Phage Endolysin Research Articles

Molecular Docking of Endolysins for Studying Peptidoglycan Binding Mechanism

Endolysins of bacteriophages, which degrade the bacterial cell wall peptidoglycan, are applicable in many industries to deal with biofilms and bacterial infections. While multi-domain endolysins have both enzymatically active and cell wall-binding domains, single-domain endolysins consist only of an enzymatically active domain, and their mechanism of peptidoglycan binding remains unexplored, for this is a challenging task experimentally. This research aimed to explore the binding mechanism of endolysins using computational approaches, namely molecular docking and bioinformatical tools, and analyze the performance of these approaches. The docking engine Autodock Vina 1.1.2 and the 3D-RISM module of AmberTools 24 were studied in the current work and used for receptor–ligand affinity and binding energy calculations, respectively. Two possible mechanisms of single-domain endolysin–ligand binding were predicted by Autodock Vina and verified by the 3D-RISM. As a result, the previously obtained experimental results on peptidoglycan binding of the isolated gamma phage endolysin PlyG enzymatically active domain were supported by molecular docking. Both methods predicted that single-domain endolysins are able to bind peptidoglycan, with Autodock Vina being able to give accurate numerical estimates of protein–ligand affinities and 3D-RISM providing comparative values.

The Synergistic and Chimeric Mechanism of Bacteriophage Endolysins: Opportunities for Application in Biotherapeutics, Food, and Health Sectors.

A major growing concern in the human and animal health sector is the emergence of antibiotic-resistant pathogenic bacteria due to the indiscriminate use of antibiotics. The exogenous application of bacteriophage endolysins causes abrupt lysis of the bacterial cell wall, which computes them as alternatives to antibiotics. Although naturally occurring endolysins may display limitations in solubility, lytic activity, and narrow lytic spectrum, novel strategies like developing chimeric endolysins and using endolysins in synergism with other antimicrobial agents are required to improve the lytic activity of natural endolysins. The modular structure of endolysins led to the development of novel chimeric endolysins via shuffling enzymatic and cell wall binding domains of different endolysins, using endolysins in a synergistic approach, and their applications in various in vitro and in vivo experiments and different applicable areas. This article aims to review the role of chimeric endolysins and their use in synergistic mode with other biofilm-reducing agents to control biofilm formation and deteriorating pre-formed biofilms in food, dairy, and medical industries. Promoting further development of phage technology and innovation in antibiotic therapy can achieve long-term sustainable development and economic returns.

Alginate binding enhances the structural stability and potentiates the lytic activity of bacteriophage endolysin's partially folded conformation

Polysaccharide polymers are increasingly being used as chaperon-like macromolecules in assisting protein folding of unfolded protein molecules. They interact with unfolded or partially folded proteins in a charge and conformation specific manner that results in the formation of stable protein-polysaccharide complexes. In most of the cases, the complex formation of protein-polysaccharide is driven via non-covalent interactions that have found to endorse the activity of proteins. T4L (18.7 kDa) and T7L (17 kDa) endolysins belong to the hydrolase and amidase class of peptidoglycan degrading enzymes. Both T4L and T7L exist in partially folded forms and are devoid of lytic activity at low pH conditions. In the current study, we assessed the binding of alginate with T4L and T7L at pH 7 and 3 using variety of biophysical and biochemical techniques. Spectroscopic studies revealed differential structural modulations of partially folded T4L and T7L upon their interaction with alginate. Further, the complex formation of alginate with partially folded T4L/T7L was confirmed by ITC and STEM. Additionally, the formed complexes of alginate with both T4L/T7L PF endolysins were found to be chemically and enzymatically stable. Moreover, such complexes were also marked with differential enhancement in their lytic activities at acidic pH conditions. This implied the potency of alginate as an excellent choice of matrix to preserve the structural and functional integrity of partially folded forms of T4L and T7L at highly acidic conditions.

The New SH3b_T Domain Increases the Structural and Functional Variability Among SH3b-Like CBDs from Staphylococcal Phage Endolysins.

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.

Bacteriophage Endolysin: Isolation, Cloning, and Antibacterial Activity

Bacteriophage Endolysin: Isolation, Cloning, and Antibacterial Activity

Phage endolysins as new therapeutic options for multidrug resistant Staphylococcus aureus: an emerging antibiotic-free way to combat drug resistant infections.

Phage endolysins as new therapeutic options for multidrug resistant Staphylococcus aureus: an emerging antibiotic-free way to combat drug resistant infections.

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.

Yeast Expressing a Phage Endolysin Reduces Endogenous Clostridium perfringens Ex Vivo in 21-Day-Old Broiler Chicken Intestinal Fluids.

The phage endolysin PlyCP41 when purified from Escherichia coli exhibits lytic activity against Clostridium perfringens (CP) in vitro. The anti-clostridial activity of PlyCP41 endolysin expressed in transgenic yeast (Saccharomyces cerevisiae) was verified in phosphate buffered saline via mixing experiments with cultured CP and transgenic yeast slurries followed by serial dilution plating and colony counts on tryptose sulfite cycloserine (CP indicator) plates. The transgenic yeast containing PlyCP41 resulted in a log10 4.5 reduction (99.997%; P < 0.01) of the cultured CP. In addition, this serial dilution plating assay was used to demonstrate that transgenic yeast slurries could reduce the endogenous CP content in fluids from three different gastrointestinal regions (proximal, medial, and distal) from 21-day-old broiler chickens. The transgenic yeast treatment of gut slurries resulted in a log 10 1.19, 4.53, and 1.28 reduction in proximal, medial, and distal gut slurries (90% to 99.99% of the endogenous CP; P < 0.01), respectively, compared to nontreatment controls. These results indicate that the phage endolysin PlyCP41 expressed in S. cerevisiae is effective at reducing the endogenous CP in gastrointestinal fluids of broiler chickens. Future studies will measure the anti-CP effect in vivo by administering transgenic yeast to broiler chickens in the feed.

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.

Split fluorescent protein-mediated multimerization of cell wall binding domain for highly sensitive and selective bacterial detection

Cell wall peptidoglycan binding domains (CBDs) of cell lytic enzymes, including bacteriocins, autolysins and bacteriophage endolysins, enable highly selective bacterial binding, and thus, have potential as biorecognition molecules for nondestructive bacterial detection. Here, a novel design for a self-complementing split fluorescent protein (FP) complex is proposed, where a multimeric FP chain fused with specific CBDs ((FP-CBD)n) is assembled inside the cell, to improve sensitivity by enhancing the signal generated upon Staphylococcus aureus or Bacillus anthracis binding. Flow cytometry shows enhanced fluorescence on the cell surface with increasing FP stoichiometry and surface plasmon resonance reveals nanomolar binding affinity to isolated peptidoglycan. The breadth of function of these complexes is demonstrated through the use of CBD modularity and the ability to attach enzymatic detection modalities. Horseradish peroxidase-coupled (FP-CBD)n complexes generate a catalytic amplification, with the degree of amplification increasing as a function of FP length, reaching a limit of detection (LOD) of 103 cells/droplet (approximately 0.1 ng S. aureus or B. anthracis) within 15 min on a polystyrene surface. These fusion proteins can be multiplexed for simultaneous detection. Multimeric split FP-CBD fusions enable use as a biorecognition molecule with enhanced signal for use in bacterial biosensing platforms.

Combined effect of SAR-endolysin LysKpV475 with polymyxin B and Salmonella bacteriophage phSE-5.

Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested in vitro and in vivo against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 μg ml-1 against Pseudomonas aeruginosa ATCC 27853, 16.25 μg ml-1 against S. enterica Typhimurium ATCC 13311, and 32.50 μg ml-1 against Klebsiella pneumoniae ATCC BAA-2146 and Enterobacter cloacae P2224. LysKpV475 showed bactericidal activity only for P. aeruginosa ATCC 27853 (32.50 μg ml-1) and P. aeruginosa P2307 (65.00 μg ml-1) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against K. pneumoniae ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and S. enterica Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against S. enterica Typhimurium was also observed when the lyophilized LysKpV475 at ⅔ MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant Salmonella reduction of 2 logs after 6 h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.

Lysin and Lytic Phages Reduce Vibrio Counts in Live Feed and Fish Larvae.

Vibrio species are naturally found in estuarine and marine ecosystems, but are also recognized as significant human enteropathogens, often linked to seafood-related illnesses. In aquaculture settings, Vibrio poses a substantial risk of infectious diseases, resulting in considerable stock losses and prompting the use of antimicrobials. However, this practice contributes to the proliferation of antimicrobial-resistant (AMR) bacteria and resistance genes. Our investigation aimed to explore the potential of biological agents such as bacteriophage CH20 and endolysin LysVPp1 in reducing Vibrio bacterial loads in both rotifer and fish larvae. LysVPp1's lytic activity was assessed by measuring absorbance reduction against various pathogenic Vibrio strains. Phage CH20 exhibited a limited host range, affecting only Vibrio alginolyticus GV09, a highly pathogenic strain. Both CH20 and LysVPp1 were evaluated for their effectiveness in reducing Vibrio load in rotifers or fish larvae through short-setting bioassays. Our results demonstrated the significant lytic effect of endolysin LysVPp1 on strains of Vibrio alginolyticus, Vibrio parahaemolyticus, and Vibrio splendidus. Furthermore, we have showcased the feasibility of reducing the load of pathogenic Vibrio in live feed and fish larvae by using a non-antibiotic-based approach, such as lytic phage and endolysin LysVPp1, thus contributing to the progress of a sustainable aquaculture from a One Health perspective.

Bacteriophage Endolysins Antibacterial Effect against S. mutans Isolated from Dental Caries: An In-Vitro Study

Background: Many bacterial infections including dental caries that caused by Streptococcus mutans (S. mutans) were difficult to be treated due to the unavailability of effective antibiotics. Therefore, it requires to focus the attention on endolysin as an antibacterial agent. This study was designed to evaluate the In-vitro effectiveness of specific bacteriophage endolysins against S. mutans isolated from dental caries.Methods: Twenty-five isolates of S. mutans were collected from patients with dental caries attended to Al-Imamain Al-Kadhimain, Medical City Hospital, and AL-Ameen Health Center, during the period from December 2021 to May 2022. The S. mutans isolates were identified by culturing them on their specific mitis salivarius sucrose bacitracin tellurite media (MS-SBT agar), morphological characteristics, gram staining, biochemical tests and VITEK 2 compact system. Seven of these S. mutans isolates were used for the in-vitro study.Results: The bacteriophage endolysin enzyme extracted from the corresponding S. mutans isolate was effective as In-vitro antibacterial agent with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) ranging between (13.63 – 17.83) µg/ml and (17.04-22.28) µg/ml respectively.Conclusion: The endolysin was effective as antibacterial agent against multi-drug resistant (MDR) S. mutans upon growing and testing on culture media.

Biofilm-disrupting effects of phage endolysins LysAm24, LysAp22, LysECD7, and LysSi3: breakdown the matrix.

The ability of most opportunistic bacteria to form biofilms, coupled with antimicrobial resistance, hinder the efforts to control widespread infections, resulting in high risks of negative outcomes and economic costs. Endolysins are promising compounds that efficiently combat bacteria, including multidrug-resistant strains and biofilms, without a low probability of subsequent emergence of stable endolysin-resistant phenotypes. However, the details of antibiofilm effects of these enzymes are poorly understood. To elucidate the interactions of bacteriophage endolysins LysAm24, LysAp22, LysECD7, and LysSi3 with bacterial films formed by Gram-negative species, we estimated their composition and assessed the endolysins' effects on the most abundant exopolymers in vitro. The obtained data suggests a pronounced efficiency of these lysins against biofilms with high (Klebsiella pneumoniae) and low (Acinetobacter baumannii) matrix contents, or dual-species biofilms, resulting in at least a twofold loss of the biomass. These peptidoglycan hydrolases interacted diversely with protective compounds of biofilms such as extracellular DNA and polyanionic carbohydrates, indicating a spectrum of biofilm-disrupting effects for bacteriolytic phage enzymes. Specifically, we detected disruption of acid exopolysaccharides by LysAp22, strong DNA-binding capacity of LysAm24, both of these interactions for LysECD7, and neither of them for LysSi3.

Decoding the Structure-Function Relationship of the Muramidase Domain in E. coli O157.H7 Bacteriophage Endolysin: A Potential Building Block for Chimeric Enzybiotics.

Bacteriophage endolysins are potential alternatives to conventional antibiotics for treating multidrug-resistant gram-negative bacterial infections. However, their structure-function relationships are poorly understood, hindering their optimization and application. In this study, we focused on the individual functionality of the C-terminal muramidase domain of Gp127, a modular endolysin from E. coli O157:H7 bacteriophage PhaxI. This domain is responsible for the enzymatic activity, whereas the N-terminal domain binds to the bacterial cell wall. Through protein modeling, docking experiments, and molecular dynamics simulations, we investigated the activity, stability, and interactions of the isolated C-terminal domain with its ligand. We also assessed its expression, solubility, toxicity, and lytic activity using the experimental data. Our results revealed that the C-terminal domain exhibits high activity and toxicity when tested individually, and its expression is regulated in different hosts to prevent self-destruction. Furthermore, we validated the muralytic activity of the purified refolded protein by zymography and standardized assays. These findings challenge the need for the N-terminal binding domain to arrange the active site and adjust the gap between crucial residues for peptidoglycan cleavage. Our study shed light on the three-dimensional structure and functionality of muramidase endolysins, thereby enriching the existing knowledge pool and laying a foundation for accurate in silico modeling and the informed design of next-generation enzybiotic treatments.

Engineering strategies and challenges of endolysin as an antibacterial agent against Gram-negative bacteria.

Bacteriophage endolysin is a novel antibacterial agent that has attracted much attention in the prevention and control of drug-resistant bacteria due to its unique mechanism of hydrolysing peptidoglycans. Although endolysin exhibits excellent bactericidal effects on Gram-positive bacteria, the presence of the outer membrane of Gram-negative bacteria makes it difficult to lyse them extracellularly, thus limiting their application field. To enhance the extracellular activity of endolysin and facilitate its crossing through the outer membrane of Gram-negative bacteria, researchers have adopted physical, chemical, and molecular methods. This review summarizes the characterization of endolysin targeting Gram-negative bacteria, strategies for endolysin modification, and the challenges and future of engineering endolysin against Gram-negative bacteria in clinical applications, to promote the application of endolysin in the prevention and control of Gram-negative bacteria.

Bacteriophages as a potential substitute for antibiotics: A comprehensive review.

Over the years, the administration of antibiotics for the purpose of addressing bacterial infections has become increasingly challenging due to the increased prevalence of antimicrobial resistance exhibited by various strains of bacteria. Multidrug-resistant(MDR) bacterial species are rising due to the unavailability of novel antibiotics, leading to higher mortality rates. With these conditions, there is a need for alternatives in which phage therapy has made promising results. Phage-derived endolysins, phage cocktails, and bioengineered phages are effective and have antimicrobial properties against MDRand extensively drug-resistant strains. Despite these, it has been observed that phages can give antimicrobial activity to more than one bacterial species. Thus, phage cocktail against resistant strains provides broad spectrum treatment and magnitude of effectivity, which is many folds higher than antibiotics. Many commercially available endolysins such as Staphefekt SA.100, Exebacase (CF-301), and N-Rephasin®SAL200 are used in biofilm penetration and treating plant diseases. The role of CMP1 phage endolysin in transgenic tomato plants in preventing Clavibacter michiganensis infection and the effectiveness of phage in protecting Atlantic salmon from vibriosis have been reported. Furthermore, phage-derived endolysin therapy, such as TSPphg phage exogenous treatment, can aid in disrupting cell walls, leading to bacterial cell lysis. As animals in aquaculture and slaughterhouses are highly susceptible to bacterial infections, effective phage therapy instead of antibiotics can help treat poultry animals, preserve them, and facilitate disease-free trade. Using bioengineered phages and phage cocktails enhances the effectiveness by providing a broad spectrum of phages and target specificity. Research is currently being conducted on clinical trials to confirm the efficacy of engineered phages and phage cocktails in humans. Although obtaining commercial approval may be time-consuming, it will be beneficial in the postantibiotic era. This review provides an overview of the significance of phage therapy as a potential alternative to antibiotics in combating resistant bacterial strains and its application to various fields and emphasizes the importance of safeguarding and ensuring treatment efficacy.

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

BackgroundStaphylococcus 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.ResultsIn 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.ConclusionsOverall, 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.

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, 88L, and 89V) 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.

Characterization of a Straboviridae phage vB_AbaM-SHI and its inhibition effect on biofilms of Acinetobacter baumannii.

Acinetobacter baumannii (A. baumannii) is a popular clinical pathogen worldwide. Biofilm-associated antibiotic-resistant A. baumannii infection poses a great threat to human health. Bacteria in biofilms are highly resistant to antibiotics and disinfectants. Furthermore, inhibition or eradication of biofilms in husbandry, the food industry and clinics are almost impossible. Phages can move across the biofilm matrix and promote antibiotic penetration. In the present study, a lytic A. baumannii phage vB_AbaM-SHI, belonging to family Straboviridae, was isolated from sauce chop factory drain outlet in Wuxi, China. The DNA genome consists of 44,180 bp which contain 93 open reading frames, and genes encoding products morphogenesis are located at the end of the genome. The amino acid sequence of vB_AbaM-SHI endolysin is different from those of previously reported A. baumannii phages in NCBI. Phage vB_AbaM-SHI endolysin has two additional β strands due to the replacement of a lysine (K) (in KU510289.1, NC_041857.1, JX976549.1 and MH853786.1) with an arginine (R) (SHI) at position 21 of A. baumannii phage endolysin. Spot test showed that phage vB_AbaM-SHI is able to lyse some antibiotic-resistant bacteria, such as A. baumannii (SL, SL1, and SG strains) and E. coli BL21 strain. Additionally, phage vB_AbaM-SHI independently killed bacteria and inhibited bacterial biofilm formation, and synergistically exerted strong antibacterial effects with antibiotics. This study provided a new perspective into the potential application value of phage vB_AbaM-SHI as an antimicrobial agent.