Sewing Up the Gaps in Phage Research with Thimble

Catherine Beazley, Jillian Edwards, Jenna Gallant, Emily Korotki, McKenzie Shaw, Emily E Whitaker

Non-tuberculosis mycobacteria (NTMs) are challenging antibiotic resistant pathogens that pose significant hurdles for treatment, particularly in immunocompromised individuals. Mycobacteriophages (phages), viruses that infect and kill mycobacteria, offer a potential alternative to traditional antibiotics. Pathogenic NTMs, including Mycobacterium abscessus, are closely related to non-pathogenic M. smegmatis which serves as a comparable fast-growing host for the isolation and characterization of therapeutically relevant mycobacteriophage. Novel mycobacteriophage Thimble was isolated from strawberry garden soil from Hampden, ME, using M. smegmatis mc2155. Thimble forms pinpoint turbid plaques and initial experiments indicate that Thimble may be able to form lysogens. This is supported by the presence of a tyrosine integrase, gp54, which facilitates integration of the phage genome into the bacterial host genome. Gp57 encodes a helix-turn-helix DNA binding domain and is potentially an immunity repressor. Thimble is a Siphoviridae phage belonging to Cluster E with a genome that is 71,466 bp in length with a GC content of 63.1%. Thimble encodes 134 protein-coding genes and two tRNAs. Thimble exhibits genome organization that is consistent with the synteny of a Siphoviridae phage, encoding structural genes on the left arm and DNA replication genes on the right arm. Analysis of the Thimble genome identified that gp39 encodes an Lsr2-like DNA binding protein. In M. tuberculosis and M. smegmatis, this protein acts as a transcriptional regulator. Additional research is needed to confirm that Thimble can form lysogens and to determine if Thimble’s gp39 can perform Lsr-2-like regulatory functions. Thimble’s ability to infect M. smegmatis makes it a candidate for further investigation in phage therapy, particularly for multidrug-resistant mycobacterial infections.