Characterization and genome analysis of B1 sub-cluster mycobacteriophage PDRPxv
Introduction
The emergence of drug-resistant strains of M. tuberculosis has created an alarming situation (WHO, 2017). Exploring alternative options to combat and manage the disease are of paramount importance. Bacteriophages, the natural killers of the bacteria, and their lytic enzymes such as endolysins, ectolysins, depolymerases are gaining traction as potential alternates/complements to antibiotics due to their antibacterial activity. It is known that about 1023 phage infections may occur per second (Hendrix et al., 1999); this high rate of infection causes extensive horizontal gene exchange between bacteriophages and their hosts, which accounts for the mosaic nature of phage genomes (Hendrix et al., 2000; Pedulla et al., 2003). Mycobacteriophages are specific to mycobacterial spp. that include fast dividing non-pathogenic M. smegmatis and slow growing pathogenic M. tuberculosis. Enormous genetic diversity is observed amongst mycobacteriophages, which are hence classified into 29 distinct clusters, where 12 of the clusters (A–D, F–H, I, K–M, P) are also further divided into sub-clusters. Additionally, there are 10 singletons that do not share homology with any of the reported phages. According to Actinobacteriophage Database (http://phagesdb.org/), so far 11,160 mycobacteriophages have been isolated from various environmental sources but only about 17 % have been sequenced so far. Their genome size ranges between 41−165 kb (with an average GC content of 50–70 %). Given the small size of phage genomes and the current low cost of DNA sequencing, the published sequence information of phage genomes represents only a tiny fraction.
In this report, we are describing whole genome analysis and annotation and one step growth curve of PDRPxv, a lytic mycobacteriophage, which we isolated from a soil sample, collected from New Delhi. The phage belongs to cluster B1 and its genome is a contig of 69171 bases, containing a total of 107 ORFs.
Section snippets
Bacterial strains, soil samples and culture media
M. smegmatis mc2155 strain was used as host for the phage isolation. The cells were grown in Middlebrook 7H9 Medium (HiMedia, India) with shaking or Middlebrook 7H10 Agar (HiMedia, India) without shaking at 37 °C supplemented with Cycloheximide and Carbenicillin (HiMedia, India) antibiotics. Soil samples were collected (wearing gloves) from the compost, hospitals/garbage dumping locations in clean 50 ml centrifuge tubes. All the other chemicals and organic solvents used in the study were
Isolation, morphological analysis and stability studies
PDRPxv mycobacteriophage is a lytic mycobacteriophage, isolated from a soil sample collected from New Delhi, using M. smegmatis mc2155 as the bacterial host. The phage isolation procedure using M. smegmatis mc2155 as host yielded plaques which were circular (diameter of approximately 4 mm after 48 h of incubation) and clear with a thin halo (Fig. 1A). TEM analysis of purified phage particles showed the phage to have an icosahedral head (52.6 ± 0.6 nm wide) and a long (295.8 ± 1.7 nm)
Conclusions
While well characterized lytic mycobacteriophages hold promise as effective antimycobacterial agents, there are certain limitations in using them as therapeutics. Since several mycobacterial species, particularly M.tuberculosis are intracellular pathogens, delivery of mycobacteriophages to enable them reach the mycobacterium is a challenge. Also, though a large number of mycobacteriophages have been isolated against M. smegmatis, only a subset can infect and kill M. tuberculosis. Thus,
Authors’ statement
The study was designed by UB. Gene prediction and annotation work was done by AS and experimental work was done by KE, AS, and PM. UB and NR analysed the data. Manuscript was written by AS, KE and UB.
CRediT authorship contribution statement
Avni Sinha: Data curation, Writing - original draft, Visualization, Investigation, Software, Validation. Kandasamy Eniyan: Data curation, Writing - original draft, Visualization, Investigation. Prasanth Manohar: Data curation, Investigation. Nachimuthu Ramesh: Supervision. Urmi Bajpai: Conceptualization, Methodology, Supervision, Validation, Writing - review & editing.
Declaration of Competing Interest
The authors have declared that no competing interests exist.
Acknowledgements
We thank Open Source Drug Discovery (OSDD) and Council of Scientific and Industrial Research (CSIR), India for supporting the project. We thank TATA-CSIR-OSDD fellowship program for providing fellowship (TCOF 21) to AS. We would like to thank, Vellore Institute of Technology, Vellore for providing resources for growth curve and stability studies. We acknowledge AIRF, Jawaharlal Nehru University (JNU), New Delhi for MALDI-MS analysis and Transmission Electron Microscopy Laboratory, VIT for TEM
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Both the authors contributed equally.