Search Results (856)

Acute hepatopancreatic necrosis disease (AHPND), caused by the bacterium Vibrio parahaemolyticus, is a major threat to global shrimp aquaculture. In this study, we evaluated the therapeutic effects of phage therapy in Litopenaeus vannamei challenged with AHPND-causing Vibrio parahaemolyticus. Phage application at various concentrations significantly improved shrimp survival, with the 1 ppm group demonstrating the highest survival rate. Enzymatic assays revealed that phage-treated shrimp exhibited enhanced immune enzyme activities, including acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LZM). In addition, antioxidant defenses such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), and total antioxidant capacity (T-AOC) significantly improved, accompanied by reduced malondialdehyde (MDA) levels. Serum biochemical analyses demonstrated marked improvements in lipid metabolism, particularly reductions in triglyceride (TG), total cholesterol (TC), and low-density lipoprotein (LDL), alongside higher levels of beneficial high-density lipoprotein (HDL). Transcriptomic analysis identified 2274 differentially expressed genes (DEGs), notably enriched in pathways involving fatty acid metabolism, peroxisome functions, lysosomes, and Toll-like receptor (TLR) signaling. Specifically, phage treatment upregulated immune and metabolic regulatory genes, including Toll-like receptor 4 (TLR4), myeloid differentiation primary response protein 88 (MYD88), interleukin-1β (IL-1β), nuclear factor erythroid 2-related factor 2 (Nrf2), and peroxisome proliferator-activated receptor (PPAR), indicating activation of innate immunity and antioxidant defense pathways. These findings suggest that phage therapy induces protective immunometabolic adaptations beyond its direct antibacterial effects, thereby providing an ecologically sustainable alternative to antibiotics for managing bacterial diseases in shrimp aquaculture. Full article

Antimicrobial resistance (AMR) has emerged as one of the most pressing global health challenges of our time. Alarming projections of increasing mortality from resistant infections highlight the urgent need for innovative solutions. While many candidates have shown promise in preliminary studies, they often encounter challenges in terms of efficacy and safety during clinical translation. This review examines cutting-edge approaches to combat AMR, with a focus on engineered antimicrobial peptides, functionalized nanoparticles, and advanced genomic therapies, including Clustered Regularly Interspaced Short Palindromic Repeats-associated proteins (CRISPR-Cas systems) and phage therapy. Recent advancements in these fields are critically analyzed, with a focus on their mechanisms of action, therapeutic potential, and current limitations. Emphasis is given to strategies targeting biofilm disruption and quorum sensing interference, which address key mechanisms of resistance. By synthesizing current knowledge, this work provides researchers with a comprehensive framework for developing next-generation antimicrobials, highlighting the most promising approaches for overcoming AMR through rational drug design and targeted therapies. Ultimately, this review aims to bridge the gap between experimental innovation and clinical application, providing valuable insights for developing effective and resistance-proof antimicrobial agents. Full article

Bacteriophages (phages), the most abundant biological entities on Earth, have long served as both model systems and therapeutic tools. Recent advances in synthetic biology and genetic engineering have revolutionized the capacity to tailor phages with enhanced functionality beyond their natural capabilities. This review outlines the current landscape of synthetic and functional engineering of phages, encompassing both in-vivo and in-vitro strategies. We describe in-vivo approaches such as phage recombineering systems, CRISPR-Cas-assisted editing, and bacterial retron-based methods, as well as synthetic assembly platforms including yeast-based artificial chromosomes, Gibson, Golden Gate, and iPac assemblies. In addition, we explore in-vitro rebooting using TXTL (transcription–translation) systems, which offer a flexible alternative to cell-based rebooting but are less effective for large genomes or structurally complex phages. Special focus is given to the design of customized phages for targeted applications, including host range expansion via receptor-binding protein modifications, delivery of antimicrobial proteins or CRISPR payloads, and the construction of biocontained, non-replicative capsid systems for safe clinical use. Through illustrative examples, we highlight how these technologies enable the transformation of phages into programmable bactericidal agents, precision diagnostic tools, and drug delivery vehicles. Together, these advances establish a powerful foundation for next-generation antimicrobial platforms and synthetic microbiology. Full article

Mesenchymal stem cells have been widely investigated in the field of regenerative medicine and also used as a model to study the differentiation-induction properties of a variety of biomaterials. This study evaluates the osteoinductive potential of novel hydroxyapatite scaffolds functionalized with a phage-displayed peptide (SC1) selected via biopanning for its similarity to bone matrix proteins. The peptide, identified through sequence alignment as a mimotope of osteonectin (SPARC), was used to functionalize scaffolds. Results from SC1 were gathered at different time points (14, 28 and 46 days) and compared with those from nonfunctionalized hydroxyapatite (HA) scaffolds. In vitro experiments, by seeding human adipose-derived stem cells (hASCs), indicated satisfactory biocompatibility for both types of scaffolds. Histochemical observations showed that SC1, better than HA scaffolds, was able to improve hASC osteogenic differentiation, as evaluated through Alizarin Red staining (showing on average a darker staining of 100%). An increase was also observed, especially at early stages (14 days), for osterix (up to 60% increase) and osteonectin immunoexpression (up to 50% increase). In in vivo experiments, cell-free scaffolds of both types were subcutaneously implanted into the backs of mice and analyzed after 2, 4, 8 and 16 weeks. Also, in this case, SC1 more effectively promoted the osteogenic differentiation of infiltrated resident cells. In particular, increased immunoexpression of osterix and osteonectin (+30% and 35%, respectively) was found already at 2 weeks. It can be concluded that SC1 scaffolds may represent a valuable tool to address critical-sized bone defects. Full article

Efficient and rapid detection of bacterial pathogens is crucial for food safety and effective disease control. While conventional methods such as PCR and ELISA are accurate, they are time-consuming, costly, and often require specialized infrastructure. Recently, electrochemical biosensors integrating graphene nanomaterials with bacteriophages—termed graphages—have emerged as promising platforms for pathogen detection, offering fast, specific, and highly responsive detection. This review critically examines all electrochemical biosensors reported to date that utilize graphene–phage hybrids. Key aspects addressed include the types of graphene nanomaterials and bacteriophages used, immobilization strategies, electrochemical transduction mechanisms, and sensor metrics—such as detection limits, linear ranges, and ability to perform in real matrices. Particular attention is given to the role of phage orientation, surface functionalization, and the use of receptor binding proteins. Finally, current limitations and opportunities for future research are outlined, including prospects for genetic engineering and sensor miniaturization. This review serves as a comprehensive reference for researchers developing phage-based biosensors, especially those interested in integrating carbon nanomaterials for improved electroanalytical performance. Full article

Background/Objectives: Antibiotic resistance presents an urgent public health threat. By developing a streamlined and effective method for studying bacteriophage induction, this research marks a step further in understanding how antibiotic-resistant genes might spread across different environments. This knowledge is essential for creating strategies to reduce the spread of antimicrobial resistance (AMR), particularly from a One Health perspective. In this study, we develop and validate a Green Fluorescent Protein (GFP)-based method as a proxy for bacteriophage induction. This method screens compounds for their potential to promote bacteriophage induction. Methods: This study utilized a recA-GFP construct in Escherichia coli to measure fluorescence as an indicator of SOS response activation. The experiments involved treating E. coli cultures with varying concentrations of the DNA-damaging chemical mitomycin C and measuring fluorescence over time. Additionally, droplet digital PCR (ddPCR) quantified bacteriophage induction in a lambda phage-carrying E. coli strain, allowing for correlation analysis between the two methods. Results: The recA-driven SOS response depended on both dose and time, with increasing concentrations of mitomycin C leading to higher fluorescence. ddPCR analysis confirmed that mitomycin C induced prophage activation, with gene ratios increasing at higher drug concentrations over time. A strong Spearman correlation (>0.7) was noted between fluorescence and ddPCR results at elevated concentrations and relevant time points, indicating the validity of the GFP-based model as a proxy for bacteriophage induction. Conclusions: The findings demonstrate a strong association between the two methods of measuring phage induction, suggesting that the GFP-based E. coli model is a reliable, cost-effective, and efficient tool for studying phage induction and its potential role in AMR spread. This method could facilitate the screening of environmental samples and specific drugs to evaluate their impact on bacteriophage induction, which opens the door for applications such as screening for antibiotic resistance dissemination. Full article

In this study, the impact of the genomic scaffold on the properties of bacteriophages was investigated by swapping the genomic scaffolds surrounding the tailspike genes between two Przondovirus phages, KP192 and KP195, which infect Klebsiella pneumoniae with different capsular types. A yeast-based transformation-associated recombination cloning technique and subsequent “rebooting” of synthetic phage genomes in bacteria were used to construct the phages. Using Klebsiella strains with K2, K64, and KL111 capsular types, it was shown that the capsular specificity of the synthetic phages is fully consistent with that of the tailspike proteins (tsp). However, the efficiency of plating and the lytic efficiency of these phages strongly depended on the genomic scaffold used and the Klebsiella strain used. Synthetic phages with swapped genomic scaffolds demonstrated superior reproduction efficiency using a number of strains compared to wild-type phages, indicating that some elements of the swapped genomic scaffold enhance phage replication efficiency, presumably by blocking some of the host anti-phage defense systems. Our findings demonstrate that even in the case of closely related phages, the selection of the genomic scaffold used for tsp gene transplantation can have a profound impact on the efficiency of phage propagation on target bacterial strains. Full article

Phage display has advanced the discovery of peptides that selectively bind to a wide variety of cell surface molecules, offering new modalities to modulate disease-related protein–protein interactions (PPIs). These cell-binding peptides occupy a unique pharmaceutical space between small molecules and large biologics, and their growing popularity has opened up new avenues for targeting cell surface proteins that were previously considered undruggable. This work provides an overview of methods for identifying cell-selective peptides using phage display combinatorial libraries, covering in vitro, ex vivo, and in vivo biopanning approaches. It addresses key considerations in library design, including the peptide conformation (linear vs. cyclic) and length, and highlights examples of clinically approved peptides developed through phage display. It also discusses the on-phage chemical cyclization of peptides to overcome the limitations of genetically encoded disulfide bridges and emphasizes advances in combining next-generation sequencing (NGS) with phage display to improve peptide selection and analysis workflows. Furthermore, due to the often suboptimal binding affinity of peptides identified in phage display selections, this article discusses affinity maturation techniques, including random mutagenesis and rational design through structure–activity relationship (SAR) studies to optimize initial peptide candidates. By integrating these developments, this review outlines practical strategies and future directions for harnessing phage display in targeting challenging cell surface proteins. Full article

A single domain antibody (SDAb) targeting canine PD-1 was developed as a potential immunotherapeutic for canine cancer. An alpaca was immunized with canine PD-1 protein, and a phage-display library was constructed using mRNA isolated from peripheral lymphocytes. Screening of the library yielded multiple SDAb candidates capable of nanomolar binding to canine PD-1. Among these, clone STX-1b5 demonstrated high expression in a yeast-based recombinant system and was selected for further characterization. Binding and competition assays using ELISA confirmed its ability to bind canine PD-1 and block PDL-1 interaction. In silico structural modeling supported the interaction of STX-1b5 with key PD-1 residues implicated in ligand binding. These findings support the feasibility of using SDAbs and cost-effective yeast expression systems to generate immunotherapeutics for veterinary use, with STX-1b5 representing a promising lead candidate for future clinical development. Full article

The increasing prevalence of multidrug-resistant (MDR) bacterial infections necessitates the exploration of alternative antimicrobial strategies, with phage therapy emerging as a viable option. However, the effectiveness of naturally occurring phages can be significantly limited by bacterial defense systems that include adsorption blocking, restriction–modification, CRISPR-Cas immunity, abortive infection, and NAD+ depletion defense systems. This review examines these bacterial defenses and their implications for phage therapy, while highlighting the potential of phages’ bioengineering to overcome these barriers. By leveraging synthetic biology, genetically engineered phages can be tailored to evade bacterial immunity through such modifications as receptor-binding protein engineering, anti-CRISPR gene incorporation, methylation pattern alterations, and enzymatic degradation of bacterial protective barriers. “Armed phages”, enhanced with antimicrobial peptides, CRISPR-based genome-editing tools, or immune-modulating factors, offer a novel therapeutic avenue. Clinical trials of bioengineered phages, currently SNIPR001 and LBP-EC01, showcase their potential to safely and effectively combat MDR infections. SNIPR001 has completed a Phase I clinical trial evaluating safety in healthy volunteers, while LBP-EC01 is in Phase II trials assessing its performance in the treatment of Escherichia coli-induced urinary tract infections in patients with a history of drug-resistant infections. As “armed phages” progress toward clinical application, they hold great promise for precision-targeted antimicrobial therapies and represent a critical innovation in addressing the global antibiotic resistance crisis. Full article

Protein kinases and phosphatases are essential for post-translational regulation, enabling bacteria to adapt to environmental stresses and modulate virulence. While prior reviews have broadly covered their roles in stress response, antibiotic resistance, and virulence, this article updates specifically on the roles of histidine kinases (HKs) and serine/threonine kinases (STKs) in mediating phage-bacteria interactions. A key aspect is phage-encoded kinases, which hijack bacterial signalling by phosphorylating and disrupting host processes to promote infection. Despite their importance, significant gaps remain in understanding these regulatory networks. This microreview highlights both the unresolved mechanisms and the therapeutic potential of targeting kinase pathways—for instance, by disrupting phage evasion strategies or enhancing phage-based antimicrobial therapies. Full article

The FIC domain-containing protein Sofic has recently been shown to provide robust protection to bacteria against phage infection. Sofic acts as a toxic protein, inducing abortive infection through the AMPylation of target proteins during phage invasion. However, the molecular mechanisms regulating Sofic’s toxic activity remain elusive. In this study, we identified a small gene encoding a short protein located downstream of Sofic in the genome, named AS1 (anti-Sofic1), which functions as an antitoxic protein to counteract Sofic’s toxicity. The crystal structure of Sofic revealed that the protein functions as a dimer in solution, with dimerization being indispensable for its toxic activity. Importantly, structural analysis indicated that ATP binding induces a conformational change in the C-terminal domain (CTD) of Sofic, underscoring the critical role of the CTD in mediating its toxic effects. In vitro colony-forming assays confirmed that the interaction between the CTD and the Amylase domain is crucial for Sofic’s toxic activity. Overall, our results provide molecular insights into the regulatory mechanisms of Sofic in antiviral immunity. Full article

In a vast variety of prokaryotes such as Escherichia coli and Streptomyces lividans, the DNA degradation (Dnd) CDE protein complex (consisting of DndC, DndD, and DndE), together with the DndA/IscS protein and the DndFGH complex, function as a defense barrier to prevent the invasion of non-self-DNA. The DndCDE complex introduces phosphorothioation (PT) modifications into DNA, and the DndFGH complex specifically cleaves non-PT DNA and, thus, restricts horizontal gene transfer and phage invasion. Despite the central importance of the DndCDE complex in DNA PT modification, which catalyzes the oxygen–sulfur swap on DNA, our understanding of this key complex remains poor. Here, we employed protein structure prediction to provide a reasonably reliable prediction of the structure of the DndCDE complex and a 23 bp DNA-DndCDE complex. We found that among the three proteins in the DndCDE complex, DndC, especially its “specificity loop”, plays a key role in recognizing the consensus PT modification sequence. In addition, the DndD protein is found to possess a highly conserved structural surface on its globular domain, presumably mediating the dimerization of DndD as well as the DndCDE complex. Furthermore, our normal mode analysis showed that there exists a dynamic transition between a closed and an open state for the DndCDE complex, facilitating its association and release of DNA. Our conclusions were corroborated by biochemical assays using purified proteins. On the whole, we provide molecular insights into the assembly and DNA-recognition mechanism of a central protein complex involved in DNA phosphorothioation. Full article

Background: Tetanus toxin, produced by Clostridium tetani, is the second deadliest known toxin. Antibodies capable of neutralizing tetanus toxin (TeNT) are vital for preventing and treating tetanus disease. Methods: Herein, we screened thirty-six single variable domains on a heavy chain (VHHs) binding to the light chain (L) and the translocation domain (HN) (L-HN) fragment of TeNT from a phage-display library. Then, the L-HN-specific clones were identified, humanized, and fused with a human fragment crystallizable region (hFc) to form humanized VHH-hFc fusion proteins. Results: The humanized VHH-hFc fusion proteins TL-16-h1-hFc, TL-25-h1-hFc, and TL-34-h1-hFc possessed potent efficacy with high binding affinity, specificity, and neutralizing activity. Only 0.3125 μg was required for TL-16-h1-hFc or TL-25-h1-hFc, and 0.625 μg was required for TL-34-h1-hFc to provide full protection against 10 × Lethal Dose 50 (LD₅₀) TeNT. In the prophylactic setting, 125 μg/kg of TL-16-h1-hFc or TL-25-h1-hFc provided full protection even when they were injected 12 days before exposure to 10 × LD₅₀ TeNT, while TL-34-h1-hFc was less effective. In the therapeutic setting, 25 μg/kg of TL-16-h1-hFc or TL-25-h1-hFc could provide complete protection when administered 24 h after exposure to 5 × LD₅₀ TeNT, while TL-34-h1-hFc required 50 μg/kg. Conclusion: Our results suggest that TL-16-h1-hFc, TL-25-h1-hFc, and TL-34-h1-hFc provide a bright future for the development of anti-TeNT preventive or therapeutic drugs. Full article

Background: Aquaculture faces significant challenges due to bacterial infections, particularly Piscirickettsia salmonis, leading to extensive antibiotic use and raising concerns about antimicrobial resistance. In this context, bacteriophages and bacterial defense systems play a critical role in the evolutionary dynamics of P. salmonis. Objective. This study aimed to investigate the genomic landscape of prophage regions and antiphage defense systems in Piscirickettsia salmonis to better understand their co-evolutionary dynamics and explore their potential role in alternative disease control strategies for aquaculture. Methods: We analyzed 79 genomes of Piscirickettsia salmonis using bioinformatic tools to identify and characterize prophage regions and antiphage defense systems. Results: At the chromosomal level, 70% of the strains contained prophage regions, with a total of 92 identified regions, most of which were classified as intact. At the plasmid level, 75% of plasmids carried prophage regions, with a total of 426 identified regions, predominantly associated with Escherichia phage RCS47, Burkholderia phage Bcep176, and Enterobacteria phage mEp235. Prophage regions were enriched in transposases, head proteins, tail proteins, and phage-like proteins. The analysis of antiphage defense systems revealed that P. salmonis predominantly harbors dGTPase, AbidD, and SoFIC at the chromosomal level, whereas MazEF was the most frequent system in plasmids. A strong positive correlation was found between the number of prophage regions and defense systems in chromosomes (ρ = 0.72, p = 6.3 × 10⁻¹⁴), while a weaker correlation was observed in plasmids. These findings highlight the complex interplay between P. salmonis and its bacteriophages, with implications for disease control in aquaculture. Conclusions: Overall, these insights into the prophage and defense system dynamics provide potential avenues for developing alternative strategies to combat P. salmonis infections and reduce reliance on antibiotics in aquaculture systems. Full article