Tailed bacteriophages typically induce lysis of infected bacteria to allow efficient escape of the virion progeny to the extracellular milieu. This review summa

Mycobacteriophages are viruses that specifically infect mycobacteria, which ultimately culminate in host cell death. Dedicated enzymes targeting the complex mycobacterial cell envelope arrangement have been identified in mycobacteriophage genomes, thus being potential candidates as antibacterial agents. These comprise lipolytic enzymes that target the mycolic acid-containing outer membrane and peptidoglycan hydrolases responsive to the atypical mycobacterial peptidoglycan layer. In the recent years, a remarkable progress has been made, particularly on the comprehension of the mechanisms of bacteriophage lysis proteins activity and regulation. Notwithstanding, information about mycobacteriophages lysis strategies is limited and is mainly represented by the studies performed with mycobacteriophage Ms6. Since mycobacteriophages target a specific group of bacteria, which include Mycobacterium tuberculosis responsible for one of the leading causes of death worldwide, exploitation of the use of these lytic enzymes demands a special attention, as they may be an alternative to tackle multidrug resistant tuberculosis. This review focuses on the current knowledge of the function of lysis proteins encoded by mycobacteriophages and their potential applications, which may contribute to increasing the effectiveness of antimycobacterial therapy.

LysB, a mycobacteriophage Ms6-encoded protein, was previously identified as a lipolytic enzyme able to hydrolyse the ester bond in lipase and esterase substrates. In the present work, we show that LysB can hydrolyse lipids containing mycolic acids from the outer membrane of the mycobacterial cell wall. LysB was shown to hydrolyse the mycolic acids from the mycolyl-arabinogalactan–peptidoglycan complex where the mycolates of the inner leaflet of the outer membrane are covalently attached to an arabinosyl head group. In addition, treatment of the extractable lipids from Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra with LysB showed that trehalose 6,6′-dimycolate (TDM), a trehalose diester of two mycolic acid molecules, was hydrolysed by the enzyme. We have also determined the structures of the mycolic acid molecules that form the M. smegmatis TDM. The identification of a phage-encoded enzyme that targets the outer membrane of the mycobacterial cell wall enhances our understanding of the mechanism of mycobacteriophage lysis.

DsDNA bacteriophages use the dual system endolysin–holin to achieve lysis of their bacterial host. In addition to these two essential genes, some bacteriophages encode additional proteins within their lysis module. In this report, we describe the activity of a protein encoded by gene lysB from the mycobacteriophage Ms6. lysB is localized within the lysis cassette, between the endolysin gene (lysA) and the holin gene (hol). Analysis of the deduced amino acid sequence of LysB revealed the presence of a conserved motif (Gly-Tyr-Ser-Gln-Gly) characteristic of enzymes with lipolytic activity. A blast search within the sequences of protein databases revealed significant similarities to other putative proteins that are encoded by mycobacteriophages only, indicating that LysB and those proteins may be specific to their mycobacterial hosts. A screening for His₆-LysB activity on esterase and lipase substrates confirmed the lipolytic activity. Examination of the kinetic parameters of recombinant His₆-LysB for the hydrolysis of p-nitrophenyl esters indicated that although this protein could use a wide range of chain length substrates (C₄–C₁₈), it presents a higher affinity for p-nitrophenyl esters of longer chain length (C₁₆ and C₁₈). Using p-nitrophenyl butyrate as a substrate, the enzyme showed optimal activity at 23 °C and pH 7.5–8.0. Activity was increased in the presence of Ca²⁺ and Mn²⁺. To the best of our knowledge, this is the first description of a protein with lipolytic activity encoded within a bacteriophage.

The mycobacteriophage Ms6 is a temperate double-stranded DNA (dsDNA) bacteriophage which, in addition to the predicted endolysin (LysA)-holin (Gp4) lysis system, encodes three additional proteins within its lysis module: Gp1, LysB, and Gp5. Ms6 Gp4 was previously described as a class II holin-like protein. By analysis of the amino acid sequence of Gp4, an N-terminal signal-arrest-release (SAR) domain was identified, followed by a typical transmembrane domain (TMD), features which have previously been observed for pinholins. A second putative holin gene ( gp5 ) encoding a protein with a predicted single TMD at the N-terminal region was identified at the end of the Ms6 lytic operon. Neither the putative class II holin nor the single TMD polypeptide could trigger lysis in pairwise combinations with the endolysin LysA in Escherichia coli . One-step growth curves and single-burst-size experiments of different Ms6 derivatives with deletions in different regions of the lysis operon demonstrated that the gene products of gp4 and gp5 , although nonessential for phage viability, appear to play a role in controlling the timing of lysis: an Ms6 mutant with a deletion of gp4 (Ms6 _{Δ gp4} ) caused slightly accelerated lysis, whereas an Ms6 _{Δ gp5} deletion mutant delayed lysis, which is consistent with holin function. Additionally, cross-linking experiments showed that Ms6 Gp4 and Gp5 oligomerize and that both proteins interact. Our results suggest that in Ms6 infection, the correct and programmed timing of lysis is achieved by the combined action of Gp4 and Gp5.