AN ABSTRACT OF THE THESIS OF Sanket S. Chiplunkar for the degree of Master of Sciences in Comparative Health Sciences presented on May 29, 2018. Title: Characterization of Membrane Vesicles Released by the Opportunistic Human Pathogen Mycobacterium avium subsp. hominissuis (MAH) in Response to an in vitro System Mimicking the Phagosomal Environment Abstract approved: __________________________________________________________________ Luiz E. Bermudez Lia Danelishvili Mycobacterium avium subsp. hominissuis (MAH) belongs to the most-clinically significant non-tuberculous mycobacterial (NTM) pathogens with constant increase in disease prevalence, mainly in several industrialized western countries where tuberculosis is less prevalent. Upon entry into the alveolar space, MAH is engulfed by resident-macrophages, where the pathogen adapts to the hostile phagosomal environment and proliferates. Mycobacteria bypass host immune defenses by secreting virulence factors. Several intracellular pathogens including mycobacteria are known to form membrane vesicles (MVs) in response to intracellular stress, helping bacteria to deliver virulence effectors in the host cell. MVs are bacterial membrane-derived spheres filled with the cargo of biologically active materials such as proteins, lipids and nucleic acids. MVs play important role in bacterial pathogenesis, nutrient acquisition, biofilm formation and immunomodulation essential for bacterial survival within the host. Macrophage phagosomal environment, partly consisting of elemental mixture and low pH, activates several virulence mechanisms of MAH. Using the in vitro model mimicking the phagosomal environment of MAH (metal mix), we characterized cargo of MVs and investigated whether MV-associated bacterial virulence effectors are delivered to the cytosol of the host macrophages. Scanning electron microscopy of MAH exposed to the in vitro phagosomal model revealed formation of MVs on bacterial surface. Further, transmission electron microscopy confirmed presence of MVs in the purified samples of isolated vesicles. Using mass spectrometric analysis, 202 proteins were identified in MVs of minimal media of starvation model and 263 proteins were found in the in vitro model mimicking phagosome environment. Out of 263 cargo proteins, 211 are unique to the metal mix and are enriched in several virulence factors including enzymes involved in lipid and fatty acid metabolism and cell wall-associated processes. Non-canonical amino acid metabolic labeling and click chemistry-based enrichment assay confirmed that at least 5 proteins found in the in vitro phagosomal model are also present in the cytosol of THP1 macrophages during MAH infection at 24h. Lipidomic analysis showed presence of outer cell wall lipids with no compositional differences between MVs of minimal media and metal mix. Using PicoGreen assay, we demonstrate that MVs of both minimal media and metal mix harbor dsDNA; however, the concentration was found to be significantly lower in the in vitro phagosomal model. Similar results are observed by laser confocal microscopy of MVs stained with lipophilic nucleic acid dye SYTO-61. Our study suggests that MAH produces MVs in phagosomes and carry important virulence factors with potential roles in bacterial intracellular survival and in the immunomodulation of host cellular defenses. ©Copyright by Sanket S. Chiplunkar May 29, 2018 All Rights Reserved Characterization of Membrane Vesicles Released by the Opportunistic Human Pathogen Mycobacterium avium subsp. hominissuis (MAH) in Response to an in vitro System Mimicking the Phagosomal Environment by Sanket S. Chiplunkar A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented May 29, 2018 Commencement June 2018 Master of Science thesis of Sanket S. Chiplunkar presented on May 29, 2018. APPROVED: Major Professor, representing Comparative Health Sciences Co-Major Professor, representing Comparative Health Sciences Dean of the College of Veterinary Medicine Dean of the Graduate School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Sanket S. Chiplunkar, Author ACKNOWLEDGEMENTS I am very grateful to Dr. Luiz Bermudez for believing in my potential and giving me this opportunity of working in his laboratory. I am also thankful to him for providing me helpful feedback and suggestions for my research and postgraduate studies. I am very grateful to Dr. Lia Danelishvili for her continuous mentorship and assistance provided during conducting experiments, writing thesis and postgraduate studies. I would like to thank the committee members Dr. Claudia Hase and Dr. Sean Newsom for agreeing to be on my committee and for providing me helpful feedback during committee meetings. I want to thank Dr. Liping Yang and Jeff Morre of OSU Mass Spectrometry Center, Teresa Sawyer of OSU Electron Microscopy Facility, Anne-Marie Girard Pohjanpelto of CGRB confocal laser scanning microscope facility and other CGRB staff for their technical assistance. I am thankful of Dr. Virginia Weis for providing me teaching assistantship. I wish to thank my lab-mates, administrative staff of department of Biomedical Sciences and Integrative Biology, TA-coordinators, friends and family who continuously supported me throughout this journey. Big thank you to my mom and dad for their continuous moral support and encouragement. This work was supported by the Oregon State University Foundation FS062E- VF01 and by the Oregon State University Incentive Programs VBS330-001100 (Lia Danelishvili). The proteomic sequencing was conducted by the Oregon State University’s Mass Spectrometry Center supported in part by OSU’s Research Office and institutional funds. The procurement of the Orbitrap Fusion Lumos was made possible by NIH grant S10 OD020111. CONTRIBUTION OF AUTHORS Sanket S. Chiplunkar contributed to experimental design, conducted experiments, analyzed data and wrote manuscript. Dr. Lia Danelishvili contributed to experimental design, conducted experiments, data analysis and manuscript preparation and funding of the project. Dr. Luiz Bermudez contributed to experimental design, manuscript preparation and project funding. TABLE OF CONTENTS Page Introduction ...…………………………………………………………………………1 Materials and Methods …………………………………………………………....…15 Results…………...…………………………………………………………………...28 Discussion ……...……………………………………………………………………59 References….………………………………………………………………………...82 Supplementary Information……...……………………………………………......…89 LIST OF FIGURES Figure Page 1. MV isolation and purification process…………………...………………………..17 2. SEM micrographs of MAH104 exposed to minimal media for 2-week and metal mix for 24h…………………………..……………………………………………….29 3. TEM micrographs of purified MV samples…………………………………….....30 4. Survival and/or viability assay of MAH104 exposed to minimal media and metal mix for 2-weeks……………………..……………………………………………….31 5. Functional classification of cargo proteins found in the MAH104 MVs of minimal media and metal mix…………………...…………………………………………….46 6. Relative categoric representation of MV cargo proteins between minimal media and metal mix………...………………………………………………………………47 7. Venn diagram showing common and unique cargo proteins between minimal media and metal mix…...…………………………………………………………….47 8. Functional classification of MAH104 AHA-labeled secreted effectors found through click chemistry-based enrichment…………………………………………..50 9. Venn diagram showing common and unique proteins between metal mix MV cargo proteins and AHA-labeled secreted effectors……….…………………….…………50 10. Minimal media and metal mix MV lipidomics……………………...…………...52 11. Minimal media and metal mix MV-associated DNA quantification………….…54 12. Laser confocal microscopic visualization of SYTO-61 stained MVs…………...54 13. PCR results of FLAG-tag incorporated MAH104 gene cloning………………...56 14. E. coli colony PCR screening for the presence of cloned construct…...………...57 15. MAH104 colony PCR screening for the presence of cloned construct………….58 16. Venn diagram showing common and unique cargo proteins between 2-week minimal media, 2-week metal mix and 24h metal mix exposure………………......105 17. Heatmap of gene ontology of host (THP1 human macrophage) proteins found in the exosomes collected 24h post MAH104 infection ……………………………...106 LIST OF FIGURES (Continued) Figure Page 18. Heatmap of gene ontology of host (THP1 human macrophage) proteins found in the exosomes collected 72h post MAH104 infection ……………………………...106 19. Venn diagram showing common and unique host cargo proteins between exosomes of uninfected THP1 human macrophages and 24h post MAH104 infection………………………………………………….…………………………107 20. Venn diagram showing common and unique host cargo proteins between exosomes of uninfected THP1 human macrophages and 72h post MAH104 infection………………………………………………….…………………………107 LIST OF TABLES Table Page 1. Contents of minimal media and metal mix ……………………………….....16 2. Primers used in the study………………………………………..….………..25 3. List of cargo proteins found in the MAH104 MVs of 2-week minimal media exposure………………………………………………………………….…..33 4. List of cargo proteins found in the MAH104 MVs of 24h metal mix exposure……………………………………………………………………...38 5. List of AHA-labeled MAH104 secreted effectors present in the cytosol of 24h post infected THP1 human macrophages, found through click chemistry-based enrichment……………………………………………………………………49