A holistic insight of mycobacteriophage induced changes in mycobacterial cells

Mycobacteriophages are phages that interact with mycobacteria resulting in their killing. Although lysis is the major mechanism by which mycobacteriophages cause cell death, other mechanisms may also be involved. The present study was initiated with the objective of investigating the changes that take place at the cellular level following the infection of mycobacterial cells by phage D29. To investigate this issue, we took recourse to performing immunofluorescence and electron microscopic studies. Transmission electron microscopic examination revealed the adsorption of phages on to the surface of mycobacteria, following which penetration of the tail through the thick mycoloic acid layer was seen. At later time points discrete populations of cells at different stages of lysis were observed, which comprised of completely lysed cells, in which the cells were fragmented and those at the early onset stage exhibited formation of membrane pores through which the phages and intracellular contents were released. SEM results also indicated that phages may come out through the entire surface of the cell, or alternatively through gaps in the surface. In some of the images we observed structures that apparently resembled membrane blebs which are normally encountered when cells undergo programmed cell death (PCD). In addition, we observed significant increase in DNA fragmentation as well as membrane depolarization, which are also indicative of occurrence of PCD. As several bacterial PCD pathways are mediated by the toxin-antitoxin (TA) modules, the expression profile of all the TA systems was examined before and after phage infection. Apart from specifically addressing the issue of PCD in mycobacteriophage infected cells, this investigation has led to the development of facile tools necessary for investigating mycobacteriophage-mycobacteria interactions by means of microscopic methods.

The therapeutic potential of these transmissible bacteriolytic entities was first identified 52 by their codiscoverer, Félix d'Hérelle (7). Since then phage research became the cradle of 53 fundamental and translational biosciences. There is an increasing interest in the studies focusing 54 on the use of bacteriophages as antibacterial agents against pathogenic bacteria. This is a 55 consequence of the ability of the phage to lyse a bacterial host (8,9). Phage D29 is one such 56 bacteriophage, which infects diverse mycobacteria such as M. smegmatis and M. tuberculosis 57 (10). Belonging to the family Siphoviridae, it typically exhibits a long non-contractile tail (11). 58 The resurgence of TB and emergence of excessive drug resistant (XDR) and totally drug 59 resistant (TDR) strains has spurred renewed interest in the therapeutic use of 60 mycobacteriophages (12). They can even serve as cornerstones for developing novel diagnostic 61 and preventive strategies (13). D29 is the prototypical model for a mycobacteriophage as it efficiently adsorbs to the host and begins DNA replication within a few minutes after infection.  Electron microscopy, even though an age old technique, was resorted to, because it is still 78 considered to be the gold standard for viral ultrastructure studies (20).

79
Several hurdles confront the utilization of phages for the curtailment of mycobacteria.

80
Rather than recruiting phages directly for treatment, they can be used as platforms for drug 81 discovery. Phages have evolved multiple strategies for interfering with bacterial growth.

82
Understanding the targets that phages use in inhibiting bacterial growth has a clear therapeutic 83 implication. In this study we focus on the interaction between mycobacteriophage and mycobacteria. Our objective was to develop cytological tools to understand the changes that 85 happen within the bacterial cell once it is attacked by mycobacteriophages. Phages were amplified by the confluent lysis method followed by suspension in SM buffer.

98
Phage purification was done by performing CsCl density gradient centrifugation, followed by 99 dialysis using a dialysis buffer (50mM Tris-Cl (pH 8.0), 10 mM NaCl, 10 mM MgCl 2 ). M. The gene encoding the major head subunit gp17, was PCR amplified using the primers D2917F 109 and D2917R (

152
Cultures were grown overnight at 37 0 C to an OD 600 between 0.2-0.3 before phage treatment.

153
Infection was done at an MOI of 1. After treatment the sample was collected, washed once and    Table 1. to contain no gp17 specific antibodies, as confirmed by Western blot and confocal microscopy.

208
Our results revealed distinct phage adsorption 30 min post-infection. After 2 hrs, several free 209 phages were observed, indicative of phage release after lysis (Fig 1). mainly of fragmented cells and cellular debris was seen, representing later stages of lysis (Fig 6).

235
SEM data correlated well with that of TEM (Fig 7). control set), indicating a loss of membrane potential (Fig 8). for PI and FITC as compared to control cells which were stained with PI only (Fig 10). found to be consistent with those obtained by the qualitative end point PCR. A dramatic decrease in gene expression was observed upon phage infection (Fig 12) whereas the expression was 297 found to increase marginally in the uninfected controls.