Exploring the Enzymatic and Antibacterial Activities of Novel Mycobacteriophage Lysin B Enzymes.

Mycobacteriophages possess different sets of lytic enzymes for disruption of the complex cell envelope of the mycobacteria host cells and release of the viral progeny. Lysin B (LysB) enzymes are mycolylarabinogalactan esterases that cleave the ester bond between the arabinogalactan and mycolic acids in the mycolylarabinogalactan-peptidoglycan (mAGP) complex in the cell envelope of mycobacteria. In the present study, four LysB enzymes were produced recombinantly and characterized with respect to their enzymatic and antibacterial activities. Examination of the kinetic parameters for the hydrolysis of para-nitrophenyl ester substrates, shows LysB-His6 enzymes to be active against a range of substrates (C4-C16), with a catalytic preference towards p-nitrophenyl laurate (C12). With p-nitrophenyl butyrate as substrate, LysB-His6 enzymes showed highest activity at 37 °C. LysB-His6 enzymes also hydrolyzed different Tween substrates with highest activity against Tween 20 and 80. Metal ions like Ca2+ and Mn2+ enhanced the enzymatic activity of LysB-His6 enzymes, while transition metal ions like Zn2+ and Cu2+ inhibited the enzymatic activity. The mycolylarabinogalactan esterase activity of LysB-His6 enzymes against mAGP complex was confirmed by LC-MS. LysB-His6 enzymes showed marginal antibacterial activity when tested alone against Mycobacterium smegmatis, however a synergetic activity was noticed when combined with outer membrane permealizers. These results confirm that LysB enzymes are lipolytic enzymes with potential application as antimycobacterials.

Functional characterization of the endolysins derived from mycobacteriophage PDRPxv.

Bacteriophage-derived endolysin enzymes play a critical role in disintegration of the host bacterial cell wall and hence have gained considerable attention as possible therapeutics for the treatment of drug-resistant infections. Endolysins can target both dividing and non-dividing cells and given the vital role peptidoglycan plays in bacterial survival, bacteria are less likely to modify it even if continuously exposed to lysins. Hence, probability of bacteria developing resistance to lysins appear bleak. Endolysins from mycobacteriophages offer great potential as alternative therapeutics for the drug-resistant TB. However, considering that a large number of mycobacteriophages have been discovered so far, the information on endolysins come from only a few mycobacteriophages. In this study, we report the structural and functional characterization of endolysins (LysinA and LysinB) encoded by mycobacteriophage PDRPxv which belongs to B1 sub cluster. On in silico analysis, we found LysinA to be a modular protein having peptidase domain at the N-terminal (104 aa), a central amidase domain (174 aa) and the peptidoglycan binding domain (62 aa) at the C-terminal. Additionally, 'H-X-H', which is a conserved motif and characteristic of peptidase domains, and the conserved residues His-His-Asp, which are characteristic of amidase domain were also observed. In LysinB enzyme, a single α/ß hydrolase domain having a catalytic triad (Ser-Asp-His) and G-X-S-X-G motif, which are characteristic of the serine esterase enzymes were predicted to be present. Both the enzymes were purified as recombinant proteins and their antimycobacterial activity against M. smegmatis was demonstrated through turbidimetric experiments and biochemical assay. Interesting observation in this study is the secretory nature of LysinA evident by its periplasmic expression in E.coli, which might explain the ability of PDRPxv to lyse the bacterial host in the absence of transmembrane Holin protein.

A DinB Ortholog Enables Mycobacterial Growth under dTTP-Limiting Conditions Induced by the Expression of a Mycobacteriophage-Derived Ribonucleotide Reductase Gene.

UNLABELLED: Mycobacterium species such as M. smegmatis and M. tuberculosis encode at least two translesion synthesis (TLS) polymerases, DinB1 and DinB2, respectively. Although predicted to be linked to DNA repair, their role in vivo remains enigmatic. M. smegmatis mc(2)155, a strain commonly used to investigate mycobacterial genetics, has two copies of dinB2, the gene that codes for DinB2, by virtue of a 56-kb chromosomal duplication. Expression of a mycobacteriophage D29 gene (gene 50) encoding a class II ribonucleotide reductase in M. smegmatis ΔDRKIN, a strain derived from mc(2)155 in which one copy of the duplication is lost, resulted in DNA replication defects and growth inhibition. The inhibitory effect could be linked to the deficiency of dTTP that resulted under these circumstances. The selective inhibition observed in the ΔDRKIN strain was found to be due solely to a reduced dosage of dinB2 in this strain. Mycobacterium bovis, which is closely related to M. tuberculosis, the tuberculosis pathogen, was found to be highly susceptible to gene 50 overexpression. Incidentally, these slow-growing pathogens harbor one copy of dinB2. The results indicate that the induction of a dTTP-limiting state can lead to growth inhibition in mycobacteria, with the effect being maximum in cells deficient in DinB2. IMPORTANCE: Mycobacterium species, such as M. tuberculosis, the tuberculosis pathogen, are known to encode several Y family DNA polymerases, one of which is DinB2, an ortholog of the DNA repair-related protein DinP of Escherichia coli. Although this protein has been biochemically characterized previously and found to be capable of translesion synthesis in vitro, its in vivo function remains unknown. Using a novel method to induce dTTP deficiency in mycobacteria, we demonstrate that DinB2 can aid mycobacterial survival under such conditions. Apart from unraveling a specific role for the mycobacterial Y family DNA polymerase DinB2 for the first time, this study also paves the way for the development of drugs that can kill mycobacteria by inducing a dTTP-deficient state.

Mycobacteriophage putative GTPase-activating protein can potentiate antibiotics.

The soaring incidences of infection by antimicrobial resistant (AR) pathogens and shortage of effective antibiotics with new mechanisms of action have renewed interest in phage therapy. This scenario is exemplified by resistant tuberculosis (TB), caused by resistant Mycobacterium tuberculosis. Mycobacteriophage SWU1 A321_gp67 encodes a putative GTPase-activating protein. Mycobacterium smegmatis with gp67 overexpression showed changed colony formation and biofilm morphology and supports the efficacy of streptomycin and capreomycin against Mycobacterium. gp67 down-regulated the transcription of genes involved in cell wall and biofilm development. To our knowledge, this is the first report to show that phage protein in addition to lysin or recombination components can synergize with existing antibiotics. Phage components might represent a promising new clue for better antibiotic potentiators.

Targeting intracellular nontuberculous mycobacteria and M. tuberculosis with a bactericidal enzymatic cocktail.

To address intracellular mycobacterial infections, we developed a cocktail of four enzymes that catalytically attack three layers of the mycobacterial envelope. This cocktail is delivered to macrophages, through a targeted liposome presented here as ENTX_001. Endolytix Cocktail 1 (EC1) leverages mycobacteriophage lysin enzymes LysA and LysB, while also including α-amylase and isoamylase for degradation of the mycobacterial envelope from outside of the cell. The LysA family of proteins from mycobacteriophages has been shown to cleave the peptidoglycan layer, whereas LysB is an esterase that hydrolyzes the linkage between arabinogalactan and mycolic acids of the mycomembrane. The challenge of gaining access to the substrates of LysA and LysB provided exogenously was addressed by adding amylase enzymes that degrade the extracellular capsule shown to be present in Mycobacterium tuberculosis. This enzybiotic approach avoids antimicrobial resistance, specific receptor-mediated binding, and intracellular DNA surveillance pathways that limit many bacteriophage applications. We show this cocktail of enzymes is bactericidal in vitro against both rapid- and slow-growing nontuberculous mycobacteria (NTM) as well as M. tuberculosis strains. The EC1 cocktail shows superior killing activity when compared to previously characterized LysB alone. EC1 is also powerfully synergistic with standard-of-care antibiotics. In addition to in vitro killing of NTM, ENTX_001 demonstrates the rescue of infected macrophages from necrotic death by Mycobacteroides abscessus and Mycobacterium avium. Here, we demonstrate shredding of mycobacterial cells by EC1 into cellular debris as a mechanism of bactericide.IMPORTANCEThe world needs entirely new forms of antibiotics as resistance to chemical antibiotics is a critical problem facing society. We addressed this need by developing a targeted enzyme therapy for a broad range of species and strains within mycobacteria and highly related genera including nontuberculous mycobacteria such as Mycobacteroides abscessus, Mycobacterium avium, Mycobacterium intracellulare, as well as Mycobacterium tuberculosis. One advantage of this approach is the ability to drive our lytic enzymes through encapsulation into macrophage-targeted liposomes resulting in attack of mycobacteria in the cells that harbor them where they hide from the adaptive immune system and grow. Furthermore, this approach shreds mycobacteria independent of cell physiology as the drug targets the mycobacterial envelope while sidestepping the host range limitations observed with phage therapy and resistance to chemical antibiotics.

Mycobacteriophage Ms6 LysA: a peptidoglycan amidase and a useful analytical tool.

Since the peptidoglycan isolated from Mycobacterium spp. is refractory to commercially available murolytic enzymes, possibly due to the presence of various modifications found on this peptidoglycan, the utility of a mycobacteriophage-derived murolytic enzyme was assessed for an analysis of peptidoglycan from mycobacteria. We cloned, expressed, and purified the lysA gene product, a protein with homology to known peptidoglycan-degrading amidases, from bacteriophage Ms6. The recombinant protein was shown to cleave the bond between l-Ala and d-muramic acid of muramyl pentapeptide and to release up to 70% of the diaminopimelic acid present in the isolated mycobacterial cell wall. In contrast to lysozyme, which, in culture, inhibits the growth of both Mycobacterium smegmatis and Mycobacterium tuberculosis, LysA had no effect on the growth of either species. However, the enzyme is useful for solubilizing the peptide chains of isolated mycobacterial peptidoglycan for analysis. The data indicate that the stem peptides from M. smegmatis are heavily amidated, containing few free carboxylic acids, regardless of the cross-linking status.

Functional analysis of the holin-like proteins of mycobacteriophage Ms6.

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(Δgp4)) caused slightly accelerated lysis, whereas an Ms6(Δgp5) 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.

Splicing of the mycobacteriophage Bethlehem DnaB intein: identification of a new mechanistic class of inteins that contain an obligate block F nucleophile.

Inteins are single turnover enzymes that splice out of protein precursors during maturation of the host protein (extein). The Cys or Ser at the N terminus of most inteins initiates a four-step protein splicing reaction by forming a (thio)ester bond at the N-terminal splice junction. Several recently identified inteins cannot perform this acyl rearrangement because they do not begin with Cys, Thr, or Ser. This study analyzes one of these, the mycobacteriophage Bethlehem DnaB intein, which we describe here as the prototype for a new class of inteins based on sequence comparisons, reactivity, and mechanism. These Class 3 inteins are characterized by a non-nucleophilic N-terminal residue that co-varies with a non-contiguous Trp, Cys, Thr triplet (WCT) and a Thr or Ser as the first C-extein residue. Several mechanistic differences were observed when compared with standard inteins or previously studied atypical KlbA Ala(1) inteins: (a) cleavage at the N-terminal splice junction in the absence of all standard N- and C-terminal splice junction nucleophiles, (b) activation of the N-terminal splice junction by a variant Block B motif that includes the WCT triplet Trp, (c) decay of the branched intermediate by thiols or Cys despite an ester linkage at the C-extein branch point, and (d) an absolute requirement for the WCT triplet Block F Cys. Based on biochemical data and confirmed by molecular modeling, we propose roles for these newly identified conserved residues, a novel protein splicing mechanism that includes a second branched intermediate, and an intein classification with three mechanistic categories.

Two-step site selection for serine-integrase-mediated excision: DNA-directed integrase conformation and central dinucleotide proofreading.

Bacteriophage-encoded serine-integrases are members of the large family of serine-recombinases and catalyze site-specific integrative recombination between a phage attP site and a bacterial attB site to form an integrated prophage. Prophage excision involves a second site-specific recombination event, in which the sites generated by integration, attL and attR, are used as substrates to regenerate attP and attB. Excision is catalyzed by integrase but also requires a phage-encoded recombination directionality factor (RDF). The Bxb1 recombination sites, attP and attB, are small (<50 bp), different in sequence, and quasisymmetrical, and they give rise to attL- and attR-recombinant products that are asymmetric but similar to each other, each being composed of B- and P-type half-sites. We show here that the determination of correct excision products is a two-step process, with a presynaptic RDF-dependent step that aligns attL and attR in the correct orientation and a postsynaptic step in which the nonpalindromic central dinucleotide confers identity to attL and attR and prevents each from recombining with itself.

Mycobacteriophage Lysin B is a novel mycolylarabinogalactan esterase.

Mycobacteriophages encounter a unique problem among phages of Gram-positive bacteria, in that lysis must not only degrade the peptidoglycan layer but also circumvent a mycolic acid-rich outer membrane covalently attached to the arabinogalactan-peptidoglycan complex. Mycobacteriophages accomplish this by producing two lysis enzymes, Lysin A (LysA) that hydrolyses peptidoglycan, and Lysin B (LysB), a novel mycolylarabinogalactan esterase, that cleaves the mycolylarabinogalactan bond to release free mycolic acids. The D29 LysB structure shows an alpha/beta hydrolase organization with a catalytic triad common to cutinases, but which contains an additional four-helix domain implicated in the binding of lipid substrates. Whereas LysA is essential for mycobacterial lysis, a Giles DeltalysB mutant mycobacteriophage is viable, but defective in the normal timing, progression and completion of host cell lysis. We propose that LysB facilitates lysis by compromising the integrity of the mycobacterial outer membrane linkage to the arabinogalactan-peptidoglycan layer.

Mycobacteriophage Ms6 LysB specifically targets the outer membrane of Mycobacterium smegmatis.

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.

The mycobacteriophage D29 gene 65 encodes an early-expressed protein that functions as a structure-specific nuclease.

The genomes of mycobacteriophages of the L5 family, which includes the lytic phage D29, contain several genes putatively linked to DNA synthesis. One such gene is 65, which encodes a protein belonging to the RecA/DnaB helicase superfamily. In this study a recombinant version of the mycobacteriophage D29 gp65 was functionally characterized. The results indicated that it is not a helicase as predicted but an exonuclease that removes 3' arms from forked structures in an ATP-dependent manner. The gp65 exonuclease acts progressively from the 3' end, until the fork junction is reached. As it goes past, its progress is stalled over a stretch of seven to eight nucleotides immediately downstream of the junction. It efficiently acts on forked structures with single stranded arms. It also acts upon 5' and 3' flaps, though with somewhat relaxed specificity, but not on double-stranded forks. Sequence comparison revealed the presence of a KNRXG motif in the C-terminal half of the protein. This is a conserved element found in the RadA/Sms family of DNA repair proteins. A mutation (R203G) in this motif led to complete loss of nuclease activity. This indicated that KNRXG plays an important role in the nuclease function of not only gp65, but possibly other RadA/Sms family proteins as well. This is the first characterization of a bacteriophage-derived RadA/Sms class protein. Given its mode of action, it is very likely that gp65 is involved in processing branched replication intermediates formed during the replication of phage DNA.

Efficient point mutagenesis in mycobacteria using single-stranded DNA recombineering: characterization of antimycobacterial drug targets.

Construction of genetically isogenic strains of mycobacteria is complicated by poor recombination rates and the lack of generalized transducing phages for Mycobacterium tuberculosis. We report here a powerful method for introducing single point mutations into mycobacterial genomes using oligonucleotide-derived single-stranded DNA recombineering and mycobacteriophage-encoded proteins. Phage Che9c gp61-mediated recombination is sufficiently efficient that single base changes can be introduced without requirement for direct selection, with isogenic mutant strains identified simply by PCR. Efficient recombination requires only short (50 nucleotide) oligonucleotides, but there is an unusually strong strand bias and an oligonucleotide targeting lagging strand DNA synthesis can recombine more than 10,000-fold efficiently than its complementary oligonucleotide. This ssDNA recombineering provides a simple assay for comparing the activities of related phage recombinases, and we find that both Escherichia coli RecET and phage lambda Red recombination proteins function inefficiently in mycobacteria, illustrating the utility of developing recombineering in new bacterial systems using host-specific bacteriophage recombinases. ssDNA mycobacterial recombineering provides a simple approach to characterizing antimycobacterial drug targets, and we have constructed and characterized single point mutations that confer resistance to isoniazid, rifampicin, ofloxacin and streptomycin.

Comparative Structural Analysis of Different Mycobacteriophage-Derived Mycolylarabinogalactan Esterases (Lysin B).

Mycobacteriophage endolysins have emerged as a potential alternative to the current antimycobacterial agents. This study focuses on mycolylarabinogalactan hydrolase (LysB) enzymes of the α/ß-hydrolase family, which disrupt the unique mycolic acid layer of mycobacterium cell wall. Multiple sequence alignment and structural analysis studies showed LysB-D29, the only enzyme with a solved three-dimensional structure, to share several common features with esterases (lacking lid domain) and lipases (acting on long chain lipids). Sequence and structural comparisons of 30 LysB homology models showed great variation in domain organizations and total protein length with major differences in the loop-5 motif harboring the catalytic histidine residue. Docking of different p-nitrophenyl ligands (C4-C18) to LysB-3D models revealed that the differences in length and residues of loop-5 contributed towards wide diversity of active site conformations (long tunnels, deep and superficial funnels, shallow bowls, and a narrow buried cave) resembling that of lipases, cutinases, and esterases. A set of seven LysB enzymes were recombinantly produced; their activity against p-nitrophenyl esters could be related to their active site conformation and acyl binding site. LysB-D29 (long tunnel) showed the highest activity with long chain p-nitrophenyl palmitate followed by LysB-Omega (shallow bowl) and LysB-Saal (deep funnel).

Antimicrobial activity of Mycobacteriophage D29 Lysin B during Mycobacterium ulcerans infection.

Buruli Ulcer (BU) is a cutaneous disease caused by Mycobacterium ulcerans. The pathogenesis of this disease is closely related to the secretion of the toxin mycolactone that induces extensive destruction of the skin and soft tissues. Currently, there are no effective measures to prevent the disease and, despite availability of antibiotherapy and surgical treatments, these therapeutic options are often associated with severe side effects. Therefore, it is important to develop alternative strategies for the treatment of BU. Endolysins (lysins) are phage encoded enzymes that degrade peptidoglycan of bacterial cell walls. Over the past years, lysins have been emerging as alternative antimicrobial agents against bacterial infections. However, mycobacteria have an unusual outer membrane composed of mycolylarabinogalactan-peptidoglycan. To overcome this complex barrier, some mycobacteriophages encode a lipolytic enzyme, Lysin B (LysB). In this study, we demonstrate for the first time that recombinant LysB displays lytic activity against M. ulcerans isolates. Moreover, using a mouse model of M. ulcerans footpad infection, we show that subcutaneous treatment with LysB prevented further bacterial proliferation, associated with IFN-γ and TNF production in the draining lymph node. These findings highlight the potential use of lysins as a novel therapeutic approach against this neglected tropical disease.

Cloning, characterization and expression analysis of nucleotide metabolism-related genes of mycobacteriophage L5.

The genomes of mycobacteriophages of the L5 family, which includes the lytic phage D29, contain several genes putatively linked to nucleotide-metabolizing functions. Two such genes, 48 and 50, encoding thymidylate synthase and ribonucleotide reductase (RNR), respectively, were overexpressed in Escherichia coli and the recombinant proteins were biochemically characterized. It was established that Gp50 was a class II RNR having properties similar to that of the corresponding enzyme from Lactobacillus leichmanni, whereas Gp48 was a flavin-dependent thymidylate synthase (ThyX) that resembled the Paramecium bursaria chlorella virus-1 ThyX enzyme in its properties. That both these proteins play a role in phage development was evident from the observation that they were detectable soon after the lytic phase of growth commenced. Gp48 and 50 were also found to coimmunoprecipitate, which indicates the possible existence of an L5 thymidylate synthase complex. Thymidylate synthase assays revealed that during the intracellular stage of phage growth, a significant decrease in the host thymidylate synthase (ThyA) activity occurred. It appears that synthesis of the viral enzyme (ThyX) is necessary to compensate for this loss in activity. In general, the results suggest that phage-encoded nucleotide metabolism-related functions play an important role in the lytic propagation of L5 and related mycobacteriophages.

The delayed early gene G23 of temperate mycobacteriophage L1 regulates the expression of deoxyribonuclease, the product of another delayed early gene of the phage.

To get clues about the genes as well as the gene regulatory circuit controlling the lytic development of temperate mycobacteriophage L1, previously we screened several conditional lethal mutants of L1 and characterized some of them to an extent. One of the mutants, L1 G23ts23, was found defective in both growth and late gene transcription at 42 degrees C but not at 32 degrees C. Here we show that the above phage mutant is also defective in the expression of phage-coded deoxyribonuclease (DNase) at 42 degrees C but not at 32 degrees C. The G23 gene however does not code for the above enzyme. Further analyses using the L1 G23ts23 mutant suggest that synthesis of DNase is also not regulated by G23 at transcriptional level. Expression of functional DNase in fact requires de novo protein synthesis. Among the 25 revertants isolated from the L1 G23ts23 mutant, which are capable of growing at 42 degrees C (by overcoming the ts defect in late transcription), two, R4 and R22, have been shown to retain the ts defect in the expression of the above enzyme and R4, to retain also the G23ts23 mutation. This suggests that R4 (R22 was not tested for the presence of G23ts23 mutation) carries an extragenic suppressor of G23ts23 mutation in a different gene (we call this putative gene as Gx), which now helps bypass the requirement of G23 for late gene transcription. Possible role of G23 on the regulation of L1-coded Gx and deoxyribonuclease has been discussed at length.

Expression, purification and crystallization of phosphoribosyl transferase from a mycobacteriophage.

Tuberculosis (TB) continues to remain a leading cause of death globally. Of particular concern is the emergence and rise in incidence of multidrug-resistant and extremely drug-resistant cases of TB. To counter this threat, it is important to explore alternative therapies, including phage therapy. Phage BTCU-1 specifically infects Mycobacterium spp. and kills the majority of them. Intriguingly, many proteins from the phage do not share high amino-acid sequence identity with proteins from species other than phages. Here, the expression, purification and crystallization of one such protein, a putative phosphoribosyl transferase from phage BTCU-1, is reported. The crystals belonged to space group C2221, with unit-cell parameters a = 59.71, b = 64.42, c = 65.32 Å, α = ß = γ = 90°. The crystals diffracted X-rays to 2.2 Šresolution.

Mycobacteriophage Lysis Enzymes: Targeting the Mycobacterial Cell Envelope.

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.

In silico analysis of mycobacteriophage Che12 genome: characterization of genes required to lysogenise Mycobacterium tuberculosis.

Che12 is a temperate Chennai phage infecting Mycobacterium tuberculosis. The nucleotide sequence of the 52,047 bp linear double stranded DNA genome has a GC content of 62.9% with 70 putative ORFs identified. Functions are assigned to 24 genes based on the similarity of the predicted products to known proteins. Che12 genome is highly similar to mycobacteriophage L5 and D29 genomes. The overall genome similarity of Che12 to L5 is 82.5% and D29 is 81.5%. The genes attributing to lysogeny such as integrase, excisionase and repressor protein are identified. The attachment site of Che12 genome attP is homologous to attB sites of Mycobacterium smegmatis and M. tuberculosis. Similarities between certain phage gene products are noted, in particular, the terminases, DNA primase and endonucleases. The complete sequence clarifies the overall transcription map of Che12 and the positions of elements involved in the maintenance of lysogeny.