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Lysogeny of Ten Mycobacteriophages for Host Mycobacterium Tuberculosis H37ra

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Lysogeny of Ten Mycobacteriophages for Host Mycobacterium Tuberculosis H37ra University of Nevada, Reno Isolation of bacteriophages against Streptococcus species; Lysogeny of Ten Mycobacteriophages for host Mycobacterium tuberculosis H37Ra. A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Environmental Sciences by Christine B. Emmons Dr. Laura Briggs/Thesis Advisor May 2020 THE GRADUATE SCHOOL We recommend that the thesis prepared under our supervision by CHRISTINE B. EMMONS ntitled Isolation of bacteriophages against Streptococcus species; Lysogeny of Ten Mycobacteriophages for host Mycobacterium tuberculosis H37Ra be accepted in partial fulfillment of the requirements for the degree of Master of Science Laura Briggs, Ph.D. Amilton de Mello, Ph.D. Co Jonathan Baker, Ph.D. May, 2020 i Abstract Bacteriophages are viruses that infect bacteria, often resulting in lysis of the bacterial host. Due to the specific nature of bacteriophages, they play an essential role in maintaining the microbial balance of ecosystems. Truckee Meadows Community College has been isolating bacteriophage from soil and water samples in conJunction with a national undergraduate research program since 2014. The focus of Truckee Meadows Community College research has primarily been on mycobacteriophages; specifically, those that infect Mycobacterium smegmatis mc2155. Two proJects are presented in this thesis. The first is the methods to capture and isolate Streptococcus bacteriophages using human saliva tested against Streptococcus mutans lab strains, and other wild-type bacteria obtained from the human oral cavity. The second is lysogeny of mycobacteriophages originally obtained from Mycobacterium smegmatis mc2155 as a host that displayed successful cross-infection with Mycobacteria tuberculosis H37Ra in previous assays. Using the previous research, the bacteriophages capable of cross-infectivity were tested for their ability to form a lysogen – to integrate viral DNA within the genome without killing or lysing the bacterium; thus, resulting in bacteriophage readiness to take over the host cell in the current bacterium, as well as future generations of the bacterium. Bacteriophages demonstrate high specificity to their host. Receptors for bacteriophage to bind to the host bacterium appear to be in the cell wall of Gram- negative and Gram-positive bacteria, through the capsule and slime layer, and ii through appendages like flagella. Though phage-specific receptors have not been identified. The tail fibers of the bacteriophage appear to be significant in finding those receptors, but the specific genes responsible also have not been identified. Developing a process for successful isolation of bacteriophage is crucial to the process of understanding specificity through characterization of the bacterial host and the bacteriophage isolated. In the Streptococcus bacteriophage study, eight lab strains of Streptococcus mutans and 11 wild-type bacteria were cultured from the human oral cavity, cultured were used as bacterial hosts. The Neisseria strains were removed from this research. Saliva was tested against the host to test for the presence of novel bacteriophage. Initial methods were derived from previously published methods for Streptococcus mutans bacteriophage isolation. Bacteriophage isolation was not successful using these initial methods, so several modifications were tested. Modifications included an increase of infection time, incubation time, limiting potential bacteriophage tail damage from centrifugation – reduced centrifugation times or elimination of centrifugation, using saliva without filtration, and using saliva without filtration and not allowing the sample to settle. While no bacteriophage was successfully isolated for characterization, these procedural changes did result in potential temperate phages; this indicates a potential for temperate bacteriophage isolation in future studies. In the mycobacteriophage study, previous research identified ten mycobacteriophages with the potential for cross-infectivity between the original iii host, Mycobacterium smegmatis mc2155, and the potential host, Mycobacterium tuberculosis H37Ra. This research confirmed that six mycobacteriophages were successful in cross-infection: Scooby Blue, Erimy, Guilian 2, Guilsminger, Old House, and Zose. Five were lysogenic, and one was lytic. The lytic bacteriophage, Zose, was not used for this study. Two of the remaining five mycobacteriophages showed an efficiency of plating (EOP) of 10-3 or better: Scooby Blue and Old House when compared to the original host. An EOP of 10-3 or better indicated productive infection of these bacteriophages, which makes these two bacteriophage potential candidates for characterization of the genomes for further understanding of cross-infectivity and lysogeny. Future work should include further adJustments to protocols for more successful isolation of Streptococcus bacteriophages. The adJustments should include longer enrichment times and sampling from other environmental sources, including soil. Future studies using the lysogenic mycobacteriophages analyzed here should include DNA sequencing of the mycobacteriophages for comparison of each genome against lytic and lysogenic bacteriophages that cross-infect original host, Mycobacterium smegmatis mc2155, and the potential host, Mycobacterium tuberculosis H37Ra, allowing for identification of lysogeny genes. The same bacteriophage genomes can be used for comparison with other mycobacteriophage genomes that do not cross-infect to specify genomic differences specific to cross-infection. iv Acknowledgments This research was made possible because of many individual contributions, some to the research and others to me personally. First, I would like to thank my Advisor, Dr. Laura Briggs. Her dedication to research, passion for teaching, and guidance through this process has been invaluable to me as a student, teacher, and researcher. I have been privileged with many opportunities throughout my career as a student, and it is in large part because she believed in me and my abilities, thank you, Dr. Briggs. I would like to thank Dr. Meeghan Gray. Her passion for teaching started me on a scientific path. Her valuable insight as a Professor, peer, and friend has helped me get to where I am today, thank you, Dr. Gray. I would like to thank Dr. Julie Ellsworth and Tina Slowan-Pomeroy for allowing me to continue to teach, research, go to school, and keep working in the lab. It has provided me the opportunity to get where I am today, thank you both. Thank you to my committee members, Dr. Amilton de Mello and Dr. Josh Baker, for guiding me through this process and always offering to help in any way that you can. My research was possible because of Nevada INBRE and Truckee Meadows Community College. Nevada INBRE provided the grant that got this research started, and Truckee Meadows Community College allowed me to do graduate research at the facility. v Finally, I would like to thank my husband, Thomas Emmons, and my son, Tristian Sommerfield. The constant support and love helped me through my education, and I am forever grateful for your belief in me, thank you. vi Table of Contents Abstract ..................................................................................................................... i Acknowledgments .................................................................................................. iv List of Abbreviations ............................................................................................ viii List of Tables ............................................................................................................ x List of Figures ......................................................................................................... xi Chapter 1 .................................................................................................................. 1 Significance ...................................................................................................................... 2 Biofilms .............................................................................................................................. 3 Streptococcus mutans .................................................................................................... 4 Infective Endocarditis ..................................................................................................... 5 Mycobacterium tuberculosis .......................................................................................... 5 Tuberculosis History ....................................................................................................... 7 Bacteriophage History .................................................................................................... 8 Bacteriophage Diversity ................................................................................................. 9 Bacteriophage Anatomy and Lifecycles .................................................................... 10 Bacteriophage Specificity and Host Range ............................................................... 12 Bacteriophage Treatment Today ................................................................................. 13 Lytic and Lysogenic Bacteriophages for Treatment of Infection ........................... 14 Summary ......................................................................................................................... 16 References ................................................................................................................
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