Characterization of Mycobacteriophage Adephagia Cytotoxic Proteins.

Mycobacterium phage Adephagia is a Cluster K phage that infects Mycobacterium smegmatis and some strains of Mycobacterium pathogens. Adephagia has a siphoviral virion morphology and is temperate. Its genome is 59,646 bp long and codes for one tRNA gene and 94 predicted protein-coding genes; most genes not associated with virion structure and assembly are functionally ill-defined. Here, we determined the Adephagia gene expression patterns in lytic and lysogenic growth and used structural predictions to assign additional gene functions. We characterized 66 non-structural genes for their toxic phenotypes when expressed in M. smegmatis, and we show that 25 of these (38%) are either toxic or strongly inhibit growth, resulting in either reduced viability or small colony sizes. Some of these genes are predicted to be involved in DNA metabolism or regulation, but others are of unknown function. We also characterize the HicAB-like toxin-antitoxin system encoded by Adephagia (gp91 and gp90, respectively) and show that the gp90 antitoxin is lysogenically expressed, abrogates gp91 toxicity, and is required for normal lytic and lysogenic growth.

Mycobacteriophage Alexphander Gene 94 Encodes an Essential dsDNA-Binding Protein during Lytic Infection.

Mycobacteriophages are viruses that specifically infect bacterial species within the genera Mycobacterium and Mycolicibacterium. Over 2400 mycobacteriophages have been isolated on the host Mycolicibacterium smegmatis and sequenced. This wealth of genomic data indicates that mycobacteriophage genomes are diverse, mosaic, and contain numerous (35-60%) genes for which there is no predicted function based on sequence similarity to characterized orthologs, many of which are essential to lytic growth. To fully understand the molecular aspects of mycobacteriophage-host interactions, it is paramount to investigate the function of these genes and gene products. Here we show that the temperate mycobacteriophage, Alexphander, makes stable lysogens with a frequency of 2.8%. Alexphander gene 94 is essential for lytic infection and encodes a protein predicted to contain a C-terminal MerR family helix-turn-helix DNA-binding motif (HTH) and an N-terminal DinB/YfiT motif, a putative metal-binding motif found in stress-inducible gene products. Full-length and C-terminal gp94 constructs form high-order nucleoprotein complexes on 100-500 base pair double-stranded DNA fragments and full-length phage genomic DNA with little sequence discrimination for the DNA fragments tested. Maximum gene 94 mRNA levels are observed late in the lytic growth cycle, and gene 94 is transcribed in a message with neighboring genes 92 through 96. We hypothesize that gp94 is an essential DNA-binding protein for Alexphander during lytic growth. We proposed that gp94 forms multiprotein complexes on DNA through cooperative interactions involving its HTH DNA-binding motif at sites throughout the phage chromosome, facilitating essential DNA transactions required for lytic propagation.

Construing the function of N-terminal domain of D29 mycobacteriophage LysA endolysin in phage lytic efficiency and proliferation.

Endolysins produced by bacteriophages hydrolyze host cell wall peptidoglycan to release newly assembled virions. D29 mycobacteriophage specifically infects mycobacteria including the pathogenic Mycobacterium tuberculosis. D29 encodes LysA endolysin, which hydrolyzes mycobacterial cell wall peptidoglycan. We previously showed that LysA harbors two catalytic domains (N-terminal domain [NTD] and lysozyme-like domain [LD]) and a C-terminal cell wall binding domain (CTD). While the importance of LD and CTD in mycobacteriophage biology has been examined in great detail, NTD has largely remained unexplored. Here, to address NTD's significance in D29 physiology, we generated NTD-deficient D29 (D29∆NTD) by deleting the NTD-coding region from D29 genome using CRISPY-BRED. We show that D29∆NTD is viable, but has a longer latent period, and a remarkably reduced burst size and plaque size. A large number of phages were found to be trapped in the host during the D29∆NTD-mediated cell lysis event. Such poor release of progeny phages during host cell lysis strongly suggests that NTD-deficient LysA produced by D29∆NTD, despite having catalytically-active LD, is unable to efficiently lyse host bacteria. We thus conclude that LysA NTD is essential for optimal release of progeny virions, thereby playing an extremely vital role in phage physiology and phage propagation in the environment.

Mycobacteriophages: therapeutic approach for mycobacterial infections.

Tuberculosis (TB) is a significant global health threat, and cases of infection with non-tuberculous mycobacteria (NTM) causing lung disease (NTM-LD) are rising. Bacteriophages and their gene products have garnered interest as potential therapeutic options for bacterial infections. Here, we have compiled information on bacteriophages and their products that can kill Mycobacterium tuberculosis or NTM. We summarize the mechanisms whereby viable phages can access macrophage-resident bacteria and not elicit immune responses, review methodologies of pharmaceutical product development containing mycobacteriophages and their gene products, mainly lysins, in the context of drug regulatory requirements and we discuss industrially relevant methods for producing pharmaceutical products comprising mycobacteriophages, emphasizing delivery of mycobacteriophages to the lungs. We conclude with an outline of some recent case studies on mycobacteriophage therapy.

Complete genome of a novel mycobacteriophage WXIN isolated in Wuhan, China.

OBJECTIVES: The rising of antibiotic resistance has sparked a renewed interest in mycobacteriophage as alternative therapeutic strategies against mycobacterial infections. So far, the vast majority of mycobacteriophages have been isolated using the model species Mycobacterium smegmatis, implying an overwhelming majority of mycobacteriophages in the environment remain uncultured, unclassified, and their specific hosts and infection strategies are still unknown. This study was undertaken to isolate and characterize novel mycobacteriophages targeting Mycobacterium septicum. DATA DESCRIPTION: Here a novel mycobacteriophage WXIN against M. septicum was isolated from soil samples in Wuhan, China. Whole genome analysis indicates that the phage genome consists of 115,158 bp with a GC content of 61.9%. Of the 260 putative open reading frames, 46 may be associated with phage packaging, structure, lysis, lysogeny, genome modification/replication, and other functional roles. The limited genome-wide similarity, along with phylogenetic trees constructed based on viral proteome and orthologous genes show that phage WXIN represents a novel cluster distantly related to cluster J mycobacteriophages (genus Omegavirus). Overall, these results provide novel insights into the genomic properties of mycobacteriophages, highlighting the great genetic diversity of mycobacteriophages in relation to their hosts.

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.

Mycobacterium tuberculosis Rv2617c is involved in stress response and phage infection resistance.

Mycobacterium tuberculosis (M. tuberculosis) is the pathogen of human tuberculosis (TB). Resistance to numerous in vivo stresses, including oxidative stress, is determinant for M. tuberculosis intracellular survival, and understanding associated mechanisms is crucial for developing new therapeutic strategies. M. tuberculosis Rv2617c has been associated with oxidative stress response when interacting with other proteins in M. tuberculosis; however, its functional promiscuity and underlying molecular mechanisms remain elusive. In this study, we investigated the phenotypic changes of Mycobacterium smegmatis (M. smegmatis) expressing Rv2617c (Ms_Rv2617c) and its behavior in the presence of various in vitro stresses and phage infections. We found that Rv2617c conferred resistance to SDS and diamide while sensitizing M. smegmatis to oxidative stress (H2O2) and altered mycobacterial phenotypic properties (single-cell clone and motility), suggestive of reprogrammed mycobacterial cell wall lipid contents exemplified by increased cell wall permeability. Interestingly, we also found that Rv2617c promoted M. smegmatis resistance to infection by phages (SWU1, SWU2, D29, and TM4) and kept phage TM4 from destroying mycobacterial biofilms. Our findings provide new insights into the role of Rv2617c in resistance to oxide and acid stresses and report for the first time on its role in phage resistance in Mycobacterium.

Genomic analysis of seven mycobacteriophages identifies three novel species with differing phenotypic stabilities.

Recently, case studies have been published regarding the application of mycobacteriophage (MP) therapy (MPT) in patients with multi-antibiotic-resistant infections. A major limitation in the development of MPT is the paucity of therapeutically useful MP. As there are approximately 10,000 MP that have yet to be sequenced, it is possible that characterization of this cohort would increase the repertoire of useful MP. This study aims to contribute to such a strategy, by characterizing a cohort of 7 mycobacteriophages. Sequencing analyses revealed that the MP have unique sequences, and subsequent gene annotation revealed differences in gene organization. Notably, MP LOCARD has the largest genome and operons encoding for glycosyltransferases. Taxonomic analysis executed with VIRIDIC, Gegenees and VICTOR revealed that LOCARD belongs to a different genus than the other phages and is the foundational member of one of three novel species identified in this study. LOCARD, LOCV2, and LOCV5 were selected as representative members of their species and subjected to phenotypic analyses to compare their stability under biologically and industrially relevant conditions. Again LOCARD stood out, as it was unaffected by the typical temperatures (37 °C) and salinity (0.9%) experienced in mammals, while the viability of LOCV2 and LOCV5 was significantly reduced. LOCARD was also tolerant to pH 10, low levels of antiviral detergent and was the least impacted by a single freeze-thaw cycle. When all these results are considered, it indicates that LOCARD in particular, has potential therapeutic and/or diagnostics applications, given its resilience towards physiological and storage conditions.

Insights into the genomic features and lifestyle of B1 subcluster mycobacteriophages.

Bacteriophages infecting Mycobacterium smegmatis mc2155 are numerous and, hence, are classified into clusters based on nucleotide sequence similarity. Analyzing phages belonging to clusters/subclusters can help gain deeper insights into their biological features and potential therapeutic applications. In this study, for genomic characterization of B1 subcluster mycobacteriophages, a framework of online tools was developed, which enabled functional annotation of about 55% of the previously deemed hypothetical proteins in B1 phages. We also studied the phenotype, lysogeny status, and antimycobacterial activity of 10 B1 phages against biofilm and an antibiotic-resistant M. smegmatis strain (4XR1). All 10 phages belonged to the Siphoviridae family, appeared temperate based on their spontaneous release from the putative lysogens and showed antibiofilm activity. The highest inhibitory and disruptive effects on biofilm were 64% and 46%, respectively. This systematic characterization using a combination of genomic and experimental tools is a promising approach to furthering our understanding of viral dark matter.

Complete genome sequences of mycobacteriophages Ageofdapage, Aubs, BABullseye, CheetoDust, ShaboiShabazz, and TomBrady.

We report genome sequences of six mycobacteriophages. Each virus was isolated from a soil sample and belongs to the siphovirus morphology. Genomes are 41,901-60,613 bp in length, contain between 62 and 103 protein-coding genes, with up to 40% of those genes having a predicted function.

Complete genome sequences of Mycobacterium smegmatis phages Ashballer and Bombitas.

We report the discovery of two mycobacteriophages isolated from soil in Rock Hill, South Carolina. Ashballer has a genome sequence length of 52,231 bp, while Bombitas is relatively larger at 110,129 bp. Both have siphovirus morphologies and have temperate lifecycles.

A novel synergistic enzyme-antibiotic therapy with immobilization of mycobacteriophage Lysin B enzyme onto Rif@UiO-66 nanocomposite for enhanced inhaled anti-TB therapy; Nanoenzybiotics approach.

The emergence of antibiotic-resistant and phage-resistant strains of Mycobacterium tuberculosis (M. tuberculosis) necessitates improving new therapeutic plans. The objective of the current work was to ensure the effectiveness of rifampicin and the mycobacteriophage LysB D29 (LysB)enzyme in the treatment of multi-drug resistant tuberculosis (MDR-TB) infection, where new and safe metal-organic framework (MOF) nanoparticles were used in combination. UiO-66 nanoparticles were synthesized under mild conditions in which the antimycobacterial agent (rifampicin) was loaded (Rif@UiO-66) and LysB D29 enzyme immobilized onto Rif@UiO-66, which were further characterized. Subsequently, the antibacterial activity of different ratios of Rif@UiO-66 and LysB/Rif@uio-66 against the nonpathogenic tuberculosis model Mycobacterium smegmatis (M. smegmatis) was evaluated by minimum inhibitory concentration (MIC) tests. Impressively, the MIC of LysB/Rif@uio-66 was 16-fold lower than that of pure rifampicin. In vitro and in vivo toxicity studies proved that LysB/Rif@UiO-66 is a highly biocompatible therapy for pulmonary infection. A biodistribution assay showed that LysB/Rif@UiO-66 showed a 5.31-fold higher drug concentration in the lungs than free rifampicin. A synergistic interaction between UiO-66, rifampicin and the mycobacteriophage lysB D29 enzyme was shown in the computational method (docking). Therefore, all results indicated that the LysB/Rif@UiO-66 nanocomposite exhibited promising innovative enzyme-antibiotic therapy for tuberculosis treatment.

Sequence analysis of Discoknowium, an A5 mycobacteriophage.

Discoknowium is a temperate A5 bacteriophage that infects the bacterial host Mycobacterium smegmatis. Isolated from a rat fecal sample, Discoknowium's genome is 50,222 bp in length, contains 84 genes and 1 tRNA, and shares 82%-98% nucleotide identity with other A5 subcluster phages.

Identification of novel genera and subcluster classifications for mycobacteriophages.

Aim: To identify novel genera amongst mycobacteriophages (MP) and verify a hypothesised correlation between the taxonomy set by the International Committee on Taxonomy of Viruses (ICTV) and the National Centre for Biotechnology Information (NCBI) with that of the Actinobacteriophage Database, which may help formalise subcluster assignment. Methods: A dataset of 721 MP genomes was analysed using VIRIDIC, a nucleotide alignment-based software that predicts genus assignments. Potentially novel genera were analysed using Gegenees and VICTOR, respectively. These genera were then compared to the subclusters assigned by the Actinobacteriophage Database to verify a hypothesis that one genus can be assigned to one subcluster (i.e., the genus-subcluster hypothesis). Results: Initially, when comparing the current genus classifications of the 721 MP dataset to the Actinobacteriophage database subcluster assignments, 83.3% of subclusters supported the genus-subcluster hypothesis. Following the sequential VIRIDIC, Gegenees and VICTOR analyses, a total of 20 novel genera were identified based on a ≥ 70% and ~ 50% similarity threshold for VIRIDIC and Gegenees, respectively, and a monophyletic nature in the VICTOR output. Interestingly, these criteria also appear to support the creation of 13 novel subclusters, which would increase the support for the genus-subcluster hypothesis to 97.6%. Conclusion: The link between genus and subcluster classifications appears robust, as most subclusters can be assigned a single genus and vice versa. By relating the taxonomic and clustering classification systems, they can be easily kept up to date to best reflect MP diversity, which could aid the rapid selection of related (or diverse) phages for research, therapeutic and diagnostic purposes.

Alginate-Encapsulated Mycobacteriophage: A Potential Approach for the Management of Intestinal Mycobacterial Disease.

Encapsulated medication is a common method of administering therapeutic treatments. As researchers explore alternative therapies, it is likely that encapsulation will remain a feature of these novel treatments, particularly when routes of delivery are considered. For instance, alginate-encapsulation is often favoured where gastric digestion poses an obstacle. When exposed to cations (namely Ca2+), alginate readily forms gels that are resilient to acidic conditions and readily dissociate in response to mid-range pH. This action can be extremely valuable for the encapsulation of phages. The efficient delivery of phages to the intestine is important when considering mycobacteriophage (MP) therapy (or MP prophylaxis) for disseminated mycobacterial infections and chronic gastroenteritis conditions. This study presents the design and in vitro validation of an alginate-encapsulated MP capable of releasing phages in a pH-dependent manner. Ultimately, it is shown that encapsulated phages pretreated with simulated gastric fluid (SGF) are capable of releasing viable phages into simulated intestinal fluid (SIF) and thereby reducing the mycobacterial numbers in spiked SIF by 90%. These findings suggest that alginate encapsulation may be a viable option for therapeutic and prophylactic approaches to the management of intestinal mycobacterial disease, such as Johne's disease.

A genome-wide overexpression screen reveals Mycobacterium smegmatis growth inhibitors encoded by mycobacteriophage Hammy.

During infection, bacteriophages produce diverse gene products to overcome bacterial antiphage defenses, to outcompete other phages, and to take over cellular processes. Even in the best-studied model phages, the roles of most phage-encoded gene products are unknown, and the phage population represents a largely untapped reservoir of novel gene functions. Considering the sheer size of this population, experimental screening methods are needed to sort through the enormous collection of available sequences and identify gene products that can modulate bacterial behavior for downstream functional characterization. Here, we describe the construction of a plasmid-based overexpression library of 94 genes encoded by Hammy, a Cluster K mycobacteriophage closely related to those infecting clinically important mycobacteria. The arrayed library was systematically screened in a plate-based cytotoxicity assay, identifying a diverse set of 24 gene products (representing ∼25% of the Hammy genome) capable of inhibiting growth of the host bacterium Mycobacterium smegmatis. Half of these are related to growth inhibitors previously identified in related phage Waterfoul, supporting their functional conservation; the other genes represent novel additions to the list of known antimycobacterial growth inhibitors. This work, conducted as part of the HHMI-supported Science Education Alliance Gene-function Exploration by a Network of Emerging Scientists (SEA-GENES) project, highlights the value of parallel, comprehensive overexpression screens in exploring genome-wide patterns of phage gene function and novel interactions between phages and their hosts.

Activity of Bacteriophage D29 Loaded on Nanoliposomes against Macrophages Infected with Mycobacterium tuberculosis.

The search for new antimicrobial agents is a continuous struggle, mainly because more and more cases of resistant strains are being reported. Mycobacterium tuberculosis (MTB) is the main microorganism responsible for millions of deaths worldwide. The development of new antimicrobial agents is generally aimed at finding strong interactions with one or more bacterial receptors. It has been proven that bacteriophages have the ability to adhere to specific and selective regions. However, their transport and administration must be carefully evaluated as an excess could prevent a positive response and the bacteriophages may be eliminated during their journey. With this in mind, the mycobacteriophage D29 was encapsulated in nanoliposomes, which made it possible to determine its antimicrobial activity during transport and its stability in the treatment of active and latent Mycobacterium tuberculosis. The antimicrobial activity, the cytotoxicity in macrophages and fibroblasts, as well as their infection and time-kill were evaluated. Phage nanoencapsulation showed efficient cell internalization to induce MTB clearance with values greater than 90%. Therefore, it was shown that nanotechnology is capable of assisting in the activity of degradation-sensitive compounds to achieve better therapy and evade the immune response against phages during treatment.

Mycobacteriophages in diagnosis and alternative treatment of mycobacterial infections.

Antimicrobial resistance is an increasing threat to human populations. The emergence of multidrug-resistant "superbugs" in mycobacterial infections has further complicated the processes of curing patients, thereby resulting in high morbidity and mortality. Early diagnosis and alternative treatment are important for improving the success and cure rates associated with mycobacterial infections and the use of mycobacteriophages is a potentially good option. Since each bacteriophage has its own host range, mycobacteriophages have the capacity to detect specific mycobacterial isolates. The bacteriolysis properties of mycobacteriophages make them more attractive when it comes to treating infectious diseases. In fact, they have been clinically applied in Eastern Europe for several decades. Therefore, mycobacteriophages can also treat mycobacteria infections. This review explores the potential clinical applications of mycobacteriophages, including phage-based diagnosis and phage therapy in mycobacterial infections. Furthermore, this review summarizes the current difficulties in phage therapy, providing insights into new treatment strategies against drug-resistant mycobacteria.

Mycobacteriophage D29 Lysin B exhibits promising anti-mycobacterial activity against drug-resistant Mycobacterium tuberculosis.

IMPORTANCE: To combat the rapidly emerging drug-resistant M. tuberculosis, it is now essential to look for alternative therapeutics. Mycobacteriophages can be considered as efficient therapeutics due to their natural ability to infect and kill mycobacteria including M. tuberculosis. Here, we have exploited the mycolyl-arabinogalactan esterase property of LysB encoded from mycobacteriophage D29. This study is novel in terms of targeting a multi-drug-resistant pathogenic strain of M. tuberculosis with LysB and also examining the combination of anti-TB drugs and LysB. All the experiments include external administration of LysB. Therefore, the remarkable lytic activity of LysB overcomes the difficulty to enter the complex cell envelope of mycobacteria. Targeting the intracellularly located M. tuberculosis by LysB and non-toxicity to macrophages take the process of the development of LysB as a drug one step ahead, and also, the interaction studies with rifampicin and isoniazid will help to form a new treatment regimen against tuberculosis.

Isolating and characterizing cluster AB Mycobacteriophage NoShow, which encodes lysis proteins shared with cluster H2 phages.

We present here Mycobacteriophage NoShow, isolated from a soil sample collected on the Maguire Campus of Saint Joseph's University in Merion Station, Pennsylvania. Even though NoShow's 52,825 bp genome is most similar to phages in cluster AB, its lysA and lysB genes are most similar to phages in cluster H2.