Search Results (67)

Antibiotic resistance has emerged as a critical global public health challenge, prompting increased interest in non-antibiotic antimicrobial strategies such as bacteriophage-derived endolysins. Although endolysins possess strong lytic potential, their application to Gram-negative bacteria remains limited due to the outer membrane barrier. PlyKp104 is a recently identified phage-derived endolysin that exhibits lytic activity against Gram-negative bacteria without the aid of membrane permeabilizers. In this study, the crystal structure of PlyKp104 was determined at a resolution of 1.85 Å. PlyKp104 consists solely of a catalytic SLT domain, and structure-based analysis revealed a putative active site and key structural features associated with substrate binding. Comparative analysis with homologous structures suggested that PlyKp104 belongs to lytic transglycosylase family 1. B-factor analysis and hydrophobic interaction mapping indicated that the domain exhibits high structural stability, supported by conserved hydrophobic residues clustered in motifs I and II. During structure determination, an unidentified electron density was consistently observed near a neutral, hydrophobic surface region. Its shape and environment suggest the presence of a lipid-like molecule, implying a potential lipid-binding site. These findings provide structural insight into PlyKp104 and contribute to the understanding of endolysin mechanisms against Gram-negative bacteria, with implications for future protein engineering efforts. Full article

Phage Lydia, a newly isolated siphovirus infecting Pseudomonas aeruginosa, was characterized with respect to its basic kinetic properties and subjected to comparative bioinformatic analysis with related phages. The phage exhibited a restricted host range, with lytic activity observed against 7 of 30 tested isolates. The genome of phage Lydia consists of a 61,986 bp dsDNA molecule and contains 89 predicted genes. Bioinformatic analysis suggests the presence of a DNA modification system, but no apparent genes associated with lysogeny or antibiotic resistance were identified. Taxonomic classification places Lydia within the Mesyanzhinovviridae family, Rabinowitzvirinae subfamily, and Yuavirus genus, with the closest relation to Pseudomonas virus M6. Comprehensive bioinformatic studies, including structural modelling and analysis of phage proteins, as well as comparative taxonomic, phylogenomic, and pangenomic analyses of the Mesyanzhinovviridae family, revealed relationships between proteins of Mesyanzhinovviridae phages, proteins from other phage groups, encapsulins, and a gene transfer agent (GTA) particle from Rhodobacter capsulatus. These analyses uncovered patterns of evolutionary history within the family, characterized by genetic exchange events alongside the maintenance of a common genomic architecture, leading to the emergence of new groups within the family. Full article

Kaposi’s Sarcoma Herpesvirus (KSHV) is the causative agent of several human diseases. There are few effective treatments available to treat infection and KSHV oncogenesis. Disrupting the KSHV infectious cycle would diminish the viral spread. The KSHV lytic phase and production of new virions require efficient copying and packaging of the KSHV genome. KSHV encodes its own lytic DNA replication machinery, including the processivity factor (PF-8), which presents itself as an attractive target for antiviral development. We characterized PF-8 at the single molecule level using transmission electron microscopy to identify key molecular interactions that mediate viral DNA replication initiation. Our results indicate that PF-8 forms oligomeric ring structures (tetramer, hexamer, and/or dodecamer) similar to the related Epstein–Barr virus processivity factor (BMRF1). Our DNA positional mapping revealed high-frequency binding locations of PF-8 within the lytic origin of replication (OriLyt). A multi-variable analysis of PF-8 DNA-binding activity with three mutant OriLyts provides new insights into the mechanisms that PF-8 associates with viral DNA and complexes to form multi-ring-like structures. Collectively, these data enhance the mechanistic understanding of the molecular interactions (protein–protein and protein-DNA) of an essential KSHV DNA replication protein. Full article

Enterotoxigenic Escherichia coli (ETEC) is a major pathogen causing diarrhea in humans and animals, with increasing antimicrobial resistance posing a growing challenge in recent years. Lytic bacteriophages (phages) offer a targeted and environmentally sustainable approach to combating bacterial infections, particularly in eliminating drug-resistant strains. In this study, ETEC strains were utilized as indicators, and a stable, high-efficiency phage, designated vB_EcoM_JE01 (JE01), was isolated from pig farm manure. The genome of JE01 was a dsDNA molecule, measuring 168.9 kb, and a transmission electron microscope revealed its characteristic T4-like Myoviridae morphology. JE01 effectively lysed multi-drug-resistant ETEC isolates. Stability assays demonstrated that JE01 retained its activity across a temperature range of 20 °C to 50 °C and a pH range of 3–11, showing resilience to ultraviolet radiation and chloroform exposure. Furthermore, JE01 effectively suppressed ETEC adhesion to porcine intestinal epithelial cells (IPEC-J2), mitigating the inflammatory response triggered by ETEC. To investigate the in vivo antibacterial efficacy of phage JE01 preparations, a diarrhea model was established using germ-free mice infected with a drug-resistant ETEC strain. The findings indicated that 12 h post-ETEC inoculation, intragastric administration of phage JE01 significantly reduced mortality, alleviated gastrointestinal lesions, decreased ETEC colonization in the jejunum, and suppressed the expression of the cytokines IL-6 and IL-8. These results demonstrate a therapeutic benefit of JE01 in treating ETEC-induced diarrhea in mice. Additionally, a fluorescent phage incorporating red fluorescent protein (RFP) was engineered, and the pharmacokinetics of phage therapy were preliminarily assessed through intestinal fluorescence imaging in mice. The results showed that the phage localized to ETEC in the jejunum rapidly, within 45 min. Moreover, the pharmacokinetics of the phage were markedly slowed in the presence of its bacterial target in the gut, suggesting sustained bacteriolytic activity in the ETEC-infected intestine. In conclusion, this study establishes a foundation for the development of phage-based therapies against ETEC. Full article

The increasing antibiotic resistance among bacteria challenges the biotech industry to search for new antibacterial molecules. Endolysin TP84_28 is a thermostable, lytic enzyme, encoded by the bacteriophage (phage) TP-84, and it effectively digests host bacteria cell wall. Biofilms, together with antibiotic resistance, are major problems in clinical medicine and industry. The challenge is to keep antibacterial molecules at the site of desired action, as their diffusion leads to a loss of efficacy. The TP84_28 endolysin gene was cloned into an expression-fusion vector, forming a fusion gene cbd_tp84_28_his with a cellulose-binding domain from the cellulase enzyme. The Cellulose-Binding Thermostable TP84_Endolysin (CBD_TP84_28_His) fusion protein was biosynthesized in Escherichia coli and purified. Thermostability and enzymatic activities against various bacterial species were measured by a turbidity reduction assay, a spot assay, and biofilm removal. Cellulose-binding properties were confirmed via interactions with microcellulose and cellulose paper-based immunoblotting. The high affinity of the CBD allows for a high concentration of the fusion enzyme at desired target sites such as cellulose-based wound dressings, artificial heart valves and food packaging. CBD_TP84_28_His exhibits a lytic effect against thermophilic bacteria Geobacillus stearothemophilus, Thermus aquaticus, Bacillus stearothermophilus, and Geobacillus ICI and minor effects against mesophilic Bacillus cereus and Bacillus subtilis. CBD_TP84_28_His retains full activity after preincubation in the temperatures of 30–65 °C and exhibits significant activity up to its melting point at 73 °C. CBD_TP84_28_His effectively reduces biofilms. These findings suggest that integrating CBDs into thermostable endolysins could enable the development of targeted antibacterial recombinant proteins with diverse clinical and industrial applications. Full article

Primary Effusion Lymphoma (PEL) cells carry Kaposi’s sarcoma-associated herpesvirus (KSHV) in a latent state, except for a small number of cells in which the virus replicates to ensure its persistence into the infected host. However, the lytic cycle can be reactivated in vitro by exposing these lymphoma cells to various treatments, leading to cell lysis. To restrict viral antigen expression, KSHV induces repressive epigenetic changes, including DNA methylation and histone modifications. Among the latter, histone deacetylation and tri-methylation of Histone H3 lisyne-27 (H3K27me3) have been reported to play a role. Here, we found that the inhibition of H3K27 tri-methylation by valemetostat DS3201 (DS), a small molecule that inhibits Enhancer of Zeste Homolog 2 (EZH2) methyltransferase, induced the KSHV lytic cycle in PEL cells, and that this effect involved the activation of the wtp53–p21 axis and autophagic dysregulation. DS also potentiated the lytic cycle activation mediated by the Histone deacetylases (HDAC) inhibitor Suberoylanilide hydroxamic acid (SAHA) and reinforced its cytotoxic effect, suggesting that such a combination could be used to unbalance the latent/lytic cycle and further impair the survival of PEL cells. Full article

Salmonella enterica is a major food-borne pathogen causing food poisoning. The use of bacteriophages as alternative biocontrol agents has gained renewed interest due to the rising issue of antibiotic-resistant bacteria. We isolated and characterized three phages targeting Salmonella: SPN3US, SPN3UB, and SPN10H. Morphological and genomic analyses revealed that they belong to the class Caudoviricetes. SPN3UB, SPN3US, and SPN10H specifically target bacterial surface molecules as receptors, including O-antigens of lipopolysaccharides, flagella, and BtuB, respectively. The phages exhibited a broad host range against Salmonella strains, highlighting their potential for use in a phage cocktail. Bacterial challenge assays demonstrated significant lytic activity of the phage cocktail consisting of the three phages against S. typhimurium UK1, effectively delaying the emergence of phage-resistant bacteria. The phage cocktail effectively reduced Salmonella contamination in foods, including milk and pork and chicken meats, during cold storage. These results indicate that a phage cocktail targeting different host receptors could serve as a promising antimicrobial strategy to control Salmonella. Full article

Antibiotics resistance is expanding amongst pathogenic bacteria. Phage therapy is a revived concept for targeting bacteria with multiple antibiotics resistances. In the present study, we isolated and characterized a novel phage from hospital treatment plant input, using Escherichia coli (E. coli) as host bacterium. Phage lytic activity was detected by using soft agar assay. Whole-genome sequencing of the phage was performed by using Next-Generation Sequencing (NGS). Host range was determined using other species of bacteria and representative genogroups of E. coli. Whole-genome sequencing of the phage revealed that Escherichia phage Ioannina is a novel phage within the Dhillonvirus genus, but significantly diverged from other Dhillonviruses. Its genome is a 45,270 bp linear double-stranded DNA molecule that encodes 61 coding sequences (CDSs). The coding sequence of CDS28, a putative tail fiber protein, presented higher similarity to representatives of other phage families, signifying a possible recombination event. Escherichia phage Ioannina lytic activity was broad amongst the E. coli genogroups of clinical and environmental origin with multiple resistances. This phage may present in the future an important therapeutic tool against bacterial strains with multiple antibiotic resistances. Full article

The Epstein–Barr virus (EBV) has a very high prevalence (>90% in adults), establishes a lifelong latency after primary infection, and exerts an oncogenic potential. This dsDNA virus encodes for various molecules, including microRNAs (miRs), which can be detected in the latent and lytic phases with different expression levels and affect, among others, immune evasion and malignant transformation. In this study, the different EBV miRs are quantified in EBV-positive lymphomas, and the impact on the host cell transcriptome of the most abundant EBV miRs will be analyzed using comparative RNA sequencing analyses. The EBV miRs ebv-miR-BART1, -BART4, -BART17, and -BHRF1-1 were most highly expressed, and their selective overexpression in EBV-negative human cells resulted in a large number of statistically significantly down- and up-regulated host cell genes. Functional analyses showed that these dysregulated target genes are involved in important cellular processes, including growth factor pathways such as WNT, EGF, FGF, and PDGF, as well as cellular processes such as apoptosis regulation and inflammation. Individual differences were observed between these four analyzed EBV miRs. In particular, ebv-miR-BHRF1-1 appears to be more important for malignant transformation and immune evasion than the other EBV miRs. Full article

Cytokinins (CKs) are a group of N⁶-substituted signaling molecules whose biosynthesis and metabolism have been documented in all kingdoms of life, including vertebrates. While their biological relevance in vertebrate systems continues to be elucidated, they have broadly been documented with therapeutic effects in exogenous applications. In this study, we evaluated the virostatic potential of four types of CKs including, N⁶-isopentenyladenine (iP), N⁶-isopentenyladenosine (iPR), N⁶-isopentenyladenosine-5′monophosphate (iPMP), and 2-methylthiol-N⁶-isopentenyladenosine (2MeSiPR) against the ranavirus type species, frog virus 3 (FV3). Following concurrent treatment and infection, iP and iPR reduced viral replication by 33.8% and 59.6%, respectively, in plaque formation assays. A decrease in viral replication was also observed when CK exposure was limited to 12 h prior to infection, where iP and iPR reduced viral replication by 31% and 23.75%, respectively. Treatment with iP and iPR was also marked by 48% and 60% decreases in viral load over 72 h, respectively, as measured in single step growth curves. Plaque morphology was altered in vitro, as iP and iPR treatment increased plaque area by 83% and 112% with lytic zone formation also becoming more prevalent in corresponding treatments. Treatment with iPMP and 2MeSiPR resulted in no effect on viral kinetics in vitro. The results of this study are the first to provide evidence of CK antiviral activity against a DNA virus and highlight the importance of their structure for therapeutic investigations. Full article

Epstein-Barr Virus (EBV) is closely linked to nasopharyngeal carcinoma (NPC), notably prevalent in southern China. Although type II latency of EBV plays a crucial role in the development of NPC, some lytic genes and intermittent reactivation are also critical for viral propagation and tumor progression. Since T cell-mediated immunity is effective in targeted killing of EBV-positive cells, it is important to identify EBV-derived peptides presented by highly prevalent human leukocyte antigen class I (HLA-I) molecules throughout the EBV life cycle. Here, we constructed an EBV-positive NPC cell model to evaluate the presentation of EBV lytic phase peptides on streptavidin-tagged specific HLA-I molecules. Utilizing a mass spectrometry (LC-MS/MS)-based immunopeptidomic approach, we characterized eleven novel EBV peptides as well as two previously identified peptides. Furthermore, we determined these peptides were immunogenic and could stimulate PBMCs from EBV VCA/NA-IgA positive donors in an NPC endemic southern Chinese population. Overall, this work demonstrates that highly prevalent HLA-I-specific EBV peptides can be captured and functionally presented to elicit immune responses in an in vitro model, which provides insight into the epitopes presented during EBV lytic cycle and reactivation. It expands the range of viral targets for potential NPC early diagnosis and treatment. Full article

Representatives of the bacterial genus Aeromonas are some of the most notorious aquaculture pathogens associated with a range of diseases in different fish species. As the world forges toward the post-antibiotic era, alternative options for combating bacterial pathogens are needed. One such alternative option is phage biocontrol. In this study, a novel podophage—JELG-KS1—infecting Aeromonas salmonicida was retrieved from wastewater along with its host strain. The genome of the JELG-KS1 phage is a 40,505 bp dsDNA molecule with a GC% of 53.42% and 185 bp direct terminal repeats and encodes 53 predicted proteins. Genomic analysis indicates that JELG-KS1 might represent a novel genus within the subfamily Studiervirinae. Podophage JELG-KS1 is a strictly lytic phage without any identifiable virulence or AMR genes that quickly adsorbs onto the surface of host cells to initiate a 48 min long infectious cycle, resulting in the release of 71 ± 12 JELG-KS1 progeny virions per infected cell. JELG-KS1 effectively lyses its host population in vitro, even at very low multiplicities of infection. However, when challenged against a panel of Aeromonas spp. strains associated with diseases in aquaculture, JELG-KS1 shows host-specificity that is confined only to its isolation strain, immediately compromising its potential for Aeromonas spp. biocontrol in aquaculture. Full article

Ardisia crenata Sims, an important ethnic medicine, is recorded in the Chinese Pharmacopoeia for treating laryngeal diseases and upper respiratory tract infections. This study aimed to evaluate the antimicrobial effect of extracts and potential antimicrobial compounds of A. crenata Sims. It was found that the roots of A. crenata Sims have a potential inhibitory effect on Candida albicans and Aspergillus flavus, with MICs of 1.56 mg/mL and 0.39 mg/mL, and the leaves of A. crenata Sims have a potential inhibitory effect on Pseudomonas aeruginosa and Staphylococcus aureus, with MICs of 3.12 mg/mL and 6.77 mg/mL, respectively. Meanwhile, five compounds including one catechin and four bergenins were obtained from roots. These components were identified on the fingerprint spectrum, representing chromatographic peaks 16, 21, 22, 23, and 25, respectively. Among these, 11-β-d-glucopyranosyl-bergenin and (−)-gallocatechin showed potential inhibition for Staphylococcus aureus and Pseudomonas aeruginosa with MIC of 0.26 and 0.33 mg/mL, respectively. The roots, stems, and leaves of A. crenata Sims are very similar in chemical composition, with large differences in content. Principal component analysis (PCA) and Hierarchical cluster analysis (HCA) showed that 16 batches of A. crenata Sims could be divided into four main production areas: Guizhou, Jiangsu, Guangxi, and Jiangxi. Furthermore, molecular docking results showed that 11-β-d-glucopyranosyl-bergenin had a better affinity for Casein lytic proteinase P (ClpP), and (−)-gallocatechin possessed a strong affinity for LasA hydrolysis protease and LasB elastase. These findings suggest catechin and bergenins from A. crenata Sims can be used as antimicrobial activity molecules. Full article

The Epstein–Barr virus (EBV) is accepted as a primary risk factor for certain nasopharyngeal carcinoma (NPC) subtypes, where the virus persists in a latent stage which is thought to contribute to tumorigenesis. Current treatments are sub-optimal, and recurrence occurs in many cases. An alternative therapeutic concept is aimed at triggering the lytic cycle of EBV selectively in tumor cells as a means to add clinical benefit. While compounds able to stimulate the lytic cascade have been identified, their clinical application so far has been limited. We are developing a novel anticancer molecule, NEO212, that was generated by covalent conjugation of the alkylating agent temozolomide (TMZ) to the naturally occurring monoterpene perillyl alcohol (POH). In the current study, we investigated its potential to trigger the lytic cycle of EBV in NPC cells in vitro and in vivo. We used the established C666.1 cell line and primary patient cells derived from the brain metastasis of a patient with NPC, both of which harbored latent EBV. Upon treatment with NEO212, there was an increase in EBV proteins Zta and Ea-D, key markers of the lytic cycle, along with increased levels of CCAAT/enhancer-binding protein homologous protein (CHOP), a marker of endoplasmic reticulum (ER) stress, followed by the activation of caspases. These effects could also be confirmed in tumor tissue from mice implanted with C666.1 cells. Towards a mechanistic understanding of these events, we used siRNA-mediated knockdown of CHOP and inclusion of anti-oxidant compounds. Both approaches blocked lytic cycle induction by NEO212. Therefore, we established a sequence of events, where NEO212 caused reactive oxygen species (ROS) production, which triggered ER stress and elevated the levels of CHOP, which was required to stimulate the lytic cascade of EBV. Inclusion of the antiviral agent ganciclovir synergistically enhanced the cytotoxic impact of NEO212, pointing to a potential combination treatment for EBV-positive cancers which should be explored further. Overall, our study establishes NEO212 as a novel agent able to stimulate EBV’s lytic cycle in NPC tumors, with implications for other virus-associated cancers. Full article

Antimicrobial peptides (AMPs) are promising molecules in diverse fields, including aquaculture. AMPs possess lytic effects on a wide range of pathogens, resulting in a potential replacement for traditional antimicrobials in aquaculture. In addition, they also have modulatory effects on host immune responses. Thus, the objective of this work was to evaluate the immunomodulatory capability of three known synthetic AMPs derived from European sea bass, NK-lysin (Nkl), hepcidin (Hamp), and dicentracin (Dic), in head-kidney cell suspensions from European sea bass and gilthead seabream. The tested peptides were neither cytotoxic for European sea bass nor gilthead seabream cells and failed to modulate the respiratory burst and phagocytosis activities. However, they modified the pattern of transcription of immune-related genes differently in both species. Peptides were able to promote the expression of marker genes for anti-inflammatory (il10), antiviral (mx, irf3), cell-mediated cytotoxicity (nccrp1, gzmb), and antibody responses (ighm) in European sea bass, with the Nkl peptide being the most effective. Contrary to this, the effects of those peptides on gilthead seabream mainly resulted in the suppression of immune responses. To conclude, European sea bass-derived peptides can be postulated as potential tools for immunostimulation in European sea bass fish farms, but more efforts are required for their universal use in other species. Full article