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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.
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Salmonella is a globally prevalent foodborne pathogen, and adverse events caused by S. Enteritidis and S. Typhimurium are extremely common. With the emergence of drug resistance, there is an urgent need for efficient and specific lytic bacteriophages as alternative to antibiotics in clinical practice. In this study, phage P6 was isolated and screened from effluent and fecal samples from duck farm environments to specifically lyse the duck sources S. Typhimurium and S. Enteritidis. Phage P6 belongs to the genus Lederbergvirus, unclassified Lederbergvirus species. The phage P6 genome did not contained non-coding RNA, virulence genes and drug resistance genes, indicating that phage P6 was biologically safe for clinical applications. Phage P6 lysed 77.78% (28/36) of multidrug-resistant Salmonella and reduced biofilms formed by S. Enteritidis CVCC 3377, 4, and 24, and S. Typhimurium 44 by 44% to 75% within 3 h, and decreased Salmonella in duckling feces by up to 1.64 orders of magnitude. Prokaryotic expression of endolysin LysP6 lysed the chloroform-treated bacterial outer membrane from different serotypes of duck-derived Salmonella and E. coli standard strain ATCC 25922. The host range was expanded compared to phage P6, and the growth of Salmonella was effectively inhibited by LysP6 in conjunction with the membrane permeabilizer EDTA within 24 h. Therefore, phage P6 and phage-derived endolysins LysP6 are suitable for application as potent biocontrol agents to improve poultry health and food safety.
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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.
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Phage lysins, bacteriophage-encoded enzymes tasked with degrading their host's cell wall, are increasingly investigated and engineered as novel antibacterials across diverse applications. Their rapid action, tuneable specificity, and low likelihood of resistance development make them particularly interesting. Despite numerous application-focused lysin studies, the art of their recombinant production remains relatively undiscussed. Here, we provide an overview of the available expression systems for phage lysin production and discuss key considerations guiding the choice of a suitable recombinant host. We systematically surveyed recent literature to evaluate the hosts used in the lysin field and cover various recombinant systems, including the well-known bacterial host Escherichia coli or yeast Saccharomyces cerevisiae, as well as plant, mammalian, and cell-free systems. Careful analysis of the limited studies expressing lysins in various hosts suggests a host-dependent effect on activity. Nonetheless, the multitude of available expression systems should be further leveraged to accommodate the growing interest in phage lysins and their expanding range of applications.
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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.
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The cell wall is an indispensable element of bacterial cells and a long-known target of many antibiotics. Penicillin, the first discovered beta-lactam antibiotic inhibiting the synthesis of cell walls, was successfully used to cure many bacterial infections. Unfortunately, pathogens eventually developed resistance to it. This started an arms race, and while novel beta-lactams, either natural or (semi)synthetic, were discovered, soon upon their application, bacteria were developing resistance. Currently, we are facing the threat of losing the race since more and more multidrug-resistant (MDR) pathogens are emerging. Therefore, there is an urgent need for developing novel approaches to combat MDR bacteria. The cell wall is a reasonable candidate for a target as it differentiates not only bacterial and human cells but also has a specific composition unique to various groups of bacteria. This ensures the safety and specificity of novel antibacterial agents that target this structure. Due to the shortage of low-molecular-weight candidates for novel antibiotics, attention was focused on peptides and proteins that possess antibacterial activity. Here, we describe proteinaceous agents of various origins that target bacterial cell wall, including bacteriocins and phage and bacterial lysins, as alternatives to classic antibiotic candidates for antimicrobial drugs. Moreover, advancements in protein chemistry and engineering currently allow for the production of stable, specific, and effective drugs. Finally, we introduce the concept of selective targeting of dangerous pathogens, exemplified by staphylococci, by agents specifically disrupting their cell walls.
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Antibacterianos , Pared Celular , Bacterias Grampositivas , Pared Celular/efectos de los fármacos , Antibacterianos/farmacología , Antibacterianos/química , Bacterias Grampositivas/efectos de los fármacos , Humanos , Bacteriocinas/farmacología , Bacteriocinas/química , Infecciones por Bacterias Grampositivas/tratamiento farmacológico , Infecciones por Bacterias Grampositivas/microbiología , BacteriófagosRESUMEN
The isolation and identification of pathogenic bacteria from a variety of samples are critical for controlling bacterial infection-related health problems. The conventional methods, such as plate counting and polymerase chain reaction-based approaches, tend to be time-consuming and reliant on specific instruments, severely limiting the effective identification of these pathogens. In this study, we employed the specificity of the cell wall-binding (CBD) domain of the Staphylococcus aureus bacteriophage 80 alpha (80α) endolysin towards the host bacteria for isolation. Amidase 3-CBD conjugated magnetic beads successfully isolated as few as 1 × 102 CFU/mL of S. aureus cells from milk, blood, and saliva. The cell wall hydrolyzing activity of 80α endolysin promoted the genomic DNA extraction efficiency by 12.7 folds on average, compared to the commercial bacterial genomic DNA extraction kit. Then, recombinase polymerase amplification (RPA) was exploited to amplify the nuc gene of S. aureus from the extracted DNA at 37 °C for 30 min. The RPA product activated Cas12a endonuclease activity to cleave fluorescently labeled ssDNA probes. We then converted the generated signal into a fluorescent readout, detectable by either the naked eye or a portable, self-assembled instrument with ultrasensitivity. The entire procedure, from isolation to identification, can be completed within 2 h. The simplicity and sensitivity of the method developed in this study make it of great application value in S. aureus detection, especially in areas with limited resource supply.
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Técnicas Biosensibles , Endopeptidasas , Staphylococcus aureus , Staphylococcus aureus/aislamiento & purificación , Staphylococcus aureus/virología , Técnicas Biosensibles/métodos , Endopeptidasas/química , Endopeptidasas/aislamiento & purificación , Endopeptidasas/genética , Bacteriófagos/química , Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Humanos , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/química , Fagos de Staphylococcus/aislamiento & purificación , Animales , Técnicas de Amplificación de Ácido Nucleico/métodos , Infecciones Estafilocócicas/microbiología , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Nucleasa Microcócica/química , Nucleasa Microcócica/metabolismo , Nucleasa Microcócica/genética , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
Pharmacokinetics and safety studies of innovative drugs is an essential part of drug development process. Previously we have developed novel drug for intravenous administration (lyophilizate) containing modified endolysin LysECD7-SMAP that showed notable antibacterial effect in different animal models of systemic infections. Here we present data on pharmacokinetics of endolysin in mice after single and multiple injections. Time-concentration curves were obtained, pharmacokinetic parameters for preparation (C0, kel t1/2, AUC0-∞, MRT, ClT, Vss) were calculated. It was shown that although endolysin is rather short-living in blood serum (t1/2â¯=â¯12.5 min) the therapeutic concentrations of LysECD7-SMAP (in degraded and non-degraded form) were detected for 60 min after injection that is sufficient for antibacterial effect. Based on the obtained data, it was proposed that endolysin distributes presumably in murine blood, degrades in blood and liver, and is eliminated via glomerular filtration. Safety profile of the preparation relating to general toxicity, immunotoxicity and allergenicity was assessed in rodents. It was demonstrated that LysECD7-SMAP in potential therapeutic (12.5 mg/kg), 10-fold (125 mg/kg) and 40-fold (500 mg/kg) doses showed no signs of intoxication and significant abnormalities after single and repeated i.v. administrations, preparation was non-immunogenic and induced minor and reversible allergic reaction in animal.
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Vast shotgun metagenomics data remain an underutilized resource for novel enzymes. Artificial intelligence (AI) has increasingly been applied to protein mining, but its conventional performance evaluation is interpolative in nature, and these trained models often struggle to extrapolate effectively when challenged with unknown data. In this study, we present a framework (DeepMineLys [deep mining of phage lysins from human microbiome]) based on the convolutional neural network (CNN) to identify phage lysins from three human microbiome datasets. When validated with an independent dataset, our method achieved an F1-score of 84.00%, surpassing existing methods by 20.84%. We expressed 16 lysin candidates from the top 100 sequences in E. coli, confirming 11 as active. The best one displayed an activity 6.2-fold that of lysozyme derived from hen egg white, establishing it as the most potent lysin from the human microbiome. Our study also underscores several important issues when applying AI to biology questions. This framework should be applicable for mining other proteins.
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Bacteriófagos , Microbiota , Humanos , Bacteriófagos/genética , Bacteriófagos/metabolismo , Minería de Datos , Proteínas Virales/metabolismo , Redes Neurales de la Computación , Animales , Muramidasa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
Endolysins (lysins), a novel class of antibacterial agents derived from bacteriophages, efficiently lyse bacteria by degrading the peptidoglycan layer within the bacterial wall. Colistin, a classic peptide antibiotic with the ability to permeabilize the outer membrane, has recently shown great promise in synergizing with lysins against gram-negative bacteria. However, the exact mechanisms responsible for their synergy remain unclear. Here, we first demonstrated the synergistic bacterial killing of various lysin and colistin combinations. With a model lysin, LysAB2, we then confirmed that there is a threshold concentration of colistin causing sufficient permeabilization of the outer membrane for lysin to access the peptidoglycan layer and subsequently exert its lytic ability. The threshold colistin concentrations were found to range 0.2-0.8 µM for the tested bacteria, with the exact value largely depending on the density of lipopolysaccharides on the outer membrane. Beyond the threshold colistin level, LysAB2 could synergize with colistin at a concentration as low as 0.31 µM. Next, we proved for the first time that lysin-induced degradation of the peptidoglycan layer facilitated the disruption of cytoplasmic membrane by colistin, elevated the level of reactive oxygen species in bacterial cells, and boosted the killing effect of colistin. Additionally, the colistin-lysin combination could effectively eliminate established biofilms due to the biofilm dispersal ability of lysin. The in-vivo efficacy was preliminary confirmed in a Galleria mellonella infection model for combination with colistin doses (≥ 1.8 µg/larvae), which could reach beyond the threshold concentration, and a fixed LysAB2 dose (10 µg/larvae). In summary, our study provided the first experimental evidence unravelling the mechanisms behind the synergy of colistin and lysins. All these findings provided important insights in guiding the dosing strategy for applying this combination in future development.
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Antibacterianos , Colistina , Farmacorresistencia Bacteriana Múltiple , Endopeptidasas , Bacterias Gramnegativas , Colistina/farmacología , Endopeptidasas/farmacología , Sinergismo Farmacológico , Bacterias Gramnegativas/efectos de los fármacos , Antibacterianos/farmacología , Humanos , Línea CelularRESUMEN
Bacterial resistance to antibiotics is a significant challenge that is associated with increased morbidity and mortality. Gram-negative bacteria are particularly problematic due to an outer membrane (OM). Current alternatives to antibiotics include antimicrobial peptides or proteins and multifunctional systems such as dendrimers. Antimicrobial proteins such as lysins can degrade the bacterial cell wall, whereas dendrimers can permeabilize the OM, enhancing the activity of endolysins against gram-negative bacteria. In this study, we present a three-stage action of endolysin combined with two different carbosilane (CBS) silver metallodendrimers, in which the periphery is modified with N-heterocyclic carbene (NHC) ligands coordinating a silver atom. The different NHC ligands contained hydrophobic methyl or N-donor pyridyl moieties. The effects of these endolysin/dendrimer combinations are based on OM permeabilization, peptidoglycan degradation, and reactive oxygen species production. The results showed that CBS possess a permeabilization effect (first action), significantly reduced bacterial growth at higher concentrations alone and in the presence of endolysin, increased ROS production (second action), and led to bacterial cell damage (third action). The complex formed between the CHAP domain of endolysin and a CBS silver metallodendrimer, with a triple mechanism of action, may represent an excellent alternative to other antimicrobials with only one resistance mechanism.
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Antibacterianos , Dendrímeros , Endopeptidasas , Bacterias Gramnegativas , Peptidoglicano , Especies Reactivas de Oxígeno , Silanos , Peptidoglicano/metabolismo , Peptidoglicano/química , Especies Reactivas de Oxígeno/metabolismo , Silanos/química , Silanos/farmacología , Antibacterianos/farmacología , Antibacterianos/química , Dendrímeros/química , Dendrímeros/farmacología , Endopeptidasas/metabolismo , Endopeptidasas/química , Bacterias Gramnegativas/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Plata/química , Plata/farmacología , Dominios Proteicos , Permeabilidad de la Membrana Celular/efectos de los fármacosRESUMEN
"An impregnable stronghold where one or more warrior clans can evade enemy attacks" may serve as a description of bacterial biofilm on a smaller level than human conflicts. Consider this hypothetical conflict: who would emerge victorious? The occupants of secure trenches or those carrying out relentless assault? Either faction has the potential for triumph; the defenders will prevail if they can fortify the trench with unwavering resolve, while the assailants will succeed if they can devise innovative means to breach the trench. Hence, bacterial biofilms pose a significant challenge and are formidable adversaries for medical professionals, often leading to the failure of antibiotic treatments in numerous hospital infections. Phage engineering has become the foundation for the targeted enhancement of various phage properties, facilitating the eradication of biofilms. Researchers across the globe have studied the impact of engineered phages and phage-derived enzymes on biofilms formed by difficult-to-treat bacteria. These novel biological agents have shown promising results in addressing biofilm-related challenges. The compilation of research findings highlights the impressive capabilities of engineered phages in combating antibiotic-resistant bacteria, superbugs, and challenging infections. Specifically, these engineered phages exhibit enhanced biofilm destruction, penetration, and prevention capabilities compared to their natural counterparts. Additionally, the engineered enzymes derived from phages demonstrate improved effectiveness in addressing bacterial biofilms. As a result, these novel solutions, which demonstrate high penetration, destruction, and inhibition of biofilms, can be regarded as a viable option for addressing infectious biofilms in the near future.
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Background: Endolysins are phage-encoded lytic enzymes that degrade bacterial peptidoglycan at the end of phage lytic cycles to release new phage particles. These enzymes are being explored as an alternative to small-molecule antibiotics. Methods: The crystal structure of KTN6 Gp46 was determined and compared with a ColabFold model. Cleavage specificity was examined using a peptidoglycan digest and reversed-phase high-performance liquid chromatography coupled to mass spectrometry (HPLC/MS). Results: The structure of KTN6 Gp46 could be determined at 1.4 Å resolution, and key differences in loops of the putative peptidoglycan binding domain were identified in comparison with its closest known homologue, the endolysin of phage SPN1S. Reversed-phase HPLC/MS analysis of the reaction products following peptidoglycan digestion confirmed the muramidase activity of Gp46, consistent with structural predictions. Conclusion: These insights into the structure and function of endolysins further expand the toolbox for endolysin engineering and explore their potential in enzyme-based antibacterial design strategies.
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The importance of the microbiota in the intestinal tract for human health has been increasingly recognized. In this perspective, microbiome modulation, a targeted alteration of the microbial composition, has gained interest. Phage lysins, peptidoglycan-degrading enzymes encoded by bacteriophages, are a promising new class of antibiotics currently under clinical development for treating bacterial infections. Due to their high specificity, lysins are considered microbiome-friendly. This review explores the opportunities and challenges of using lysins as microbiome modulators. First, the high specificity of endolysins, which can be further modulated using protein engineering or targeted delivery methods, is discussed. Next, obstacles and possible solutions to assess the microbiome-friendliness of lysins are considered. Finally, lysin delivery to the intestinal tract is discussed, including possible delivery methods such as particle-based and probiotic vehicles. Mapping the hurdles to developing lysins as microbiome modulators and identifying possible ways to overcome these hurdles can help in their development. In this way, the application of these innovative antimicrobial agents can be expanded, thereby taking full advantage of their characteristics.
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Bacteriófagos , Endopeptidasas , Microbioma Gastrointestinal , Humanos , Bacteriófagos/fisiología , Animales , Endopeptidasas/metabolismo , Bacterias/genética , Bacterias/metabolismo , Bacterias/virología , Bacterias/clasificación , Probióticos , Antibacterianos/farmacología , Infecciones Bacterianas/microbiología , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/terapia , Proteínas Virales/metabolismo , Proteínas Virales/genética , Peptidoglicano/metabolismoRESUMEN
Development of novel antibacterial agents is imperative due to the increasing threat of antibiotic-resistant pathogens. This study aimed to develop the enhanced antibacterial activity and in-vivo efficacy of a novel truncated endolysin, CHAPSAP26-161, derived from the endolysin LysSAP26, against multidrug-resistant bacteria. CHAPSAP26-161 exhibited higher protein purification efficiency in E. coli and antibacterial activity than LysSAP26. Moreover, CHAPSAP26-161 showed the higher lytic activity against A. baumannii with minimal bactericidal concentrations (MBCs) of 5-10 µg/ml, followed by Staphylococcus aureus with MBCs of 10-25 µg/ml. Interestingly, CHAPSAP26-161 could lyse anaerobic bacteria, such as Clostridioides difficile, with MBCs of 25-50 µg/ml. At pH 4-8 and temperatures of 4°C-45°C, CHAPSAP26-161 maintained antibacterial activity without remarkable difference. The lytic activity of CHAPSAP26-161 was increased with Zn2+. In vivo tests demonstrated the therapeutic effects of CHAPSAP26-161 in murine systemic A. baumannii infection model. In conclusion, CHAPSAP26-161, a truncated endolysin that retains only the CHAP domain from LysSAP26, demonstrated enhanced protein purification efficiency and antibacterial activity compared to LysSAP26. It further displayed broad-spectrum antibacterial effects against S. aureus, A. baumannii, and C. difficile. Our in vitro and in-vivo results of CHAPSAP26-161 highlights its promise as an innovative therapeutic option against those bacteria with multiple antibiotic resistance.
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Acinetobacter baumannii , Antibacterianos , Clostridioides difficile , Modelos Animales de Enfermedad , Endopeptidasas , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus , Animales , Endopeptidasas/farmacología , Endopeptidasas/química , Endopeptidasas/metabolismo , Antibacterianos/farmacología , Antibacterianos/química , Ratones , Clostridioides difficile/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Acinetobacter baumannii/efectos de los fármacos , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Infecciones por Acinetobacter/tratamiento farmacológico , Infecciones por Acinetobacter/microbiología , Concentración de Iones de Hidrógeno , Farmacorresistencia Bacteriana Múltiple , Femenino , Ratones Endogámicos BALB C , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , TemperaturaRESUMEN
Bacterial ghosts (BGs) are described as bacterial cell envelopes that retain their structure but lack cytoplasmic contents. The study of BGs spans multiple disciplinary domains, and the development of BG production techniques to obtain ample and stable BG samples holds significant implications for probing the biological characteristics of BGs, devising novel disease treatment strategies, and leveraging their industrial applications. Numerous products encoded within bacteriophage (phage) genomes possess the capability to lyse bacteria, thereby inducing BG formation primarily via disruption of bacterial cell wall integrity. This review comprehensively surveys the utilization of phage-encoded proteins in BG production techniques, encompassing methodologies such as phage E protein-mediated lysis, perforin protein-induced lysis, and strategies combining E protein with holin-endolysin systems. Additionally, discussions and summaries are provided on the current applications, challenges, and modification strategies associated with different techniques. Through a focused exploration of BG production techniques, with an emphasis on precise manipulation of BG formation using phage-encoded protein technologies, this study aims to furnish robust tools and methodologies for delving into the mechanisms underlying BG formation, as well as for the development of novel therapeutic strategies and applications based on BGs.
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Bacterias , Bacteriófagos , Proteínas Virales , Bacteriófagos/genética , Bacteriófagos/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Bacterias/virología , Bacterias/metabolismo , Bacterias/genética , Pared Celular/metabolismo , Endopeptidasas/metabolismo , BacteriólisisRESUMEN
Endolysins, proteins encoded by phages to lyse their hosts and release their progeny, have evolved to adapt to the structural features of each host. The endolysins from Staphylococcus-infecting phages typically feature complex architectures with two enzymatically active domains (EADs) and one cell wall-binding domain (CBD) belonging to the bacterial SH3 (SH3b) superfamily. This study focuses on three SH3b-like CBDs from representative staphylococcal phage endolysins (LysRODI, LysC1C and LysIPLA5) that were structurally and functionally characterized. While RODI_CBD and C1C_CBD were assigned to the well-known SH3_5 family, a new family, SH3b_T (PF24246), was identified using the CBD from LysIPLA5 as a model. GFP-fused CBDs were created to assess their differential binding to a collection of staphylococcal strains. IPLA5_CBD showed enhanced binding to Staphylococcus epidermidis, while RODI_CBD and C1C_CBD exhibited distinct binding profiles, with RODI_CBD targeting Staphylococcus aureus specifically and C1C_CBD displaying broad binding. Sequence comparisons suggested that a few differences in key amino acids could be responsible for the latter binding difference. The CBDs modulated the activity spectrum of synthetic EAD-CBD combinations in accordance with the previous binding profiles, but in a manner that was also dependent on the EAD present in the fusion protein. These results serve as a context for the diversity and versatility of SH3b domains in staphylococcal endolysins, providing insights on how (i) the CBDs from this superfamily have diverged to adapt to diverse bacterial ligands in spite of sharing a common fold; and (ii) the evolution of specificity relies on the EAD-CBD combination rather than solely the CBD.
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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.
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Antibacterianos , Biopelículas , Endopeptidasas , Escherichia coli , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Endopeptidasas/farmacología , Endopeptidasas/genética , Endopeptidasas/metabolismo , Antibacterianos/farmacología , Concentración de Iones de Hidrógeno , Plancton/efectos de los fármacos , Plancton/virología , Colifagos/genética , Colifagos/fisiología , Ácido Láctico/farmacología , Bacteriófagos/genética , Temperatura , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , Proteínas Virales/genética , Proteínas Virales/farmacología , Proteínas Virales/metabolismoRESUMEN
Salmonella enterica (S. enterica) is the most common food and waterborne pathogen worldwide. The growing trend of antibiotic-resistant S. enterica poses severe healthcare threats. As an alternative antimicrobial agent, bacteriophage-encoded endolysins (endolysins) are a potential agent in controlling S. enterica infection. Endolysins are enzymes that particularly target the peptidoglycan layer of bacterial cells, leading to their rupture and destruction. However, the application of endolysins against Gram-negative bacteria is limited due to the presence of the outer membrane in the cell wall, which hinders the permeation of externally applied endolysins. This study aimed the prokaryotic expression system to produce the recombinant endolysin ENDO-1252, encoded by the Salmonella bacteriophage-1252 associated with S. Enteritidis. Subsequently, ENDO-1252 had strong lytic activity not only against S. Enteritidis but also against S. Typhimurium. In addition, ENDO-1252 showed optimal thermostability and lytic activity at 25°C with a pH of 7.0. In combination with 0.1 mM EDTA, the effect of 120 µg of ENDO-1252 for 6 hours exhibited the highest lytic activity, resulting in a reduction of 1.15 log or 92.87% on S. Enteritidis. These findings suggest that ENDO-1252 can be used as a potential and innovative antibacterial agent for controlling the growth of S. Enteritidis.
Asunto(s)
Endopeptidasas , Fagos de Salmonella , Salmonella enterica , Endopeptidasas/farmacología , Endopeptidasas/genética , Endopeptidasas/metabolismo , Fagos de Salmonella/genética , Salmonella enterica/efectos de los fármacos , Salmonella enterica/genética , Antibacterianos/farmacología , Salmonella enteritidis/efectos de los fármacos , Salmonella enteritidis/genética , Salmonella enteritidis/crecimiento & desarrolloRESUMEN
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.