Modular and integrative activity reporters enhance biochemical studies in the yeast ER.

The yeast endoplasmic reticulum sequestration and screening (YESS) system is a broadly applicable platform to perform high-throughput biochemical studies of post-translational modification enzymes (PTM-enzymes). This system enables researchers to profile and engineer the activity and substrate specificity of PTM-enzymes and to discover inhibitor-resistant enzyme mutants. In this study, we expand the capabilities of YESS by transferring its functional components to integrative plasmids. The YESS integrative system yields uniform protein expression and protease activities in various configurations, allows one to integrate activity reporters at two independent loci and to split the system between integrative and centromeric plasmids. We characterize these integrative reporters with two viral proteases, Tobacco etch virus (TEVp) and 3-chymotrypsin like protease (3CLpro), in terms of coefficient of variance, signal-to-noise ratio and fold-activation. Overall, we provide a framework for chromosomal-based studies that is modular, enabling rigorous high-throughput assays of PTM-enzymes in yeast.

Fibroblast Activation Protein Inhibitor Tracers and Their Preclinical, Translational, and Clinical Status in China.

Quinoline-based fibroblast activation protein (FAP) inhibitors (FAPIs) have recently emerged as a focal point in global nuclear medicine, underscored by their promising applications in cancer theranostics and the diagnosis of various nononcological conditions. This review offers an in-depth summary of the existing literature on the evolution and use of FAPI tracers in China, tracing their journey from preclinical to clinical research. Moreover, this review also assesses the diagnostic accuracy of FAPI PET for the most common cancers in China, analyzes its impact on oncologic management paradigms, and investigates the potential of FAP-targeted radionuclide therapy in patients with advanced or metastatic cancer. This review also summarizes studies using FAPI PET for nononcologic disorders in China. Thus, this qualitative overview presents a snapshot of China's engagement with FAPI tracers, aiming to guide future research endeavors.

Peritumoral stroma and systemic inflammatory response in cervical cancer.

OBJECTIVE: To compare cervical stroma in advanced cervical cancer with the control group; to compare, in the pre-treatment period, hemogram parameters in patients with advanced cervical cancer with the same parameters as the control group; and to verify if there is an association of stromal markers with prognostic factors in cervical cancer. MATERIALS AND METHODS: We prospectively evaluated 16 patients diagnosed with advanced invasive cervical cancer. A control group of 22 patients was used (uterine leiomyoma). Immunohistochemistry was performed to verify the stromal immunostaining of alpha-smooth muscle actin (SMA) and fibroblast activation protein alpha (FAP). Immunostainings and hemogram parameters were compared using Fisher's exact and Mann-Whitney Test, respectively. RESULTS: Strong FAP immunostaining was more frequent in patients with cervical cancer when compared with patients with leiomyoma (P = 0.0002). Regarding SMA, strong immunostaining was also found more in the group of cancer patients compared to the control group (P < 0.00001). The neutrophil-lymphocyte ratio (NLR) values were higher in the cancer patient group compared to the control group (P = 0.0019). There was no association of the parameters studied with prognostic factors. CONCLUSIONS: Strong FAP and SMA immunostaining was found more in patients with cervical cancer when compared to the control group. NLR values were also higher in cervical cancer.

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.

USP19 regulates DNA methylation damage repair and confers temozolomide resistance through MGMT stabilization.

OBJECTIVE: To elucidate the relationship between USP19 and O(6)-methylguanine-DNA methyltransferase (MGMT) after temozolomide treatment in glioblastoma (GBM) patients with chemotherapy resistance. METHODS: Screening the deubiquitinase pannel and identifying the deubiquitinase directly interacts with and deubiquitination MGMT. Deubiquitination assay to confirm USP19 deubiquitinates MGMT. The colony formation and tumor growth study in xenograft assess USP19 affects the GBM sensitive to TMZ was performed by T98G, LN18, U251, and U87 cell lines. Immunohistochemistry staining and survival analysis were performed to explore how USP19 is correlated to MGMT in GBM clinical management. RESULTS: USP19 removes the ubiquitination of MGMT to facilitate the DNA methylation damage repair. Depletion of USP19 results in the glioblastoma cell sensitivity to temozolomide, which can be rescued by overexpressing MGMT. USP19 is overexpressed in glioblastoma patient samples, which positively correlates with the level of MGMT protein and poor prognosis in these patients. CONCLUSION: The regulation of MGMT ubiquitination by USP19 plays a critical role in DNA methylation damage repair and GBM patients' temozolomide chemotherapy response.

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

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

Circulating soluble fibroblast activation protein (FAP) levels are independent of cardiac and extra-cardiac FAP expression determined by targeted molecular imaging in patients with myocardial FAP activation.

INTRODUCTION: Tissue Fibroblast Activation Protein alpha (FAP) is overexpressed in various types of acute and chronic cardiovascular disease. A soluble form of FAP has been detected in human plasma, and low circulating FAP concentrations are associated with increased risk of death in patients with acute coronary syndrome. However, little is known about the regulation and release of FAP from fibroblasts, and whether circulating FAP concentration is associated with tissue FAP expression. This study characterizes the release of FAP in human cardiac fibroblasts (CF) and analyzes the association of circulating FAP concentrations with in vivo tissue FAP expression in patients with acute (ST-segment elevation myocardial infarction, STEMI) and chronic (severe aortic stenosis, AS) myocardial FAP expression. METHODS AND RESULTS: FAP was released from CF in a time- and concentration-dependent manner. FAP concentration was higher in supernatant of TGFß-stimulated CF, and correlated with cellular FAP concentration. Inhibition of metallo- and serine-proteases diminished FAP release in vitro. Median FAP concentrations of patients with acute (77 ng/mL) and chronic (75 ng/mL, p = 0.50 vs. STEMI) myocardial FAP expression did not correlate with myocardial nor extra-myocardial nor total FAP volume (P ≥ 0.61 in all cases) measured by whole-body FAP-targeted positron emission tomography. CONCLUSION: We describe a time- and concentration dependent, protease-mediated release of FAP from cardiac fibroblasts. Circulating FAP concentrations were not associated with increased in vivo tissue FAP expression determined by molecular imaging in patients with both chronic and acute myocardial FAP expression. These data suggest that circulating FAP and tissue FAP expression provide complementary, non-interchangeable information.

DoUBLing up: ubiquitin and ubiquitin-like proteases in genome stability.

Maintaining stability of the genome requires dedicated DNA repair and signalling processes that are essential for the faithful duplication and propagation of chromosomes. These DNA damage response (DDR) mechanisms counteract the potentially mutagenic impact of daily genotoxic stresses from both exogenous and endogenous sources. Inherent to these DNA repair pathways is the activity of protein factors that instigate repair processes in response to DNA lesions. The regulation, coordination, and orchestration of these DDR factors is carried out, in a large part, by post-translational modifications, such as phosphorylation, ubiquitylation, and modification with ubiquitin-like proteins (UBLs). The importance of ubiquitylation and UBLylation with SUMO in DNA repair is well established, with the modified targets and downstream signalling consequences relatively well characterised. However, the role of dedicated erasers for ubiquitin and UBLs, known as deubiquitylases (DUBs) and ubiquitin-like proteases (ULPs) respectively, in genome stability is less well established, particularly for emerging UBLs such as ISG15 and UFM1. In this review, we provide an overview of the known regulatory roles and mechanisms of DUBs and ULPs involved in genome stability pathways. Expanding our understanding of the molecular agents and mechanisms underlying the removal of ubiquitin and UBL modifications will be fundamental for progressing our knowledge of the DDR and likely provide new therapeutic avenues for relevant human diseases, such as cancer.

Serratia montpellierensis sp. nov., Isolated from Laboratory-Reared Parasitic Wasps Psyttalia lounsburyii Silvestri and Psyttalia ponerophaga Silvestri (Hymenoptera: Braconidae).

Two strains of bacteria, PsyLou2AT and PsyPon4B, were isolated from adult braconid wasps Psyttalia lounsburyii and Psyttalia ponerophaga, respectively. These laboratory-reared wasps were investigated as agents for biological control of the olive fruit fly, Bactrocera oleae. Analysis of 16S rRNA genes of the two isolates demonstrated that they were highly related and belonged to the genus Serratia. Genomic sequencing of these isolates revealed genomes of 5,152,551 bp and 5,154,385 bp for PsyLou2AT and PsyPon4B, respectively, and both genomes had a mol% G+C content of 59.6%. Phylogenetic analyses using BLAST-based average nucleotide identity (ANIb), and digital DNA-DNA hybridization methods indicated that PsyLou2AT was most closely related to Serratia nevei S15T, producing ANIb and dDDH values of 96.11% and 70.2%, respectively. Since these values were literally on the species cutoff threshold, additional S. nevei genome assemblies were analyzed using ANIb and dDDH calculations. This revealed that among assemblies that were clearly identifiable as S. nevei, S. nevei S15T was the most closely related to PsyLou2AT, and that a majority of assemblies produced dDDH values of 68.3-68.7% relative to PsyLou2AT. Additionally, PsyLou2AT differed biochemically from S. nevei S15T in that it produced positive Voges Proskauer tests, produced protease, lacked arginine dihydrolase, and did not utilize D-lactose. Hence, PsyLou2AT represents a novel taxon within the Serratia, for which we propose the name Serratia montpellierensis sp. nov. The type strain is PsyLou2AT (=LMG 32817T =NRRL B-65689T).

Advances in Small Molecular Agents against Oral Cancer.

Oral cancer is a common malignancy with a high mortality rate. Although surgery is the best treatment option for patients with cancer, this approach is ineffective for advanced metastases. Molecular agents are irreplaceable in preventing and treating distant metastases. This review aims to summarise the molecular agents used for the treatment of oral cancer in the last decade and describe their sources and curative effects. These agents are classified into phenols, isothiocyanates, anthraquinones, statins, flavonoids, terpenoids, and steroids. The mechanisms of action of these agents include regulating the expression of cell signalling pathways and related proteases to affect the proliferation, autophagy, migration, apoptosis, and other biological aspects of oral cancer cells. This paper may serve as a reference for subsequent studies on the treatment of oral cancer.

Assessing Sarcocornia as a Salt Substitute: Effects on Lipid Profile and Gelatinase Activity.

Sodium, although essential for life, is a key factor in changes in vascular function and cardiovascular disease when consumed in excess. Sarcocornia spp., a halophyte plant with many nutritional benefits, presents itself as a promising substitute for the consumption of purified salt. Matrix metalloproteinases (MMPs) 2 and 9 are widely studied due to their action in physiological processes and as biomarkers at the diagnostic level due to their increased expression in inflammatory processes. This study aimed to evaluate whether replacing salt with Sarcocornia perennis (S. perennis) powder in healthy young people leads to an improvement in biochemical profiles and the attenuation of MMP-2 and MMP-9 activity. In the present study, 30 participants were randomized into a control group that consumed salt and an intervention group that replaced salt with powdered S. perennis. The evaluation of the biochemical parameters was carried out by the spectrophotometry method, and the evaluation of MMP activity was carried out by zymography. A significant decrease was observed in the intervention group in total cholesterol, high-density lipoprotein cholesterol (HDL-c), and creatinine (p-value ≤ 0.05), along with lower but not significantly different mean values of triglycerides. Regarding MMP activity after the intervention, a lower mean value was observed for MMP-9 activity, with there being higher mean values for MMP-2 activity, both with p-values ≥ 0.05. The results confirmed that the consumption of S. perennis is a beneficial choice for health regarding the lipid profile. The evaluation of MMP activity indicated the potential of S. perennis in the regulation of MMP-9 activity in healthy individuals, along with the need for the further study of these proteases in individuals with pathologies.

Reducing Immunoreactivity of Gluten Peptides by Probiotic Lactic Acid Bacteria for Dietary Management of Gluten-Related Diseases.

Immunoreactive gluten peptides that are not digested by peptidases produced by humans can trigger celiac disease, allergy and non-celiac gluten hypersensitivity. The aim of this study was to evaluate the ability of selected probiotic strains to hydrolyze immunoreactive gliadin peptides and to identify peptidase-encoding genes in the genomes of the most efficient strains. Residual gliadin immunoreactivity was measured after one- or two-step hydrolysis using commercial enzymes and bacterial peptidase preparations by G12 and R5 immunoenzymatic assays. Peptidase preparations from Lacticaseibacillus casei LC130, Lacticaseibacillus paracasei LPC100 and Streptococcus thermophilus ST250 strains significantly reduced the immunoreactivity of gliadin peptides, including 33-mer, and this effect was markedly higher when a mixture of these strains was used. In silico genome analyses of L. casei LC130 and L. paracasei LPC100 revealed the presence of genes encoding peptidases with the potential to hydrolyze bonds in proline-rich peptides. This suggests that L. casei LC130, L. paracasei LPC100 and S. thermophilus ST250, especially when used as a mixture, have the ability to hydrolyze immunoreactive gliadin peptides and could be administered to patients on a restricted gluten-free diet to help treat gluten-related diseases.

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 MDR and 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.

Genetic interaction mapping reveals functional relationships between peptidoglycan endopeptidases and carboxypeptidases.

Peptidoglycan (PG) is the main component of the bacterial cell wall; it maintains cell shape while protecting the cell from internal osmotic pressure and external environmental challenges. PG synthesis is essential for bacterial growth and survival, and a series of PG modifications are required to allow expansion of the sacculus. Endopeptidases (EPs), for example, cleave the crosslinks between adjacent PG strands to allow the incorporation of newly synthesized PG. EPs are collectively essential for bacterial growth and must likely be carefully regulated to prevent sacculus degradation and cell death. However, EP regulation mechanisms are poorly understood. Here, we used TnSeq to uncover novel EP regulators in Vibrio cholerae. This screen revealed that the carboxypeptidase DacA1 (PBP5) alleviates EP toxicity. dacA1 is essential for viability on LB medium, and this essentiality was suppressed by EP overexpression, revealing that EP toxicity both mitigates, and is mitigated by, a defect in dacA1. A subsequent suppressor screen to restore viability of ΔdacA1 in LB medium identified hypomorphic mutants in the PG synthesis pathway, as well as mutations that promote EP activation. Our data thus reveal a more complex role of DacA1 in maintaining PG homeostasis than previously assumed.

Impacts of AlaAT3 transgenic poplar on rhizosphere soil chemical properties, enzyme activity, bacterial community, and metabolites under two nitrogen conditions.

Poplar stands as one of the primary afforestation trees globally. We successfully generated transgenic poplar trees characterized by enhanced biomass under identical nutrient conditions, through the overexpression of the pivotal nitrogen assimilation gene, pxAlaAT3. An environmental risk assessment was conducted for investigate the potential changes in rhizosphere soil associated with these overexpressing lines (OL). The results show that acid phosphatase activity was significantly altered under ammonium in OL compared to the wild-type control (WT), and a similar difference was observed for protease under nitrate. 16SrDNA sequencing indicated no significant divergence in rhizosphere soil microbial community diversity between WT and OL. Metabolomics analysis revealed that the OL caused minimal alterations in the metabolites of the rhizosphere soil, posing no potential harm to the environment. With these findings in mind, we anticipate that overexpressed plants will not adversely impact the surrounding soil environment.

Structure-based virtual screening of novel USP5 inhibitors targeting the zinc finger ubiquitin-binding domain.

The equilibrium of cellular protein levels is pivotal for maintaining normal physiological functions. USP5 belongs to the deubiquitination enzyme (DUBs) family, controlling protein degradation and preserving cellular protein homeostasis. Aberrant expression of USP5 is implicated in a variety of diseases, including cancer, neurodegenerative diseases, and inflammatory diseases. In this paper, a multi-level virtual screening (VS) approach was employed to target the zinc finger ubiquitin-binding domain (ZnF-UBD) of USP5, leading to the identification of a highly promising candidate compound 0456-0049. Molecular dynamics (MD) simulations were then employed to assess the stability of complex binding and predict hotspot residues in interactions. The results indicated that the candidate stably binds to the ZnF-UBD of USP5 through crucial interactions with residues ARG221, TRP209, GLY220, ASN207, TYR261, TYR259, and MET266. Binding free energy calculations, along with umbrella sampling (US) simulations, underscored a superior binding affinity of the candidate relative to known inhibitors. Moreover, US simulations revealed conformational changes of USP5 during ligand dissociation. These insights provide a valuable foundation for the development of novel inhibitors targeting USP5.

Early recovery of proteasome activity in cells pulse-treated with proteasome inhibitors is independent of DDI2.

Rapid recovery of proteasome activity may contribute to intrinsic and acquired resistance to FDA-approved proteasome inhibitors. Previous studies have demonstrated that the expression of proteasome genes in cells treated with sub-lethal concentrations of proteasome inhibitors is upregulated by the transcription factor Nrf1 (NFE2L1), which is activated by a DDI2 protease. Here, we demonstrate that the recovery of proteasome activity is DDI2-independent and occurs before transcription of proteasomal genes is upregulated but requires protein translation. Thus, mammalian cells possess an additional DDI2 and transcription-independent pathway for the rapid recovery of proteasome activity after proteasome inhibition.

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

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

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.