Biology subjects

Nadar, K.

Publications and source records attributed to Nadar, K..

5 recordsLinked to original sources

bioRxiv2026

Genomic and Functional Insights into the 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.

Molecular Biology↗

bioRxiv2024

A novel LysinB from an F2 sub-cluster mycobacteriophage RitSun

With the growing antibiotic resistance in mycobacterial species posing a significant threat globally, there is an urgent need to find alternative solutions. Bacteriophage-derived endolysins aid in releasing phage progeny from the host bacteria by attacking the cell wall at the end of their life cycle. Endolysins are attractive antibacterial candidates due to their rapid lytic action, specificity and low risk of resistance development. In mycobacteria, owing to the complex, hydrophobic cell wall, mycobacteriophages usually synthesize two endolysins: LysinA, which hydrolyzes peptidoglycan; LysinB, which delinks mycolylarabinogalactan from peptidoglycan and releases mycolic acid. In this study, we conducted domain analysis and functional characterization of a recombinant LysinB from RitSun, an F2 sub-cluster mycobacteriophage. Several properties of RitSun LysinB are important as an antimycobacterial agent: its ability to lyse Mycobacterium smegmatis from without, a specific activity of 1.36 U/mg, higher than the reported ones and its inhibitory effect on biofilm formation. Given the impervious nature of the mycobacterial cell envelope, native endolysins ability to damage cells on exogenous applications warrants further investigation. A molecular dissection of RitSun LysinB to identify its cell wall destabilizing sequence could be utilized to engineer other native lysins as fusion proteins and expand their activity profile.

molecular biology↗

bioRxiv2024

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

Mycobacterial infections are accountable for some of the most challenging to treat diseases in humans and animals. With the rise in drug-resistant clinical isolates of Mycobacterium spp., phage therapy is emerging as a promising alternative to antibiotic therapy. Isolation of genetically diverse phages that can infect pathogenic mycobacterium species is an important exercise. Here, we present the complete genome sequences of five mycobacteriophages isolated from environmental samples using Mycobacterium smegmatis Mc2 155 as the bacterial host. These phages belong to different clusters, including F, G, K and P and contribute to the diversity of sequenced mycobacteriophages.

genomics↗

bioRxiv2024

Unlocking prophage potential: In silico and experimental analysis of a novel Mycobacterium fortuitum LysinB containing a peptidoglycan-binding domain

Endolysins are highly evolved bacteriophage-encoded lytic enzymes produced to damage the bacterial cell wall for phage progeny release. They offer promising potential as highly specific lytic proteins with a low chance of bacterial resistance. The diversity in lysin sequences and domain organization can be staggering. In silico analysis of bacteriophage and prophage genomes can help identify endolysins exhibiting unique features and high antibacterial activity, hence feeding the pipeline of narrow-spectrum protein antibiotics. Mycobacteriophage lysis cassettes mostly have two lytic enzymes, LysinA and LysinB. The enzyme LysinA targets peptidoglycan in the cell wall and possesses a modular architecture. LysinB typically contains a single domain and acts upon the mycolyl ester linkages in mycolyl-arabinogalactan-peptidoglycan (Payne et al., 2010). This study aimed to find novel LysinBs against Mycobacterium fortuitum. After a detailed in silico characterization of lysis cassettes from three M. fortuitum prophages, we chose to work on a LysinB (hereafter described as LysinB_MF) found in an incomplete prophage (phiE1336, 9.4 kb in strain E1336). LysinB_MF showed low sequence similarity with any other endolysins in the database and formed a separate clade on phylogenetic analysis. LysinB_MFs structure, extracted from the AlphaFold Protein Structure Database, demonstrated a modular architecture with two structurally distinct domains: a peptidoglycan-binding domain (PGBD) at the N-terminal and the characteristic alpha/beta hydrolase domain connected via a linker peptide. We found the alpha/beta hydrolase domain, which is the enzyme-active domain (EAD), contains the conserved Ser-Asp-His catalytic triad with a tunnel-like topology and forms intermolecular hydrogen bonds. The PGBD shows structural similarity to the cell-wall binding domain of an amidase from Clostridium acetobutylicum, hinting at its acquisition due to domain mobility. Our in silico electrostatic potential analysis suggested that PGBD might be essential to the enzyme activity. Based on our analysis, PGBD emerged as an integral constituent of enzymes with diverse functional properties and is predicted to be conserved cross-kingdom. Overall, this study highlights the importance of mining mycobacterial prophages as a novel endolysin source. It also provides unique insights into the diverse architecture of mycobacteriophage-encoded endolysins and the importance of functional domains for their catalytic activities.

molecular biology↗

bioRxiv2023

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

BackgroundA large number (about 1200) of mycobacteriophages (phages) have been isolated on Mycobacterium smegmatis mc2155. 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-resistant M. 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 lysogenize M. smegmatis and 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 of Phages 1-10 on M. smegmatis biofilm was found to be potent; the highest inhibitory and disruptive effect of phages (at a fixed titre of 108 pfu/ml) was 64% and 46%, respectively. Also, all ten phages could kill 4XR1 (the isoniazid-resistant M. smegmatis strain). 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.

microbiology↗