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 /{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.