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.

Elucidating the molecular properties and anti-mycobacterial activity of cysteine peptidase domain of D29 mycobacteriophage endolysin.

Emergence of antibiotic resistance in pathogenic Mycobacterium tuberculosis (Mtb) has elevated tuberculosis to a serious global threat, necessitating alternate solutions for its eradication. D29 mycobacteriophage can infect and kill several mycobacterial species including Mtb. It encodes an endolysin LysA to hydrolyze host bacteria peptidoglycan for progeny release. We previously showed that out of the two catalytically active domains of LysA [N-terminal domain (NTD) and lysozyme-like domain], NTD, when ectopically expressed in Mycobacterium smegmatis (Msm), is able to kill the bacterium nearly as efficiently as full-length LysA. Here, we dissected the functioning of NTD to develop it as a phage-derived small molecule anti-mycobacterial therapeutic. We performed a large-scale site-directed mutagenesis of the conserved residues in NTD and examined its structure, stability, and function using molecular dynamic simulations coupled with biophysical and biochemical experiments. Our data show that NTD functions as a putative cysteine peptidase with a catalytic triad composed of Cys41, His112, and Glu137, acting as nucleophile, base, and acid, respectively, and showing characteristics similar to the NlpC/P60 family of cysteine peptidases. Additionally, our peptidoglycan hydrolysis assays suggested that NTD hydrolyzes only mycobacterial peptidoglycan and does not act on Gram-positive and Gram-negative bacterial peptidoglycans. More importantly, the combined activity of exogenously added NTD and sub-lethal doses of anti-mycobacterial drugs kills Msm in vitro and exhibits disruption of pre-formed mycobacterial biofilm. We additionally show that NTD treatment increases the permeability of antibiotics in Msm, which reduces the minimum inhibitory concentration of the antibiotics. Collectively, we present NTD as a promising phage-derived therapeutic against mycobacteria.IMPORTANCEMycobacteriophages are the viruses that use mycobacteria as host for their progeny production and, in the process, kill them. Mycobacteriophages are, therefore, considered as promising alternatives to antibiotics for killing pathogenic Mycobacterium tuberculosis. The endolysin LysA produced by mycobacteriophage D29 plays an important role in host cell lysis and virion release. Our work presented here highlights the functioning of LysA's N-terminal catalytic domain (NTD) in order to develop it as phage-derived small molecule therapeutics. We show that combined treatment of exogenously added NTD and sub-lethal doses of anti-mycobacterial drugs kills M. smegmatis, shows synergism by reducing the minimum inhibitory concentration of these antibiotics, and exhibits disruption of pre-formed mature biofilm. These outcomes and our detailed biochemical and biophysical dissection of the protein further pave the way toward engineering and development of NTD as a promising therapeutic against mycobacterial infections such as tuberculosis.

X-ray structure and mutagenesis analyses of Clostridioides difficile endolysin Ecd09610 glucosaminidase domain.

Clostridioides difficile endolysin (Ecd09610) consists of an unknown domain at its N terminus, followed by two catalytic domains, a glucosaminidase domain and endopeptidase domain. X-ray structure and mutagenesis analyses of the Ecd09610 catalytic domain with glucosaminidase activity (Ecd09610CD53) were performed. Ecd09610CD53 was found to possess an α-bundle-like structure with nine helices, which is well conserved among GH73 family enzymes. The mutagenesis analysis based on X-ray structures showed that Glu405 and Asn470 were essential for enzymatic activity. Ecd09610CD53 may adopt a neighboring-group mechanism for a catalytic reaction in which Glu405 acted as an acid/base catalyst and Asn470 helped to stabilize the oxazolinium ion intermediate. Structural comparisons with the newly identified Clostridium perfringens autolysin catalytic domain (AcpCD) in the P1 form and a zymography analysis demonstrated that AcpCD was 15-fold more active than Ecd09610CD53. The strength of the glucosaminidase activity of the GH73 family appears to be dependent on the depth of the substrate-binding groove.

Antibacterial synergy between a phage endolysin and citric acid against the Gram-negative kiwifruit pathogen Pseudomonas syringae pv. actinidiae.

Horticultural diseases caused by bacterial pathogens provide an obstacle to crop production globally. Management of the infection of kiwifruit by the Gram-negative phytopathogen Pseudomonas syringae pv. actinidiae (Psa) currently includes copper and antibiotics. However, the emergence of bacterial resistance and a changing regulatory landscape are providing the impetus to develop environmentally sustainable antimicrobials. One potential strategy is the use of bacteriophage endolysins, which degrade peptidoglycan during normal phage replication, causing cell lysis and the release of new viral progeny. Exogenous use of endolysins as antimicrobials is impaired by the outer membrane of Gram-negative bacteria that provides an impermeable barrier and prevents endolysins from accessing their target peptidoglycan. Here, we describe the synergy between citric acid and a phage endolysin, which results in a reduction of viable Psa below detection. We show that citric acid drives the destabilization of the outer membrane via acidification and sequestration of divalent cations from the lipopolysaccharide, which is followed by the degradation of the peptidoglycan by the endolysin. Scanning electron microscopy revealed clear morphological differences, indicating cell lysis following the endolysin-citric acid treatment. These results show the potential for citric acid-endolysin combinations as a possible antimicrobial approach in agricultural applications. IMPORTANCE: The phytopathogen Pseudomonas syringae pv. actinidiae (Psa) causes major impacts to kiwifruit horticulture, and the current control strategies are heavily reliant on copper and antibiotics. The environmental impact and increasing resistance to these agrichemicals are driving interest in alternative antimicrobials including bacteriophage-derived therapies. In this study, we characterize the endolysin from the Otagovirus Psa374 which infects Psa. When combined with citric acid, this endolysin displays an impressive antibacterial synergy to reduce viable Psa below the limit of detection. The use of citric acid as a synergistic agent with endolysins has not been extensively studied and has never been evaluated against a plant pathogen. We determined that the synergy involved a combination of the chelation activity of citric acid, acidic pH, and the specific activity of the ΦPsa374 endolysin. Our study highlights an exciting opportunity for alternative antimicrobials in agriculture.

A novel endolysin from an Enterococcus faecalis phage and application.

Enterococcus faecalis is the primary species detected in cases of secondary persistent infection resulting from root canal therapy failure. Due to the overuse of antibacterial agents, E. faecalis has developed resistance to these drugs, making it challenging to treat clinical diseases caused by E. faecalis infection. Therefore, there is an urgent need to explore new alternative drugs for treating E. faecalis infections. We aimed to clone and express the genes of phage endolysins, purify the recombinant proteins, and analyze their antibacterial activity, lysis profile, and ability to remove biofilm. The crude enzyme of phage endolysin pEF51 (0.715 mg/mL), derived from phage PEf771 infecting E. faecalis, exhibited superior bacterial inhibitory activity and a broader bactericidal spectrum than its parental phage PEf771. Furthermore, pEF51 demonstrated high efficacy in eliminating E. faecalis biofilm. Therapeutic results of the infected Sprague-Dawley (SD) rat model indicated that among 10 SD rats, only one developed a thoracic peritoneal abscess and splenic peritoneal abscess after 72 h of treatment with pEF51. This suggests that pEF51 could provide protection against E. faecalis infection in SD rats. Based on the 16S rDNA metagenomic data of the intestinal microbial community of SD rats, endolysin pEF51 exerted a certain influence on the diversity of intestinal microorganisms at the genus level. Thus, pEF51 may serve as a promising alternative to antibiotics in the management of E. faecalis infection.

Phage therapy: A renewed approach against oral diseases caused by Enterococcus faecalis infections.

Antibiotics play an important role in the treatment of infectious diseases. Long-term overuse or misuse of antibiotics, however, has triggered the global crisis of antibiotic resistance, bringing challenges to treating clinical infection. Bacteriophages (phages) are the viruses infecting bacterial cells. Due to high host specificity, high bactericidal activity, and good biosafety, phages have been used as natural alternative antibacterial agents to fight against multiple drug-resistant bacteria. Enterococcus faecalis is the main species detected in secondary persistent infection caused by failure of root canal therapy. Due to strong tolerance and the formation of biofilm, E. faecalis can survive the changes in pH, temperature, and osmotic pressure in the mouth and thus is one of the main causes of periapical lesions. This paper summarizes the advantages of phage therapy, its applications in treating oral diseases caused by E. faecalis infections, and the challenges it faces. It offers a new perspective on phage therapy in oral diseases.

Antibacterial activity of bacteriophage-encoded endolysins against planktonic and biofilm cells of pathogenic Escherichia coli.

This study was designed to assess the possibility of using bacteriophage-encoded endolysins for controlling planktonic and biofilm cells. The endolysins, LysEP114 and LysEP135, were obtained from plasmid vectors containing the endolysin genes derived from Escherichia coli phages. The high identity (>96 %) was observed between LysEP114 and LysEP135. LysEP114 and LysEP135 were characterized by pH, thermal, and lactic acid stability, lytic spectrum, antibacterial activity, and biofilm eradication. The molecular masses of LysEP114 and LysEP135 were 18.2 kDa, identified as muramidases. LysEP114 and LysEP135 showed high lytic activity against the outer membrane-permeabilized E. coli KCCM 40405 at below 37 °C, between pH 5 to 11, and below 70 mM of lactic acid. LysEP114 and LysEP135 showed the broad rang of lytic activity against E. coli KACC 10115, S. Typhimurium KCCM 40253, S. Typhimurium CCARM 8009, tetracycline-resistant S. Typhimurium, polymyxin B-resistant S. Typhimurium, chloramphenicol-resistant S. Typhimurium, K. pneumoniae ATCC 23357, K. pneumoniae CCARM 10237, and Shigella boydii KACC 10792. LysEP114 and LysEP135 effectively reduced the numbers of planktonic E. coli KCCM by 1.7 and 2.1 log, respectively, when treated with 50 mM lactic acid. The numbers of biofilm cells were reduced from 7.3 to 4.1 log CFU/ml and 2.2 log CFU/ml, respectively, when treated with LysEP114- and LysEP135 in the presence of 50 mM lactic acid. The results suggest that the endolysins in combination with lactic acid could be potential alternative therapeutic agents for controlling planktonic and biofilm cells.

Bacteriophage therapy against ESKAPE bacterial pathogens: Current status, strategies, challenges, and future scope.

The ESKAPE pathogens are the primary threat due to their constant spread of drug resistance worldwide. These pathogens are also regarded as opportunistic pathogens and could potentially cause nosocomial infections. Most of the ESKAPE pathogens have developed resistance to almost all the antibiotics that are used against them. Therefore, to deal with antimicrobial resistance, there is an urgent requirement for alternative non-antibiotic strategies to combat this rising issue of drug-resistant organisms. One of the promising alternatives to this scenario is implementing bacteriophage therapy. This under-explored mode of treatment in modern medicine has posed several concerns, such as preferable phages for the treatment, impact on the microbiome (or gut microflora), dose optimisation, safety, etc. The review will cover a rationale for phage therapy, clinical challenges, and propose phage therapy as an effective therapeutic against bacterial coinfections during pandemics. This review also addresses the expected uncertainties for administering the phage as a treatment against the ESKAPE pathogens and the advantages of using lytic phage over temperate, the immune response to phages, and phages in combinational therapies. The interaction between bacteria and bacteriophages in humans and countless animal models can also be used to design novel and futuristic therapeutics like personalised medicine or bacteriophages as anti-biofilm agents. Hence, this review explores different aspects of phage therapy and its potential to emerge as a frontline therapy against the ESKAPE bacterial pathogen.

Advances in the development of phage-mediated cyanobacterial cell lysis.

Cyanobacteria, the only oxygenic photoautotrophs among prokaryotes, are developing as both carbon building blocks and energetic self-supported chassis for the generation of various bioproducts. However, one of the challenges to optimize it as a more sustainable platform is how to release intracellular bioproducts for an easier downstream biorefinery process. To date, the major method used for cyanobacterial cell lysis is based on mechanical force, which is energy-intensive and economically unsustainable. Phage-mediated bacterial cell lysis is species-specific and highly efficient and can be conducted under mild conditions; therefore, it has been intensively studied as a bacterial cell lysis weapon. In contrast to heterotrophic bacteria, biological cell lysis studies in cyanobacteria are lagging behind. In this study, we reviewed cyanobacterial cell envelope features that could affect cell strength and elicited a thorough presentation of the necessary phage lysin components for efficient cell lysis. We then summarized all bioengineering manipulated pipelines for lysin component optimization and further revealed the challenges for each intent-oriented application in cyanobacterial cell lysis. In addition to applied biotechnology usage, the significance of phage-mediated cyanobacterial cell lysis could also advance sophisticated biochemical studies and promote biocontrol of toxic cyanobacteria blooms.

Antimicrobial peptide thanatin fused endolysin PA90 (Tha-PA90) for the control of Acinetobacter baumannii infection in mouse model.

BACKGROUND: This study addresses the urgent need for infection control agents driven by the rise of drug-resistant pathogens such as Acinetobacter baumannii. Our primary aim was to develop and assess a novel endolysin, Tha-PA90, designed to combat these challenges. METHODS: Tha-PA90 incorporates an antimicrobial peptide (AMP) called thanatin at its N-terminus, enhancing bacterial outer membrane permeability and reducing host immune responses. PA90 was selected as the endolysin component. The antibacterial activity of the purified Tha-PA90 was evaluated using an in vitro colony-forming unit (CFU) reduction assay and a membrane permeability test. A549 cells were utilized to measure the penetration into the cytosol and the cytotoxicity of Tha-PA90. Finally, infection control was monitored in A. baumannii infected mice following the intraperitoneal administration of Tha-PA90. RESULTS: Tha-PA90 demonstrated remarkable in vitro efficacy, completely eradicating A. baumannii strains, even drug-resistant variants, at a low concentration of 0.5 µM. Notably, it outperformed thanatin, achieving only a < 3-log reduction at 4 µM. Tha-PA90 exhibited 2-3 times higher membrane permeability than a PA90 and thanatin mixture or PA90 alone. Tha-PA90 was found within A549 cells' cytosol with no discernible cytotoxic effects. Furthermore, Tha-PA90 administration extended the lifespan of A. baumannii-infected mice, reducing bacterial loads in major organs by up to 3 logs. Additionally, it decreased proinflammatory cytokine levels (TNF-α and IL-6), reducing the risk of sepsis from rapid bacterial lysis. Our findings indicate that Tha-PA90 is a promising solution for combating drug-resistant A. baumannii. Its enhanced efficacy, low cytotoxicity, and reduction of proinflammatory responses render it a potential candidate for infection control. CONCLUSIONS: This study underscores the significance of engineered endolysins in addressing the pressing challenge of drug-resistant pathogens and offers insights into improved infection management strategies.

Development of novel antimicrobials with engineered endolysin LysECD7-SMAP to combat Gram-negative bacterial infections.

BACKGROUND: Among the non-traditional antibacterial agents in development, only a few targets critical Gram-negative bacteria such as carbapenem-resistant Pseudomonas aeruginosa, Acinetobacter baumannii or cephalosporin-resistant Enterobacteriaceae. Endolysins and their genetically modified versions meet the World Health Organization criteria for innovation, have a novel mode of antibacterial action, no known bacterial cross-resistance, and are being intensively studied for application against Gram-negative pathogens. METHODS: The study presents a multidisciplinary approach, including genetic engineering of LysECD7-SMAP and production of recombinant endolysin, its analysis by crystal structure solution following molecular dynamics simulations and evaluation of antibacterial properties. Two types of antimicrobial dosage forms were formulated, resulting in lyophilized powder for injection and hydroxyethylcellulose gel for topical administration. Their efficacy was estimated in the treatment of sepsis, and pneumonia models in BALB/c mice, diabetes-associated wound infection in the leptin receptor-deficient db/db mice and infected burn wounds in rats. RESULTS: In this work, we investigate the application strategies of the engineered endolysin LysECD7-SMAP and its dosage forms evaluated in preclinical studies. The catalytic domain of the enzyme shares the conserved structure of endopeptidases containing a putative antimicrobial peptide at the C-terminus of polypeptide chain. The activity of endolysins has been demonstrated against a range of pathogens, such as Klebsiella pneumoniae, A. baumannii, P. aeruginosa, Staphylococcus haemolyticus, Achromobacter spp, Burkholderia cepacia complex and Haemophylus influenzae, including those with multidrug resistance. The efficacy of candidate dosage forms has been confirmed in in vivo studies. Some aspects of the interaction of LysECD7-SMAP with cell wall molecular targets are also discussed. CONCLUSIONS: Our studies demonstrate the potential of LysECD7-SMAP therapeutics for the systemic or topical treatment of infectious diseases caused by susceptible Gram-negative bacterial species and are critical to proceed LysECD7-SMAP-based antimicrobials trials to advanced stages.

The therapeutic effect of engineered phage, derived protein and enzymes against superbug bacteria.

Defending against antibiotic-resistant infections is similar to fighting a war with limited ammunition. As the new century unfolded, antibiotic resistance became a significant concern. In spite of the fact that phage treatment has been used as an effective means of fighting infections for more than a century, researchers have had to overcome many challenges of superbug bacteria by manipulating phages and producing engineered enzymes. New enzymes and phages with enhanced properties have a significant impact on the ability to fight antibiotic-resistant infections, which is considered a window of hope for the future. This review, therefore, illustrates not only the challenges caused by antibiotic resistance and superbug bacteria but also the engineered enzymes and phages that are being developed to solve these issues. Our study found that engineered phages, phage proteins, and enzymes can be effective in treating superbug bacteria and destroying the biofilm caused by them. Combining these engineered compounds with other antimicrobial substances can increase their effectiveness against antibiotic-resistant bacteria. Therefore, engineered phages, proteins, and enzymes can be used as a substitute for antibiotics or in combination with antibiotics to treat patients with superbug infections in the future.

Prediction of key amino acids of Salmonella phage endolysin LysST-3 and detection of its mutants' activity.

Salmonella Typhimurium, a zoonotic pathogen, causes systemic and localized infection. The emergence of drug-resistant S. Typhimurium has increased; treating bacterial infections remains challenging. Phage endolysins derived from phages have a broader spectrum of bacteriolysis and better bacteriolytic activity than phages, and are less likely to induce drug resistance than antibiotics. LysST-3, the endolysin of Salmonella phage ST-3, was chosen in our study for its high lytic activity, broad cleavage spectrum, excellent bioactivity, and moderate safety profile. LysST-3 is a promising antimicrobial agent for inhibiting the development of drug resistance in Salmonella. The aim of this study is to investigate the molecular characteristics of LysST-3 through the prediction of key amino acid sites of LysST-3 and detection of its mutants' activity. We investigated its lytic effect on Salmonella and identified its key amino acid sites of interaction with substrate. LysST-3 may be a Ca2+, Mg2+ - dependent metalloenzyme. Its concave structure of the bottom "gripper" was found to be an important part of its amino acid active site. We identified its key sites (29P, 30T, 86D, 88 L, and 89 V) for substrate binding and activity using amino acid-targeted mutagenesis. Alterations in these sites did not affect protein secondary structure, but led to a significant reduction in the cleavage activity of the mutant proteins. Our study provides a basis for phage endolysin modification to target drug-resistant bacteria. Identifying the key amino acid site of the endolysin LysST-3 provides theoretical support for the functional modification of the endolysin and the development of subsequent effective therapeutic solutions.

Assessing the synergistic potential of bacteriophage endolysins and antimicrobial peptides for eradicating bacterial biofilms.

Phage-encoded endolysins have emerged as a potential substitute to conventional antibiotics due to their exceptional benefits including host specificity, rapid host killing, least risk of resistance. In addition to their antibacterial potency and biofilm eradication properties, endolysins are reported to exhibit synergism with other antimicrobial agents. In this study, the synergistic potency of endolysins was dissected with antimicrobial peptides to enhance their therapeutic effectiveness. Recombinantly expressed and purified bacteriophage endolysin [T7 endolysin (T7L); and T4 endolysin (T4L)] proteins have been used to evaluate the broad-spectrum antibacterial efficacy using different bacterial strains. Antibacterial/biofilm eradication studies were performed in combination with different antimicrobial peptides (AMPs) such as colistin, nisin, and polymyxin B (PMB) to assess the endolysin's antimicrobial efficacy and their synergy with AMPs. In combination with T7L, polymyxin B and colistin effectively eradicated the biofilm of Pseudomonas aeruginosa and exhibited a synergistic effect. Further, a combination of T4L and nisin displayed a synergistic effect against Staphylococcus aureus biofilms. In summary, the obtained results endorse the theme of combinational therapy consisting of endolysins and AMPs as an effective remedy against the drug-resistant bacterial biofilms that are a serious concern in healthcare settings.

Characterization of a marine endolysin LysVPB against Vibrio parahaemolyticus.

Currently, there is an urgent to develop safe and environmentally friendly alternatives to antibiotics for combating Vibrio parahaemolyticus. Endolysins are considered promising antibacterial agents due to their desirable range of action and ability to deal with antibiotic-resistant bacteria. While numerous Vibrio phages have been identified, the research on their endolysins is still in its infancy. In this study, a novel endolysin called LysVPB was cloned and expressed in Pichia pastoris. Phylogenetic analysis revealed that LysVPB bears little resemblance to other known endolysins, highlighting its unique nature. Homology modeling identified a putative calcium-binding site in LysVPB. The recombinant LysVPB achieved a lytic activity of 64.8 U/mL and had a molecular weight of approximately 17 kDa. LysVPB exhibited enhanced efficacy at pH 9.0, with 60% of its maximum activity observed within the broad pH range of 6.0-10.0. The catalytic efficiency of LysVPB peaked at 30 °C but significantly declined beyond 50 °C. Ba2+, Co2+, and Cu2+ showed inhibitory effects on the activity of LysVPB, while Ca2+ can boost it to 126.8%. Furthermore, LysVPB exhibited satisfactory efficacy against strains of V. parahaemolyticus. LysVPB is an innovative phage lysin with good characteristics that are specific to certain hosts. The modular nature of LysVPB allows for efficient domain exchange with alternative lysins as antimicrobial components and fusion with antimicrobial peptides. This opens up possibilities for engineering chimeric lysins in a broader range of target hosts with high antimicrobial effectiveness and strong activity under physiological conditions.

Enzymatic and antibacterial activity of the recombinant endolysin PVP-SE1gp146 expressed in Hansenula polymorpha.

Antibiotic resistance, a major global concern, highlights the need for discovering alternative therapies. Recently, endolysins have garnered attention as antibacterial tools with a lower resistance development rate compared to conventional antibiotics, and their production in various expression hosts holds significance. Given its generally recognized as safe (GRAS) status and other advantages, Hansenula polymorpha offers a promising host for endolysin production. PVP-SE1gp146 originates from the Salmonella Enteritidis-specific phage PVP-SE1, which has been previously characterized. We inserted the PVP-SE1gp146 coding gene into the H. polymorpha expression vector pHIPX4. The resulting recombinant, pHIPX4-PVP-SE1gp146, was then introduced into H. polymorpha NCYC495 to facilitate the production of the endolysin PVP-SE1gp146. The expression level of the PVP-SE1gp146 protein was assessed, and it was determined to be approximately 43 mg/l of yeast culture medium. The enzymatic (muralytic) activity of this endolysin was also evaluated, corresponding to the version produced by the E. coli Bl21 strain. The endolysin exhibited admissible antibacterial activity against several gram-negative species, including P. aeruginosa, E. coli, and A. baumannii, while showing an almost negligible impact on K. pneumoniae. Endolysin production within GRAS-approved hosts holds potential for combating antibiotic-resistant bacteria. Challenges involve optimizing concentrations, targeting gram-negative species and improving attachment to bacterial cell walls. Addressing these issues requires dedicated research in endolysin engineering and a comprehensive evaluation of their production in diverse expression hosts.

LysSYL: a broad-spectrum phage endolysin targeting Staphylococcus species and eradicating S. aureus biofilms.

BACKGROUND: Staphylococcus aureus and its single or mixed biofilm infections seriously threaten global public health. Phage therapy, which uses active phage particles or phage-derived endolysins, has emerged as a promising alternative strategy to antibiotic treatment. However, high-efficient phage therapeutic regimens have yet to be established. RESULTS: In this study, we used an enrichment procedure to isolate phages against methicillin-resistant S. aureus (MRSA) XN108. We characterized phage SYL, a new member of the Kayvirus genus, Herelleviridae family. The phage endolysin LysSYL was expressed. LysSYL demonstrated stability under various conditions and exhibited a broader range of efficacy against staphylococcal strains than its parent phage (100% vs. 41.7%). Moreover, dynamic live/dead bacterial observation demonstrated that LysSYL could completely lyse MRSA USA300 within 10 min. Scan and transmission electron microscopy revealed evident bacterial cell perforation and deformation. In addition, LysSYL displayed strong eradication activity against single- and mixed-species biofilms associated with S. aureus. It also had the ability to kill bacterial persisters, and proved highly effective in eliminating persistent S. aureus when combined with vancomycin. Furthermore, LysSYL protected BALB/c mice from lethal S. aureus infections. A single-dose treatment with 50 mg/kg of LysSYL resulted in a dramatic reduction in bacterial loads in the blood, liver, spleen, lungs, and kidneys of a peritonitis mouse model, which resulted in rescuing 100% of mice challenged with 108 colony forming units of S. aureus USA300. CONCLUSIONS: Overall, the data provided in this study highlight the strong therapeutic potential of endolysin LysSYL in combating staphylococcal infections, including mono- and mixed-species biofilms related to S. aureus.

Bacteriophage endolysin treatment for systemic infection of Streptococcus iniae in hybrid striped bass.

Streptococcus iniae, a zoonotic Gram-positive pathogen, poses a threat to finfish aquaculture, causing streptococcosis with an annual economic impact exceeding $150 million globally. As aquaculture trends shift towards recirculating systems, the potential for horizontal transmission of S. iniae among fish intensifies. Current vaccine development provides only short-term protection, driving the widespread use of antibiotics like florfenicol. However, this practice raises environmental concerns and potentially contributes to antibiotic resistance. Thus, alternative strategies are urgently needed. Endolysin therapy, derived from bacteriophages, employs hydrolytic endolysin enzymes that target bacterial peptidoglycan cell walls. This study assesses three synthetic endolysins (PlyGBS 90-1, PlyGBS 90-8, and ClyX-2) alongside the antibiotic carbenicillin in treating S. iniae-infected hybrid striped bass (HSB). Results demonstrate that ClyX-2 exhibits remarkable bacteriolytic potency, with lytic activity detected at concentrations as low as ∼15 µg/mL, approximately 8-fold more potent than the PlyGBS derivatives. In therapeutic effectiveness assessments, both carbenicillin and ClyX-2 treatments achieved significantly higher survival rates (85 % and 95 %, respectively) compared to placebo and PlyGBS-based endolysin treatments. Importantly, no statistical differences were observed between ClyX-2 and carbenicillin treatments. This highlights ClyX-2 as a promising alternative for combating S. iniae infections in aquaculture, offering potent bacteriolytic activity and high survival rates.

Characterization and complete genome analysis of Klebsiella phage Kp109 with lytic activity against Klebsiella pneumoniae.

Klebsiella pneumonia is a serious pathogen involved in a range of infections. The increasing frequency of infection associated with K. pneumoniae and accelerated development of antimicrobial resistance has limited the available options of antibiotics for the treatment of infection. Bacteriophages are an attractive substitute to alleviate the problem of antibiotic resistance. In this study, isolation, microbiological and genomic characterization of bacteriophage Kp109 having the ability to infect K. pneumoniae has been shown. Phage Kp109 showed good killing efficiency and tolerance to a broad range of temperatures (4-60 °C) and pH (3-9). Transmission electron microscopy and genomic analysis indicated that phage Kp109 belongs to the genus Webervirus and family Drexlerviridae. Genomic analysis showed that the Kp109 has a 51,630 bp long double-stranded DNA genome with a GC content of 51.64%. The absence of known lysogenic, virulence, and antibiotic-resistant genes (ARGs) in its genome makes phage Kp109 safer to be used as a biocontrol agent for different purposes including phage therapy. The computational analysis of the putative endolysin gene revealed a binding energy of - 6.23 kcal/mol between LysKp109 and ligand NAM-NAG showing its potential to be used as an enzybiotic. However, future research is required for experimental validation of the in silico work to further corroborate the results obtained in the present study. Overall, phenotypic, genomic, and computational characterization performed in the present study showed that phages Kp109 and LysKp109 are promising candidates for future in vivo studies and could potentially be used for controlling K. pneumoniae infection.

Lysins as a powerful alternative to combat Bacillus anthracis.

This review gathers all, to the best of our current knowledge, known lysins, mainly bacteriophage-derived, that have demonstrated activity against Bacillus anthracis strains. B. anthracis is a spore-forming, toxin-producing bacteria, naturally dwelling in soil. It is best known as a potential biowarfare threat, an etiological agent of anthrax, and a severe zoonotic disease. Anthrax can be treated with antibiotics (ciprofloxacin, penicillin, doxycycline); however, their administration may take up even to 60 days, and different factors can compromise their effectiveness. Bacterial viruses, bacteriophages (phages), are natural enemies of bacteria and use their lytic enzymes, endolysins (lysins), to specifically kill bacterial cells. Harnessing the potential of lysins to combat bacterial infections holds promise for diminishing antibiotic usage and, consequently, addressing the escalating antibiotic resistance in bacteria. In this context, we list the lysins with the activity against B. anthracis, providing a summary of their lytic properties in vitro and the outcomes observed in animal models. Bacillus cereus strain ATCC 4342/RSVF1, a surrogate for B. anthracis, was also included as a target bacteria. KEY POINTS: • More than a dozen different B. anthracis lysins have been identified and studied. • They fall into three blocks regarding their amino acid sequence similarity and most of them are amidases. • Lysins could be used in treating B. anthracis infections.