Insights into the genomic features, lifestyle and therapeutic potential of B1 sub-cluster mycobacteriophages

Abstract

BackgroundA large number (about 1200) of mycobacteriophages (phages) have been isolated onMycobacterium smegmatismc2155. Their genome analysis shows high sequence diversity; therefore, based on nucleotide sequence similarity and genomic architecture, the related phages have been grouped in clusters and sub-clusters. However, a deeper study of mycobacteriophages has been conducted only for a few clusters. This study explores the traits of phages belonging to the B1 sub-cluster. We have attempted to functionally annotate and experimentally characterize B1 phages to get an insight into their biology and explore their therapeutic potential.MethodsAnalysis of B1 sub-cluster phage genomes to understand their key characteristics & lifestyle and to determine the putative function of hypothetical proteins (HPs), we developed a framework with a specific set of computational tools available online. For the experimental characterization, mycobacteriophages were isolated from environmental samples and were examined for their morphology, lysogeny status, effect on biofilm and activity against drug-resistantM. smegmatis. The B1 sub-cluster phages were identified by PCR using the specific primers.ResultsWe have predicted the function of about 55% of the 77 representative proteins in B1 phages, which were previously deemed hypothetical. We studied ten B1 phages (Phages 1-10)which included their morphological characteristics, lysogeny status and antibiofilm activity. TEM analysis, showing an average head & tail size of 65 nm and 202.12 nm, respectively. The turbid morphology of several plaques suggested these phages to be temperate. To verify, we tested their potential to lysogenizeM. smegmatisand later found the spontaneous release from the putative lysogens. Interestingly, a putative RepA-like protein was identified in B1 phage genomes, indicating a possibility of extrachromosomal replication of prophages. Further, the impact ofPhages 1-10onM. smegmatisbiofilm was found to be potent; the highest inhibitory and disruptive effect of phages (at a fixed titre of 108pfu/ml) was 64% and 46%, respectively. Also, all ten phages could kill 4XR1 (the isoniazid-resistantM. smegmatisstrain).ConclusionWe believe this combination of experimental analysis and exploration of genomic features of mycobacteriophages belonging to a sub-cluster can provide deeper insights into mycobacteriophage biology and also help in understanding their therapeutic potential.

Beyond antibiotics: phage-encoded lysins against Gram-negative pathogens

“…Viruses that target and replicate inside a bacterial host are known as phages ( Das and Bajpai, 2022 ; Das et al, 2023 ). They require escape strategies to exit the bacterium to disseminate their progenies.…”

Genome announcement and analysis of five new mycobacteriophages belonging to the F, G, K, and P clusters

“…Environmental samples were collected from various regions of Delhi-NCR and Mumbai, India. The phages were isolated, purified and amplified on M. smegmatis Mc 2 155 as the host, using the double agar overlay method previously described by Das et al, 2023. Each phage was subjected to three rounds of purification and amplified to a titre >10 10 pfu/ml. The high-titre lysates were used for genomic DNA isolation using the PCI method described previously by Sinha et al, 2020. Whole genome sequencing was outsourced, and the genomes were sequenced using the Illumina platform (150x2 PE chemistry).…”