CRISPRi-mediated characterization of novel anti-tuberculosis targets: Mycobacterial peptidoglycan modifications promote beta-lactam resistance and intracellular survival.

The lack of effective therapeutics against emerging multi-drug resistant strains of Mycobacterium tuberculosis (Mtb) prompts the identification of novel anti-tuberculosis targets. The essential nature of the peptidoglycan (PG) layer of the mycobacterial cell wall, which features several distinctive modifications, such as the N-glycolylation of muramic acid and the amidation of D-iso-glutamate, makes it a target of particular interest. To understand their role in susceptibility to beta-lactams and in the modulation of host-pathogen interactions, the genes encoding the enzymes responsible for these PG modifications (namH and murT/gatD, respectively) were silenced in the model organism Mycobacterium smegmatis using CRISPR interference (CRISPRi). Although beta-lactams are not included in TB-therapy, their combination with beta-lactamase inhibitors is a prospective strategy to treat MDR-TB. To uncover synergistic effects between the action of beta-lactams and the depletion of these PG modifications, knockdown mutants were also constructed in strains lacking the major beta-lactamase of M. smegmatis BlaS, PM965 (M. smegmatis ΔblaS1) and PM979 (M. smegmatis ΔblaS1 ΔnamH). The phenotyping assays affirmed the essentiality of the amidation of D-iso-glutamate to the survival of mycobacteria, as opposed to the N-glycolylation of muramic acid. The qRT-PCR assays confirmed the successful repression of the target genes, along with few polar effects and differential knockdown level depending on PAM strength and target site. Both PG modifications were found to contribute to beta-lactam resistance. While the amidation of D-iso-glutamate impacted cefotaxime and isoniazid resistance, the N-glycolylation of muramic acid substantially promoted resistance to the tested beta-lactams. Their simultaneous depletion provoked synergistic reductions in beta-lactam MICs. Moreover, the depletion of these PG modifications promoted a significantly faster bacilli killing by J774 macrophages. Whole-genome sequencing revealed that these PG modifications are highly conserved in a set of 172 clinical strains of Mtb, demonstrating their potential as therapeutic targets against TB. Our results support the development of new therapeutic agents targeting these distinctive mycobacterial PG modifications.

Identification of drivers of mycobacterial resistance to peptidoglycan synthesis inhibitors.

Beta-lactams have been excluded from tuberculosis therapy due to the intrinsic resistance of Mycobacterium tuberculosis (Mtb) to this antibiotic class, usually attributed to a potent beta-lactamase, BlaC, and to an unusually complex cell wall. In this pathogen, the peptidoglycan is cross-linked by penicillin-binding proteins (PBPs) and L,D-transpeptidases, the latter resistant to inhibition by most beta-lactams. However, recent studies have shown encouraging results of beta-lactam/beta-lactamase inhibitor combinations in clinical strains. Additional research on the mechanisms of action and resistance to these antibiotics and other inhibitors of peptidoglycan synthesis, such as the glycopeptides, is crucial to ascertain their place in alternative regimens against drug-resistant strains. Within this scope, we applied selective pressure to generate mutants resistant to amoxicillin, meropenem or vancomycin in Mtb H37Rv or Mycolicibacterium smegmatis (Msm) mc2-155. These were phenotypically characterized, and whole-genome sequencing was performed. Mutations in promising targets or orthologue genes were inspected in Mtb clinical strains to establish potential associations between altered susceptibility to beta-lactams and the presence of key genomic signatures. The obtained isolates had substantial increases in the minimum inhibitory concentration of the selection antibiotic, and beta-lactam cross-resistance was detected in Mtb. Mutations in L,D-transpeptidases and major PBPs, canonical targets, or BlaC were not found. The transcriptional regulator PhoP (Rv0757) emerged as a common denominator for Mtb resistance to both amoxicillin and meropenem, while Rv2864c, a lipoprotein with PBP activity, appears to be specifically involved in decreased susceptibility to the carbapenem. Nonetheless, the mutational pattern detected in meropenem-resistant mutants was different from the yielded by amoxicillin-or vancomycin-selected isolates, suggesting that distinct pathways may participate in increased resistance to peptidoglycan inhibitors, including at the level of beta-lactam subclasses. Cross-resistance between beta-lactams and antimycobacterials was mostly unnoticed, and Msm meropenem-resistant mutants from parental strains with previous resistance to isoniazid or ethambutol were isolated at a lower frequency. Although cell-associated nitrocefin hydrolysis was increased in some of the isolates, our findings suggest that traditional assumptions of Mtb resistance relying largely in beta-lactamase activity and impaired access of hydrophilic molecules through lipid-rich outer layers should be challenged. Moreover, the therapeutical potential of the identified Mtb targets should be explored.

Chemical Characterization and Bioactivity of Commercial Essential Oils and Hydrolates Obtained from Portuguese Forest Logging and Thinning.

Essential oils (EOs) and hydrolates (Hds) are natural sources of biologically active ingredients with broad applications in the cosmetic industry. In this study, nationally produced (mainland Portugal and Azores archipelago) EOs (11) and Hds (7) obtained from forest logging and thinning of Eucalyptus globulus, Pinus pinaster, Pinus pinea and Cryptomeria japonica, were chemically evaluated, and their bioactivity and sensorial properties were assessed. EOs and Hd volatiles (HdVs) were analyzed by GC-FID and GC-MS. 1,8-Cineole was dominant in E. globulus EOs and HdVs, and α- and ß-pinene in P. pinaster EOs. Limonene and α-pinene led in P. pinea and C. japonica EOs, respectively. P. pinaster and C. japonica HVs were dominated by α-terpineol and terpinen-4-ol, respectively. The antioxidant activity was determined by DPPH, ORAC and ROS. C. japonica EO showed the highest antioxidant activity, whereas one of the E. globulus EOs showed the lowest. Antimicrobial activity results revealed different levels of efficacy for Eucalyptus and Pinus EOs while C. japonica EO showed no antimicrobial activity against the selected strains. The perception and applicability of emulsions with 0.5% of EOs were evaluated through an in vivo sensory study. C. japonica emulsion, which has a fresh and earthy odour, was chosen as the most pleasant fragrance (60%), followed by P. pinea emulsion (53%). In summary, some of the studied EOs and Hds showed antioxidant and antimicrobial activities and they are possible candidates to address the consumers demand for more sustainable and responsibly sourced ingredients.

The Mycobacteriophage Ms6 LysB N-Terminus Displays Peptidoglycan Binding Affinity.

Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N-terminus. The N-terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N-terminus of Ms6 LysB binds to the PG.

Host miRNA-21 promotes liver dysfunction by targeting small intestinal Lactobacillus in mice.

New evidence shows that host-microbiota crosstalk can be modulated via endogenous miRNAs. We have previously reported that miR-21 ablation protects against liver injury in cholestasis. In this study, we investigated the role of miR-21 in modulating the gut microbiota during cholestasis and its effects in liver dysfunction. Mice lacking miR-21 had reduced liver damage and were protected against small intestinal injury as well as from gut microbiota dysbiosis when subjected to bile duct ligation surgery. The unique microbiota profile of miR-21KO mice was characterized by an increase in Lactobacillus, a key microbiome genus for gut homeostasis. Interestingly, in vitro incubation of synthetic miR-21 directly reduced Lactobacillus load. Moreover, supplementation with Lactobacillus reuteri revealed reduced liver fibrosis in acute bile duct-ligated mice, mimicking the protective effects in miR-21 knockout mice. D-lactate, a main product of Lactobacillus, regulates gut homeostasis that may link with reduced liver fibrosis. Altogether, our results demonstrate that miR-21 promotes liver dysfunction through direct modulation of the gut microbiota and highlight the potential therapeutic effects of Lactobacillus supplementation in gut and liver homeostasis.

Cryo-EM Structures and Regulation of Arabinofuranosyltransferase AftD from Mycobacteria.

Mycobacterium tuberculosis causes tuberculosis, a disease that kills over 1 million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides-arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9-Å resolution structure of M. abscessus AftD, determined by single-particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate-binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4- and 3.5-Å structures of a mutant with impaired ACP binding reveal a conformational change, suggesting that ACP may regulate AftD function. Mutagenesis experiments using a conditional knockout constructed in M. smegmatis confirm the essentiality of the putative active site and the ACP binding for AftD function.

Transient transfection of Babesia ovis using heterologous promoters.

Babesia species, etiological agents of babesiosis, a recognized emerging tick-borne disease, are a significant animal and human health concern with a worldwide socio-economic impact. The development of genetic manipulation techniques, such as transfection technology, is pivotal to improve knowledge regarding the biology of these poorly studied parasites towards better disease control strategies. For Babesia ovis, responsible for ovine babesiosis, a tick-borne disease of small ruminants, these tools are not yet available. The present study was based on the existence of interchangeable cross-species functional promoters between Babesia species. Herein, we describe for the first time B. ovis transient transfection using two heterologous promoters, the ef-1α-B intergenic regions from B. bovis and B. ovata. Their ability to drive expression of a reporter luciferase in B. ovis supports their cross-species functionality. Also, the ef-1α-B promoter region from B. ovata resulted in statistically significantly higher luminescence values in comparison to the control, thus a possibly suitable promoter for stable gene expression. Evaluation of transfection efficiency using qPCR demonstrated that higher luminescence levels were due to promoter strength rather than a higher transfection efficiency. These findings represent a step forward in the development of methods for B. ovis genetic manipulation, an undoubtedly necessary tool to study this parasite basic biology, including its life cycle, the parasite interactions with host cells and virulence factors.

Revisiting Anti-tuberculosis Therapeutic Strategies That Target the Peptidoglycan Structure and Synthesis.

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), is one of the leading cause of death by an infectious diseases. The biosynthesis of the mycobacterial cell wall (CW) is an area of increasing research significance, as numerous antibiotics used to treat TB target biosynthesis pathways of essential CW components. The main feature of the mycobacterial cell envelope is an intricate structure, the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex responsible for its innate resistance to many commonly used antibiotics and involved in virulence. A hallmark of mAGP is its unusual peptidoglycan (PG) layer, which has subtleties that play a key role in virulence by enabling pathogenic species to survive inside the host and resist antibiotic pressure. This dynamic and essential structure is not a target of currently used therapeutics as Mtb is considered naturally resistant to most ß-lactam antibiotics due to a highly active ß-lactamase (BlaC) that efficiently hydrolyses many ß-lactam drugs to render them ineffective. The emergence of multidrug- and extensive drug-resistant strains to the available antibiotics has become a serious health threat, places an immense burden on health care systems, and poses particular therapeutic challenges. Therefore, it is crucial to explore additional Mtb vulnerabilities that can be used to combat TB. Remodeling PG enzymes that catalyze biosynthesis and recycling of the PG are essential to the viability of Mtb and are therefore attractive targets for novel antibiotics research. This article reviews PG as an alternative antibiotic target for TB treatment, how Mtb has developed resistance to currently available antibiotics directed to PG biosynthesis, and the potential of targeting this essential structure to tackle TB by attacking alternative enzymatic activities involved in Mtb PG modifications and metabolism.

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.

The Ms6 Mycolyl-Arabinogalactan Esterase LysB is Essential for an Efficient Mycobacteriophage-Induced Lysis.

All dsDNA phages encode two proteins involved in host lysis, an endolysin and a holin that target the peptidoglycan and cytoplasmic membrane, respectively. Bacteriophages that infect Gram-negative bacteria encode additional proteins, the spanins, involved in disruption of the outer membrane. Recently, a gene located in the lytic cassette was identified in the genomes of mycobacteriophages, which encodes a protein (LysB) with mycolyl-arabinogalactan esterase activity. Taking in consideration the complex mycobacterial cell envelope that mycobacteriophages encounter during their life cycle, it is valuable to evaluate the role of these proteins in lysis. In the present work, we constructed an Ms6 mutant defective on lysB and showed that Ms6 LysB has an important role in lysis. In the absence of LysB, lysis still occurs but the newly synthesized phage particles are deficiently released to the environment. Using cryo-electron microscopy and tomography to register the changes in the lysis phenotype, we show that at 150 min post-adsorption, mycobacteria cells are incompletely lysed and phage particles are retained inside the cell, while cells infected with Ms6wt are completely lysed. Our results confirm that Ms6 LysB is necessary for an efficient lysis of Mycobacterium smegmatis, acting, similarly to spanins, in the third step of the lysis process.

EC300: a phage-based, bacteriolysin-like protein with enhanced antibacterial activity against Enterococcus faecalis.

Bacteriophage lytic enzymes, either endolysins or virion-associated lysins, have been receiving considerable attention as potential antibacterial agents, particularly for the combat of antibiotic-resistant Gram-positive pathogens. A conclusion that easily emerges from the careful analysis of a great number of reports on the field is that the activity of phage lytic enzymes is rarely studied in conditions that support robust growth of the target bacteria. Here, we report the construction and study of a chimerical lysin, EC300, which was designed to target and kill Enterococcus faecalis in conditions supporting vigorous bacterial growth. EC300 resulted from the fusion of a predicted M23 endopeptidase domain of a virion-associated lysin to the putative cell wall binding domain of a previously characterized amidase endolysin, both produced by the E. faecalis phage F170/08. This bacteriolysin-like protein exhibited a clear enhanced lytic activity over the parental endolysin when both were assayed in a rich bacterial growth medium. We demonstrate the killing efficacy of EC300 against growing cells of a panel of typed E. faecalis clinical strains with high level of antibiotic resistance. The possible reasons for the marked difference between the lytic performance of EC300 and that of the amidase are discussed.

A two-component, multimeric endolysin encoded by a single gene.

Bacteriophage endolysins are bacterial cell wall degrading enzymes whose potential to fight bacterial infections has been intensively studied. Endolysins from Gram-positive systems are typically described as monomeric and as having a modular structure consisting of one or two N-terminal catalytic domains (CDs) linked to a C-terminal region responsible for cell wall binding (CWB). We show here that expression of the endolysin gene lys170 of the enterococcal phage F170/08 results in two products, the expected full length endolysin (Lys170FL) and a C-terminal fragment corresponding to the CWB domain (CWB170). The latter is produced from an in-frame, alternative translation start site. Both polypeptides interact to form the fully active endolysin. Biochemical data strongly support a model where Lys170 is made of one monomer of Lys170FL associated with up to three CWB170 subunits, which are responsible for efficient endolysin binding to its substrate. Bioinformatics analysis indicates that similar secondary translation start signals may be used to produce and add independent CWB170-like subunits to different enzymatic specificities. The particular configuration of endolysin Lys170 uncovers a new mode of increasing the number of CWB motifs associated to CD modules, as an alternative to the tandem repetition typically found in monomeric cell wall hydrolases.

In vitro design of a novel lytic bacteriophage cocktail with therapeutic potential against organisms causing diabetic foot infections.

In patients with diabetes mellitus, foot infections pose a significant risk. These are complex infections commonly caused by Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii, all of which are potentially susceptible to bacteriophages. Here, we characterized five bacteriophages that we had determined previously to have antimicrobial and wound-healing potential in chronic S. aureus, P. aeruginosa and A. baumannii infections. Morphological and genetic features indicated that the bacteriophages were lytic members of the family Myoviridae or Podoviridae and did not harbour any known bacterial virulence genes. Combinations of the bacteriophages had broad host ranges for the different target bacterial species. The activity of the bacteriophages against planktonic cells revealed effective, early killing at 4 h, followed by bacterial regrowth to pre-treatment levels by 24 h. Using metabolic activity as a measure of cell viability within established biofilms, we found significant cell impairment following bacteriophage exposure. Repeated treatment every 4 h caused a further decrease in cell activity. The greatest effects on both planktonic and biofilm cells occurred at a bacteriophage : bacterium input multiplicity of 10. These studies on both planktonic cells and established biofilms allowed us to better evaluate the effects of a high input multiplicity and a multiple-dose treatment protocol, and the findings support further clinical development of bacteriophage therapy.

Genetics of Phage Lysis.

We have been witnessing an increased interest in bacteriophage studies focused on their use as antibacterial agents to fight pathogenic bacteria. This interest is a consequence of the phages' ability to lyse a bacterial host. Until recently, little was known about the mechanisms used by mycobacteriophages to induce lysis of their complex hosts. However, studies on Ms6-induced lysis have changed this scenario and provided new insights into the mechanisms of bacteriophage-induced lysis. Specific lysis protein genes have been identified in mycobacteriophage genomes, reflecting the particular mycobacterial cell envelope composition. These include enzymes that target mycolic acid-containing lipids and proteins that participate in the secretion of the phage endolysin, functioning as chaperone-like proteins. This chapter focuses on the current knowledge of mycobacteriophage-induced lysis, starting with an overview of phage lysis and basic features of the lysis players.

High throughput phenotypic selection of Mycobacterium tuberculosis mutants with impaired resistance to reactive oxygen species identifies genes important for intracellular growth.

Mycobacterium tuberculosis has the remarkable capacity to survive within the hostile environment of the macrophage, and to resist potent antibacterial molecules such as reactive oxygen species (ROS). Thus, understanding mycobacterial resistance mechanisms against ROS may contribute to the development of new anti-tuberculosis therapies. Here we identified genes involved in such mechanisms by screening a high-density transposon mutant library, and we show that several of them are involved in the intracellular lifestyle of the pathogen. Many of these genes were found to play a part in cell envelope functions, further strengthening the important role of the mycobacterial cell envelope in protection against aggressions such as the ones caused by ROS inside host cells.

Diversity in bacterial lysis systems: bacteriophages show the way.

Bacteriophages have developed multiple host cell lysis strategies to promote release of descendant virions from infected bacteria. This review is focused on the lysis mechanisms employed by tailed double-stranded DNA bacteriophages, where new developments have recently emerged. These phages seem to use a least common denominator to induce lysis, the so-called holin-endolysin dyad. Endolysins are cell wall-degrading enzymes whereas holins form 'holes' in the cytoplasmic membrane at a precise scheduled time. The latter function was long viewed as essential to provide a pathway for endolysin escape to the cell wall. However, recent studies have shown that phages can also exploit the host cell secretion machinery to deliver endolysins to their target and subvert the bacterial autolytic arsenal to effectively accomplish lysis. In these systems the membrane-depolarizing holin function still seems to be essential to activate secreted endolysins. New lysis players have also been uncovered that promote degradation of particular bacterial cell envelopes, such as that of mycobacteria.

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.

Phage endolysins with broad antimicrobial activity against Enterococcus faecalis clinical strains.

Increasing antibiotic resistance of bacterial pathogens has drawn the attention to the potential use of bacteriophage endolysins as alternative antibacterial agents. Here we have identified, characterized, and studied the lytic potential of two endolysins, Lys168 and Lys170, from phages infecting Enterococcus faecalis. Lys168 and Lys170 belong to the cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) and amidase-2 protein families, respectively. Lys168 is quite a unique enterococcal phage endolysin. It shares 95% amino acidic identity with the endolysin of Staphylococcus aureus phage SAP6, which in turn is distantly related to all known CHAP endolysins of S. aureus phages. Lys170 seems to be a natural chimera assembling catalytic and cell-wall-binding domains of different origin. Both endolysins showed a clear preference to act against E. faecalis and they were able to lyse a high proportion of clinical isolates of this species. Specifically, Lys168 and Lys170 lysed more than 70% and 90% of the tested isolates, respectively, which included a panel of diverse and typed strains representative of highly prevalent clonal complexes. Lys170 was active against all tested E. faecalis VRE strains. The quasi specificity toward E. faecalis is discussed considering the nature of the enzymes' functional domains and the structure of the cell wall peptidoglycan.

Novel chimerical endolysins with broad antimicrobial activity against methicillin-resistant Staphylococcus aureus.

Due to their bacterial lytic action, bacteriophage endolysins have recently gained great attention as a potential alternative to antibiotics in the combat of Gram-positive pathogenic bacteria, particularly those displaying multidrug resistance. However, large-scale production and purification of endolysins is frequently impaired due to their low solubility. In addition, a large number of endolysins appear to exhibit reduced lytic efficacy when compared with their action during phage infection. Here, we took advantage of the high solubility of two recently characterized enterococcal endolysins to construct chimeras targeting Staphylococcus aureus. The putative cell wall binding domain of these endolysins was substituted by that of a staphylococcal endolysin that showed poor solubility. Under appropriate conditions the resulting chimeras presented the high solubility of the parental enterococcal endolysins. In addition, they proved to be broadly active against a collection of the most relevant methicillin-resistant S. aureus epidemic clones and against other Gram-positive pathogens. Thus, fusion of endolysin domains of heterologous origin seems to be a suitable approach to design new potent endolysins with changed and/or extended lytic spectrum that are amenable to large-scale production.

The endolysin-binding domain encompasses the N-terminal region of the mycobacteriophage Ms6 Gp1 chaperone.

The intermolecular interactions of the mycobacteriophage Ms6 secretion chaperone with endolysin were characterized. The 384-amino-acid lysin (lysin(384))-binding domain was found to encompass the N-terminal region of Gp1, which is also essential for a lysis phenotype in Escherichia coli. In addition, a GXXXG-like motif involved in Gp1 homo-oligomerization was identified within the C-terminal region.