Analysis of a Novel Cluster V Mycobacteriophage EniyanLRS and its Therapeutically Relevant LysB

Multidrug-resistant tuberculosis and the rising burden of nontuberculous mycobacterial diseases demand innovative treatment strategies. Mycobacteriophages and endolysins offer promising therapeutic potential due to their targeted bacterial cell wall damaging activity, vast diversity, and lack of antibiotic cross-resistance. Here, we report the genomic and functional characterisation of a V Cluster mycobacteriophage, 'EniyanLRS', and its endolysins. Some genomic features: genome size (78.53 kbp), a relatively low GC content (56.9%), a long Tape Measure Protein gene (5.97 kbp), and a high number of tRNAs (24), suggest distinct adaptation and translational efficiency of our phage. Phenotypically, EniyanLRS exhibits a siphovirus morphology, lytic life cycle and infects drug resistant Mycobacterium fortuitum. While its endolysin LysA, harbouring a lysozyme-chitinase-amidase domain, showed moderate lysozyme like activity without significant antibacterial activity, LysB, an alpha beta hydrolase, exhibited superior in vitro esterase activity and showed pronounced cell wall disruption in M. smegmatis and M. fortuitum, along with considerable antibiofilm activity (62.77% and 41.91% inhibition, respectively). Collectively, our analysis reveals key functional properties of EniyanLRS phage and its LysB, demonstrating their suitability as effective biological candidates for mycobacterial therapy.