular mechanism of Leishmania pathogenesis. 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Thesis/Dissertation
eng
Experimental Medicine
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Reiner, Neil E.
Leishmania donovani upregulates host macrophage Argonaute 1 to persist inside infected host, and quantitative proteomic analysis of RNA-induced silencing complex isolated from infected host
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