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.

Exploring the therapeutic potential of DV-B-120 as an inhibitor of dengue virus infection.

Dengue fever, an infectious disease prevalent in subtropical and tropical regions, currently lacks effective small-molecule drugs as treatment. In this study, we used a fluorescence peptide cleavage assay to screen seven compounds to assess their inhibition of the dengue virus (DENV) NS2B-NS3 protease. DV-B-120 demonstrated superior inhibition of NS2B-NS3 protease activity and lower toxicity compared to ARDP0006. The selectivity index of DV-B-120 was higher than that of ARDP0006. In vivo assessments of the antiviral efficacy of DV-B-120 against DENV replication demonstrated delayed mortality of suckling mice treated with the compound, with 60-80% protection against life-threatening effects, compared to the outcomes of DENV-infected mice treated with saline. The lower clinical scores of DENV-infected mice treated with DV-B-120 indicated a reduction in acute-progressive illness symptoms, underscoring the potential therapeutic impact of DV-B-120. Investigations of DV-B-120's ability to restore the antiviral type I IFN response in the brain tissue of DENV-infected ICR suckling mice demonstrated its capacity to stimulate IFN and antiviral IFN-stimulated gene expression. DV-B-120 not only significantly delayed DENV-2-induced mortality and illness symptoms but also reduced viral numbers in the brain, ultimately restoring the innate antiviral response. These findings strongly suggest that DV-B-120 holds promise as a therapeutic agent against DENV infection and highlight its potential contribution in addressing the current lack of effective treatments for this infectious disease.IMPORTANCEThe prevalence of dengue virus (DENV) infection in tropical and subtropical regions is escalating due to factors like climate change and mosquito vector expansion. With over 300 million annual infections and potentially fatal outcomes, the urgent need for effective treatments is evident. While the approved Dengvaxia vaccine has variable efficacy, there are currently no antiviral drugs for DENV. This study explores seven compounds targeting the NS2B-NS3 protease, a crucial protein in DENV replication. These compounds exhibit inhibitory effects on DENV-2 NS2B-NS3, holding promise for disrupting viral replication and preventing severe manifestations. However, further research, including animal testing, is imperative to assess therapeutic efficacy and potential toxicity. Developing safe and potent treatments for DENV infection is critical in addressing the rising global health threat posed by this virus.

SP-CHAP, an endolysin with enhanced activity against biofilm pneumococci and nasopharyngeal colonization.

Streptococcus pneumoniae (Spn), a Gram-positive bacterium, is responsible for causing a wide variety of invasive infections. The emergence of multi-drug antibiotic resistance has prompted the search for antimicrobial alternatives. Phage-derived peptidoglycan hydrolases, known as endolysins, are an attractive alternative. In this study, an endolysin active against Spn, designated SP-CHAP, was cloned, produced, purified, biochemically characterized, and evaluated for its antimicrobial properties. Cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domains are widely represented in bacteriophage endolysins but have never previously been reported for pneumococcal endolysins. Here, we characterize the first pneumococcal endolysin with a CHAP catalytic domain. SP-CHAP was antimicrobial against all Spn serovars tested, including capsular and capsule-free pneumococci, and it was found to be more active than the most widely studied pneumococcal endolysin, Cpl-1, while not affecting various oral or nasal commensal organisms tested. SP-CHAP was also effective in eradicating Spn biofilms at concentrations as low as 1.56 µg/mL. In addition, a Spn mouse nasopharyngeal colonization model was employed, which showed that SP-CHAP caused a significant reduction in Spn colony-forming units, even more than Cpl-1. These results indicate that SP-CHAP may represent a promising alternative to combating Spn infections. IMPORTANCE: Considering the high rates of pneumococcal resistance reported for several antibiotics, alternatives are urgently needed. In the present study, we report a Streptococcus pneumoniae-targeting endolysin with even greater activity than Cpl-1, the most characterized pneumococcal endolysin to date. We have employed a combination of biochemical and microbiological assays to assess the stability and lytic potential of SP-CHAP and demonstrate its efficacy on pneumococcal biofilms in vitro and in an in vivo mouse model of colonization. Our findings highlight the therapeutic potential of SP-CHAP as an antibiotic alternative to treat Streptococcus pneumoniae infections.

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.

Gram-negative endolysins: overcoming the outer membrane obstacle.

Our ability to control the growth of Gram-negative bacterial pathogens is challenged by rising antimicrobial resistance and requires new approaches. Endolysins are phage-derived enzymes that degrade peptidoglycan and therefore offer potential as antimicrobial agents. However, the outer membrane (OM) of Gram-negative bacteria impedes the access of externally applied endolysins to peptidoglycan. This review highlights recent advances in the discovery and characterization of natural endolysins that can breach the OM, as well as chemical and engineering approaches that increase antimicrobial efficacy of endolysins against Gram-negative pathogens.

Characterization of a novel bacteriophage endolysin (LysAB1245) with extended lytic activity against distinct capsular types associated with Acinetobacter baumannii resistance.

Capsular polysaccharides are considered as major virulence factors associated with the ability of multidrug-resistant (MDR) Acinetobacter baumannii to cause severe infections. In this study, LysAB1245, a novel bacteriophage-encoded endolysin consisting of a lysozyme-like domain from phage T1245 was successfully expressed, purified, and evaluated for its antibacterial activity against distinct capsular types associated with A. baumannii resistance. The results revealed a broad spectrum activity of LysAB1245 against all clinical MDR A. baumannii isolates belonging to capsular type (KL) 2, 3, 6, 10, 47, 49, and 52 and A. baumannii ATCC 19606. At 2 h following the treatment with 1.7 unit/reaction of LysAB1245, more than 3 log reduction in the numbers of bacterial survival was observed. In addition, LysAB1245 displayed rapid bactericidal activity within 30 min (nearly 3 log CFU/mL of bacterial reduction). Thermostability assay indicated that LysAB1245 was stable over a broad range of temperature from 4 to 70°C, while pH sensitivity assay demonstrated a wide range of pH from 4.5 to 10.5. Furthermore, both minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of LysAB1245 against all MDR A. baumannii isolates and A. baumannii ATCC 19606 were 4.21 µg/mL (0.1 unit/reaction). Conclusively, these results suggest that LysAB1245 possesses potential application for the treatment of nosocomial MDR A. baumannii infections.

A polypeptide derived from pilose antler ameliorates CUMS-induced depression-like behavior by SENP2-PLCß4 signaling axis.

Neurogenesis is known to be closely associated with depression. We aimed to investigate whether a polypeptide monomer derived from pilose antler (polypeptide sequence LSALEGVFYP, PAP) exerts an antidepressant effect by influencing neurogenesis, and to elucidate the mechanism of its antidepressant action. Behavioral tests were performed to observe the antidepressant effect of PAP. Neurogenesis in the dentate gyrus (DG) region of hippocampus was observed by immunofluorescence. The expression of key proteins of Sentrin/SUMO-specific proteases 2 (SENP2)- Phosphoinositide-specific phospholipase C beta 4 (PLCß4) pathway was accessed by co-immunoprecipitation (Co-IP), and the calcium homeostasis associated proteins were observed via Western blot (WB). Subsequently, temozolomide (TMZ) pharmacologically blocked neurogenesis to verify the antidepressant effect of PAP on neurogenesis. The mechanism of PAP antidepressant effect was verified by constructing a sh-SENP2 virus vector to silence SENP2 protein. Finally, corticosterone (CORT)-induced PC12 cell model was used to verify whether PAP was involved in the process of deconjugated PLCß4 SUMOylated. The results showed that PAP improved depression-like behavior and neurogenesis induced by chronic unpredictable mild stimulation (CUMS). In addition, PAP acted on SENP2-PLCß4 pathway to deconjugate the SUMOylation of PLCß4 and affect calcium homeostasis. Pharmacological blockade of neurogenesis by TMZ treatment impaired the antidepressant efficacy of PAP. Knockout of SENP2 in the CUMS model attenuated the antidepressant response of PAP, and the impaired neurogenesis was not ameliorated by PAP treatment. In summary, PAP acted on the SENP2-PLCß4 signaling pathway to inhibit the SUMOylation of PLCß4 and maintain calcium homeostasis, thereby protecting neurogenesis and playing an antidepressant role.

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.

Peptidoglycan biosynthesis-associated enzymatic kinetic characteristics and ß-lactam antibiotic inhibitory effects of different Streptococcus pneumoniae penicillin-binding proteins.

Penicillin-binding proteins (PBPs) include transpeptidases, carboxypeptidases, and endopeptidases for biosynthesis of peptidoglycans in the cell wall to maintain bacterial morphology and survival in the environment. Streptococcus pneumoniae expresses six PBPs, but their enzymatic kinetic characteristics and inhibitory effects on different ß-lactam antibiotics remain poorly understood. In this study, all the six recombinant PBPs of S. pneumoniae displayed transpeptidase activity with different substrate affinities (Km = 1.56-9.11 mM) in a concentration-dependent manner, and rPBP3 showed a greater catalytic efficiency (Kcat = 2.38 s-1) than the other rPBPs (Kcat = 3.20-7.49 × 10-2 s-1). However, only rPBP3 was identified as a carboxypeptidase (Km = 8.57 mM and Kcat = 2.57 s-1). None of the rPBPs exhibited endopeptidase activity. Penicillin and cefotaxime inhibited the transpeptidase and carboxypeptidase activity of all the rPBPs but imipenem did not inhibited the enzymatic activities of rPBP3. Except for the lack of binding of imipenem to rPBP3, penicillin, cefotaxime, and imipenem bound to all the other rPBPs (KD = 3.71-9.35 × 10-4 M). Sublethal concentrations of penicillin, cefotaxime, and imipenem induced a decrease of pneumococcal pbps-mRNA levels (p < 0.05). These results indicated that all six PBPs of S. pneumoniae are transpeptidases, while only PBP3 is a carboxypeptidase. Imipenem has no inhibitory effect on pneumococcal PBP3. The pneumococcal genes for encoding endopeptidases remain to be determined.

LysZX4-NCA, a new endolysin with broad-spectrum antibacterial activity for topical treatment.

The prevalence of multidrug-resistant highly virulent Klebsiella pneumoniae (MDR-hvKP) requires the development of new therapeutic agents. Herein, a novel lytic phage vB_KpnS_ZX4 against MDR-hvKP was discovered in hospital sewage. Phage vB_KpnS_ZX4 had a short latent period (5 min) and a large burst size (230 PFU/cell). It can rapidly reduce the number of bacteria in vitro and improve survival rates of bacteremic mice in vivo from 0 to 80 % with a single injection of 108 PFU. LysZX4, an endolysin derived from vB_KpnS_ZX4, exhibits potent antimicrobial activity in vitro in combination with ethylenediaminetetraacetic acid (EDTA). The antimicrobial activity of LysZX4 was further enhanced by the fusion of KWKLFKI residues from cecropin A (LysZX4-NCA). In vitro antibacterial experiments showed that LysZX4-NCA exerts broad-spectrum antibacterial activity against clinical Gram-negative bacteria, including MDR-hvKP. Moreover, in the mouse model of MDR-hvKP skin infection, treatment with LysZX4-NCA resulted in a three-log reduction in bacterial burden on the skin compared to the control group. Therefore, the novel phages vB_KpnS_ZX4 and LysZX4-NCA are effective reagents for the treatment of systemic and local MDR-hvKP infections.

Antimicrobial activity of prophage endolysins against critical Enterobacteriaceae antibiotic-resistant bacteria.

Enterobacteriaceae species are part of the 2017 World Health Organization antibiotic-resistant priority pathogens list for development of novel medicines. Multidrug-resistant Klebsiella pneumoniae is an increasing threat to public health and has become a relevant human pathogen involved in life-threatening infections. Phage therapy involves the use of phages or their lytic endolysins as bioagents for the treatment of bacterial infectious diseases. Gram-negative bacteria have an outer membrane, making difficult the access of endolysins to the peptidoglycan. Here, three endolysins from prophages infecting three distinct Enterobacterales species, Kp2948-Lys from K. pneumoniae, Ps3418-Lys from Providencia stuartii, and Kaer26608-Lys from Klebsiella aerogenes, were purified and exhibited antibacterial activity against their specific bacterium species verified by zymogram assays. These three endolysins were successfully associated to liposomes composed of dimyristoyl phosphatidyl choline (DMPC), dioleoyl phosphatidyl ethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) at a molar ratio (4:4:2), with an encapsulation efficiency ranging from 24 to 27%. Endolysins encapsulated in liposomes resulted in higher antibacterial activity compared to the respective endolysin in the free form, suggesting that the liposome-mediated delivery system enhances fusion with outer membrane and delivery of endolysins to the target peptidoglycan. Obtained results suggest that Kp2948-Lys appears to be specific for K. pneumoniae, while Ps3418-Lys and Kaer26608-Lys appear to have a broader antibacterial spectrum. Endolysins incorporated in liposomes constitute a promising weapon, applicable in the several dimensions (human, animals and environment) of the One Health approach, against multidrug-resistant Enterobacteriaceae.

Ovarian Tumor Domain-Containing 7B Attenuates Pathological Cardiac Hypertrophy by Inhibiting Ubiquitination and Degradation of Krüppel-Like Factor 4.

BACKGROUND: Cardiac hypertrophy (CH) is a well-established risk factor for many cardiovascular diseases and a primary cause of mortality and morbidity among older adults. Currently, no pharmacological interventions have been specifically tailored to treat CH. OTUD7B (ovarian tumor domain-containing 7B) is a member of the ovarian tumor-related protease (OTU) family that regulates many important cell signaling pathways. However, the role of OTUD7B in the development of CH is unclear. Therefore, we investigated the role of OTUD7B in CH. METHODS AND RESULTS: OTUD7B knockout mice were used to assay the role of OTUD7B in CH after transverse aortic coarctation surgery. We further assayed the specific functions of OTUD7B in isolated neonatal rat cardiomyocytes. We found that OTUD7B expression decreased in hypertrophic mice hearts and phenylephrine-stimulated neonatal rat cardiomyocytes. Furthermore, OTUD7B deficiency exacerbated transverse aortic coarctation surgery-induced myocardial hypertrophy, abnormal cardiac function, and fibrosis. In cardiac myocytes, OTUD7B knockdown promoted phenylephrine stimulation-induced myocardial hypertrophy, whereas OTUD7B overexpression had the opposite effect. An immunoprecipitation-mass spectrometry analysis showed that OTUD7B directly binds to KLF4 (Krüppel-like factor 4). Additional molecular experiments showed that OTUD7B impedes KLF4 degradation by inhibiting lysine residue at 48 site-linked ubiquitination and suppressing myocardial hypertrophy by activating the serine/threonine kinase pathway. CONCLUSIONS: These results demonstrate that the OTUD7B-KLF4 axis is a novel molecular target for CH treatment.

Development of engineered endolysins with in vitro intracellular activity against streptococcal bovine mastitis-causing pathogens.

Bacteriophage-derived endolysins are a novel class of antimicrobials known to rapidly kill bacteria, including antibiotic-resistant strains. We here engineered endolysins against the bovine mastitis pathogens Streptococcus uberis, Streptococcus agalactiae and Streptococcus dysgalactiae, also targeting intracellular survival and biofilm formation. For this purpose, high-throughput DNA assembly was used to create a library with >80,000 theoretical endolysin variants for screening of their bacteriolytic activity against Gram-positive isolates from (sub)clinically affected cows. This lytic activity was evaluated by turbidity reduction and time-kill assays in phosphate-buffered saline and pasteurized whole cow's milk to allow a rank up of the most potent leading candidates. A top candidate was selected with a 4.0 log killing efficacy against S. uberis, also showing similar activity against S. agalactiae and S. dysgalactiae. This top candidate eradicated S. uberis biofilm and showed intracellular activity in two bovine mammary epithelial cell lines as was confirmed by confocal microscopy. A potentiating effect on cloxacillin, a beta-lactam penicillin used to intramammarily treat bovine Gram-positive mastitis, was observed for this top candidate endolysin in raw cow's milk from (sub)clinically infected udders. Our in vitro results indicate that engineered endolysins may have a future role as add-on in the treatment of bovine streptococcal mastitis.

Characterization of the Clostridium perfringens phage endolysin cpp-lys and its application on lettuce.

Clostridium perfringens is an important foodborne pathogen that can have severe consequences, including mortality and economic losses. In this study, the gene encoding cpp-lys, an endolysin from the C. perfringens phage cpp has been cloned and overexpressed. The encoded protein was characterized, and then its efficacy in controlling C. perfringens on lettuce was evaluated. The endolysin cpp-lys presented lytic activity against seven strains of C. perfringens that produce different types of toxins. It maintained stability across a wide range of temperatures (4 °C - 50 °C), and demonstrated tolerance to varying pH levels (4-9). Storage of endolysin cpp-lys under room-temperature conditions (16 °C-25 °C) and cold-temperature conditions (4 °C, -20 °C, and -80 °C) for 30 days did not affect its lytic activity. However, the lytic activity of cpp-lys decreased by 40 % and 18 % after storage for 30 d at 42 °C and 37 °C, respectively. The endolysin cpp-lys did not display cytotoxic activity against normal eukaryotic cells. The bacterial viability on lettuce was significantly lower in the group treated with endolysin cpp-lys than in the PBS group, and >4-log of C. perfringens J1 were removed within 15 min. Cpp-lys plus Zn2+ inhibited the activity of cpp-lys. The EDTA-treated cpp-lys significantly reduced the number of bacteria by up to 0.6-log CFU compared with the endolysin cpp-lys group. The findings of this study demonstrated that endolysin cpp-lys has potential applications in controlling C. perfringens in the food industry.

Evaluating the efficacy of endolysins and membrane permeabilizers against Vibrio parahaemolyticus in marine conditions.

Endolysins have garnered significant attention as a potential alternative to antibiotics in aquaculture, mainly for combating Vibrio spp., Gram-negative pathogens responsible for infectious outbreaks. However, endolysin effectiveness against Gram-negative bacteria is limited due to the outer membrane's poor permeability. The combat against marine pathogens poses an additional challenge of finding endolysins that retain their activity in high ionic strength conditions. Thus, this study aimed to demonstrate that certain endolysins retain muralytic activity in seawater and also evaluated outer membrane permeabilizers as endolysin adjuvants. The effectiveness of KZ144 and LysPA26 endolysins, along with EDTA and oregano essential oil, was evaluated against Vibrio parahaemolyticus ATCC-17802 in natural seawater. Results revealed the muralytic activity of both endolysins in seawater. However, the endolysins appeared to counteract the permeabilizers' effect during the initial bactericidal assays. Further investigations revealed that the observed effect was not antagonistic. After the permeabilizer action, V. parahaemolyticus likely used endolysins as a growth substrate. Endolysins may not play an indifferent role if they fail to exert a bactericidal effect. Instead, they can serve as a substrate for fast-growing bacteria, such as V. parahaemolyticus, increasing bacterial density. It should be considered a potential drawback of endolysins' proteinaceous nature as bactericidal agents.

Design, Screening, and Characterization of Engineered Phage Endolysins with Extracellular Antibacterial Activity against Gram-Negative Bacteria.

Phage-encoded endolysins are emerging antibacterial agents based on their ability to efficiently degrade peptidoglycan on Gram-positive bacteria, but the envelope characteristics of Gram-negative bacteria limit their application. Engineering modifications of endolysins can improve the optimization of their penetrative and antibacterial properties. This study constructed a screening platform to screen for engineered Artificial-Bp7e (Art-Bp7e) endolysins with extracellular antibacterial activity against Escherichia coli. An oligonucleotide of 20 repeated NNK codons was inserted upstream of the endolysin gene Bp7e to construct a chimeric endolysin library in the pColdTF vector. The chimeric Art-Bp7e proteins were expressed by transforming the plasmid library into E. coli BL21 and released by chloroform fumigation, and the protein activities were evaluated by the spotting method and the colony-counting method to screen for promising proteins. Sequence analysis showed that all screened proteins with extracellular activities had a chimeric peptide with a positive charge and an α-helical structure. Also, a representative protein, Art-Bp7e6, was further characterized. It exhibited broad antibacterial activity against E. coli (7/21), Salmonella enterica serovar Enteritidis (4/10), Pseudomonas aeruginosa (3/10), and even Staphylococcus aureus (1/10). In the transmembrane process, the chimeric peptide of Art-Bp7e6 depolarized the host cell envelope, increased the permeability of the cell, and facilitated the movement of Art-Bp7e6 across the envelope to hydrolyze the peptidoglycan. In conclusion, the screening platform successfully screened for chimeric endolysins with extracellular antibacterial activities against Gram-negative bacteria, which provides methodological support for the further screening of engineered endolysins with high extracellular activities against Gram-negative bacteria. Also, the established platform showed broad application prospects and can be used to screen various proteins. IMPORTANCE The presence of the envelope in Gram-negative bacteria limits the use of phage endolysins, and engineering endolysins is an efficient way to optimize their penetrative and antibacterial properties. We built a platform for endolysin engineering and screening. A random peptide was fused with the phage endolysin Bp7e to construct a chimeric endolysin library, and engineered Artificial-Bp7e (Art-Bp7e) endolysins with extracellular activity against Gram-negative bacteria were successfully screened from the library. The purposeful Art-Bp7e had a chimeric peptide with an abundant positive charge and an α-helical structure, which led Bp7e to acquire the ability for the extracellular lysis of Gram-negative bacteria and showed a broad lysis spectrum. The platform provides a huge library capacity without the limitations of reported proteins or peptides. It can be utilized for the further screening of optimal endolysins against Gram-negative bacteria as well as for the screening of additional proteins with specific modifications.

Acid protease functionalized novel silver nanoparticles (APTs-AgNPs): A new approach towards photocatalytic and biological applications.

Herein, we described for the first time, an efficient biogenic synthesis of APTs-AgNPs using acid protease from Melilotus indicus leaf extract. The acid protease (APTs) has an essential role in the stabilization, reduction, and capping of APTs-AgNPs. The crystalline nature, size, and surface morphology of APTs-AgNPs were examined using different techniques such as XRD, UV, FTIR, SEM, EDS, HRTEM, and DLS analysis. The generated APTs-AgNPs demonstrated notable performance as dual functionality (photocatalyst and antibacterial disinfection). By destroying 91 % of methylene blue (MB) in <90 min of exposure, APTs-AgNPs demonstrated remarkable photocatalytic activity. APTs-AgNPs also showed remarkable stability as a photocatalyst after five test cycles. Furthermore, the APTs-AgNPs was found to be a potent antibacterial agent with inhibition zones of 30(±0.5 mm), 27(±0.4 mm), 16(±0.1 mm), and 19(±0.7 mm) against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria, respectively, under both light and dark conditions. Furthermore, APTs-AgNPs effectively scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, demonstrating their potent antioxidant activity. The outcomes of this study thus demonstrates the dual functionality of APTs-AgNPs produced using the biogenic approach method as a photocatalyst and an antibacterial agent for effective microbial and environmental control.

Characterization of a broad-spectrum endolysin rLysJNwz and its utility against Salmonella in foods.

Salmonella is a common foodborne pathogen worldwide. The use of bacteriophage-encoded endolysins as antimicrobial agents is a promising approach for controlling pathogenic contamination. In this context, a recombinant endolysin named rLysJNwz, consisting of a single domain falling with the L-alanogyl-D-glutamate peptidase-like family, was cloned, expressed, and characterized. The yield of rLysJNwz was about 25 mg/L. Synergy between 7.5 µg/mL rLysJNwz and 0.5 mmol/L EDTA could decrease the viable counts of Salmonella NCTC 8271 by 93.28%. A synergistic effect between rLysJNwz and polymyxin B was demonstrated, exhibiting the MIC of polymyxin B decreased by twofold. Specifically, rlysJNwz had strong thermostability at temperatures (4-95 °C) and maintained high activity at pHs from 5.0 to 11.0. rlysJNwz was a metal ion-dependent peptidase, which activated by divalent metal ions such as Zn2+, Mn2+, or Ca2+. Moreover, it was also found that the synergism of rlysJNwz and EDTA had bactericidal activities against a broad range of Gram-negative bacteria, including several multidrug-resistant bacteria. The application of rLysJNwz combined with EDTA was evaluated on contaminated eggs and lettuce for 60 min, displaying more than 86.7% and 86.5% reduction of viable Salmonella, respectively. Hence, these results suggest that rLysJNwz is a potential antibacterial agent to control Salmonella, especially antibiotic-resistant pathogen contamination in the field of food safety. KEY POINTS: • rLysJNwz shows lytic activities against a broad range of Gram-negative bacteria. • Endolysin rLysJNwz is a stable metalloenzyme and has high thermostability. • rLysJNwz and 0.5 mmol/L EDTA synergistically inactivate Salmonella on eggs and lettuce.

The Magic of Proteases: From a Procoagulant and Anticoagulant Factor V to an Equitable Treatment of Its Inherited Deficiency.

Proteostasis, i.e., the homeostasis of proteins, responsible for ensuring protein turnover, is regulated by proteases, which also participate in the etiopathogenesis of multiple conditions. The magic of proteases is such that, in blood coagulation, one same molecule, such as coagulation factor V, for example, can perform both a procoagulant and an anticoagulant function as a result of the activity of proteases. However, this magic has an insidious side to it, as it may also prevent the completion of the clinical value chain of factor V deficiency. This value chain encompasses the discovery of knowledge, the transfer of this knowledge, and its translation to clinical practice. In the case of rare and ultra-rare diseases like factor V deficiency, this value chain has not been completed as the knowledge acquisition phase has dragged out over time, holding up the transfer of knowledge to clinical practice. The reason for this is related to the small number of patients afflicted with these conditions. As a result, new indications must be found to make the therapies cost-effective. In the case of factor V, significant research efforts have been directed at developing a recombinant factor V capable of resisting the action of the proteases capable of inactivating this factor. This is where bioethics and health equity considerations come into the equation.

Characterization of Three Different Endolysins Effective against Gram-Negative Bacteria.

Genes encoding endolysins were identified and cloned from three different Escherichia coli bacteriophages, 10-24(13), PBEC30, and PBEC56. Putative antimicrobial peptide (AMP)-like C-terminal alpha helix structures with amphipathic natures were predicted from the three endolysins. Each gene was cloned and expressed as hexahistidine-tagged forms, and the products were purified and characterized. The purified endolysins exhibited antibacterial activities against a variety of Gram-negative bacteria including Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumonia. Their antibacterial activities were improved by N-terminal fusion with an antimicrobial peptide, cecropin A. Minimum inhibitory concentrations (MIC) were as low as 4 µg/mL, depending on the targeted strain. The endolysins' enzymatic activities were not affected by changes in pH at ranges from 5 to 10 and were stable at temperatures between 4 and 65 °C. The in vivo efficacies of the three endolysins were also demonstrated using Galleria melonella for infection models.