Lytic Efficiency of Mycobacteriophages

Abstract

Mycobacteriophages D29 and TM4 are the two virulent phages widely used for the study of mycobacterial genetics. Both the phages are capable of killing Mycobacterium tuberculosis but the efficiency of these phages in killing has not been evaluated and compared. There are reports based on codon usage analysis where TM4 is predicted to be a better killing phage over D29 which corroborated with the whole genome in silico analysis. In addition a kill assay using 5 wild type virulent mycobacteriophages viz. D29, TM4, I3, Che7 and Che11 was performed to study the killing efficiency of these phages using LRP assay. Based on the results, D29 was found to infect all the 10 clinical strains of M. tuberculosis and significantly reduced RLU at 3 hours and this effect continued up to 24 hours. Thus, D29 is found to have better killing efficiency than TM4 contradicting the earlier predictions. In silico analysis of holin and lysin genes of TM4 and D29 substantiated our findings.

Mycobacteriophage D29 holin C‐terminal region functionally assists in holin aggregation and bacterial cell death

“…The mycobacteriophage D29 contains three genes in its lytic cassette : gp10 , gp11 and gp12 . Gp10 works as a peptidoglycan hydrolase and Gp12 harbors esterase activity that is required to breakdown the mycolic acid layer .…”

“…Mycobacteriophage Ms6 has two genes, gp4 and gp5 , both of which are required for the timed lysis of the host cell and subsequent release of phages . However, D29 possesses only one holin‐coding gene . It is conceivable that the function and regulation of gp11 ‐encoded holin occurs in a manner that is very different from the proposed models derived from other proteins of this family and therefore requires thorough investigation.…”

“…In mycobacteriophage D29, the lytic cassette includes the gp11 gene that codes for a putative holin. gp11 is flanked by two endolysin genes: gp10 and gp12 . Gp11 has been predicted to be a class II holin having two transmembrane helices, with a short N‐terminal region, and a long highly charged C‐terminal domain .…”

Molecular Dissection of Phage Endolysin

“…Indeed, D29 phage is known to infect M. tuberculosis (20). We are, therefore, tempted to add that Lysin A will function in M. tuberculosis as well and, consequently, can be developed as a selective inhibitor for this pathogen.…”

“…Two of these genes encode Lysin A and Lysin B that sandwich one other gene coding for a membrane pore-forming protein, holin (20). The Lysin A (coded by gp10) is responsible for the hydrolysis of PG, 3 whereas Lysin B (coded by gp12) acts at the junction between mycolic acid and the PG (21,22).…”