Preliminary investigation of cluster M lysogeny and prophage-mediated defense mechanisms
- WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
- Montana Jo Henson (Creator)
- Institution
- Western Carolina University (WCU )
- Web Site: http://library.wcu.edu/
- Advisor
- Maria Gainey
Abstract: Bacteriophages are viruses that infect bacteria and are characteristically specific for their bacterial hosts with preferences rarely traversing genus boundaries. Mycobacteriophages – bacteriophages that infect Mycobacterial hosts – are the largest collection of genetically characterized phages known to infect a single host and are used to study phage biology and evolution. Mycobacteriophages have been divided into 32+ genetic clusters based on nucleotide sequence similarity and shared gene content. When a bacteriophage infects a bacterial cell, it usually enters the lytic cycle. Lytic replication results in the production of virus particles, and lysis of the host cell. Temperate bacteriophages may also enter the lysogenic cycle. During lysogeny, the bacteriophage integrates its genome into the host cell’s chromosome. After integration, the viral genome is referred to as a prophage, and the bacterial cell is called a lysogen. To defend the lysogen cell against bacteriophages in the environment, some prophages have evolved to contain prophage defense genes. These genes can allow a prophage to prevent homotypic and/or the heterotypic bacteriophages from establishing a productive infection in the lysogen cell. Mycobacteriophage treatment of antibiotic resistant human Mycobacterium abscessus and Mycobacterium tuberculosis infections has recently shown great promise. However, patient Mycobacterium abscessus isolates are only susceptible to a narrow range of mycobacteriophages due to the presence of prophages carrying uncharacterized defense systems. Cluster M-like prophages have been identified in patient isolates. Though cluster M-like prophages may play a significant role in resistance to bacteriophage therapy, little is known about how they establish lysogeny and what prophage defense mechanisms they may contain. During this study, the integration site and defense profile of the IPhane7 (subcluster M1) prophage was investigated. The location of the bacterial integration site (attB) was determined by full genome Illumina sequencing of the IPhane7 lysogen. Prophage-host junction specific PCR, followed by Sanger sequencing was used to confirm the attB/attP sites. The defense profile of cluster M lysogens was determined by the creation of cluster M lysogens and subsequent challenge with a genetically diverse panel of Mycobacteriophages. While the overall defense profile of cluster M prophages is unknown, previous work from the Gainey laboratory revealed that expression of only gene products (gp) 1 and 2 can prevent infection of cluster M bacteriophages. Gene 1 is unique to cluster M bacteriophages and is predicted to code for a 97 amino acid protein with a Sec-signal sequence. Gene 2 is found in other bacteriophages and sequenced Actinobacterial genomes. Select cluster M gp1 or gp1/gp2 were introduced into M. smegmatis cells using high and low plasmid expression vectors. The defense profile against cluster M and other heterotypic mycobacteriophages was then determined via viral challenges, and the results displayed high specificity amongst phage phenotypes. Additionally, we investigated how the synergistic actions of gp1/gp2 cluster M defense may be overcome by using comparative genomics to analyze gp1/2 defense escape mutants.
Preliminary investigation of cluster M lysogeny and prophage-mediated defense mechanisms
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Created on 3/1/2023
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Additional Information
- Publication
- Thesis
- Language: English
- Date: 2023
- Keywords
- Bacteriophages, Biochemistry, Bioinformatics, Genomics, Microbiology, Virology
- Subjects
- Bacteriophages
- Biochemistry
- Bioinformatics
- Genomics
- Microbiology
- Virology
- Lysogeny
- Mycobacterium