Search Results (60)

Background/Objectives: The escalating crisis of antibiotic resistance and the inherent limitations of conventional antibiotics necessitate the development of innovative therapeutic strategies. Targeted drug delivery (TDD) offers a powerful approach to enhance efficacy, minimize systemic toxicity, and circumvent bacterial resistance. This systematic review aims to evaluate the potential of unique bacterial transport systems (BTSs), surface specific receptors and intracellular enzymes as platforms for TDD via the “Trojan Horse” strategy (THS). Methods: A comprehensive literature review was conducted, focusing on studies that investigated the specificity and mechanisms of BTSs responsible for the uptake of metabolites that are essential for and unique to bacteria. This includes an analysis of transport systems for siderophores, bacteria-specific sugars, cell wall components, D-amino acids, and vitamins. We assessed preclinical and clinical examples of drug conjugates utilizing these pathways, as well as emerging platforms such as bacteriophage-derived proteins, antibody–antibiotic conjugates, and bacterial extracellular vesicles (EVs). Results: BTSs demonstrate high specificity for their cognate substrates, providing effective molecular gateways for TDD of drugs photosensitizers and diagnostic probes in form of conjugates. The siderophore–cephalosporin conjugate cefiderocol represents a clinically validated example, having received FDA approval. Preclinical studies further reveal that conjugates utilizing sugars (e.g., maltose, trehalose) and vitamins (e.g., B12) can significantly enhance antibiotic uptake and activity against both Gram-positive and Gram-negative pathogens, including drug-resistant strains. Emerging platforms like bacteriophage endolysins and engineered EVs show promise for overcoming biological barriers such as bacterial outer membranes and intracellular host niches. Conclusions: The THS leveraging BTSs represents a clinically viable and promising avenue for next-generation antibacterial therapies. Advantages of BTS include overcoming bacterial resistance, such as reduced membrane permeability and efflux pumps, enabling the “revival” of antibiotics that are poorly permeable or toxic, increasing their local concentration at the target site and reducing side effects on host cells. While significant progress has been made, a striking disconnect persists between the hundreds of conjugates demonstrating potent in vitro activity and the limited agent that has achieved clinical use. This in vitro–in vivo gap reflects, in large part, the early stage of this field rather than a fundamental failure. Further research is critically needed not only to identify novel BTSs and optimize drug-linker chemistry, but also to systematically address the translational barriers—including poor pharmacokinetics, immunogenicity, and unexpected toxicity—that have prevented most promising candidates from advancing beyond preclinical evaluation. Full article

The escalation of bacterial resistance to existing antibiotics represents a growing global health challenge, exacerbated by the widespread misuse of antimicrobial agents. As a result, alternative antibacterial strategies are increasingly being explored, including phage-derived lytic proteins. In this study, we report a preliminary characterisation of a novel phage-derived lytic protein identified through computational screening of bacteriophage genome sequences. A putative open reading frame, designated SM07 (1383 bp), was selected from bacteriophage sequences contributed by the University of KwaZulu-Natal to a global phage repository. The gene was synthesised, sub-cloned into the pET-30b(+) vector with an N-terminal histidine tag, and recombinantly expressed in Escherichia coli BL-21(AI) cells. The protein was purified using affinity and ion-exchange chromatography. Purified SM07 exhibited in vitro antimicrobial activity against Pseudomonas aeruginosa, with a minimum inhibitory concentration of 4 µg/mL, while no significant cytotoxic effects were observed in Vero kidney cells at concentrations substantially above the effective dose. Together, these findings provide initial evidence supporting the antimicrobial potential of SM07 and highlight phage-derived lytic proteins as candidates for further investigation as alternative agents against P. aeruginosa-associated infections. Full article

Chronic Kidney Disease (CKD) and Colorectal Cancer (CRC) share a profound epidemiological link, supported by Mendelian randomization studies suggesting causality. This review articulates a refined Gut–Kidney Axis, focusing on the pathophysiology of indole-derived uremic toxins. CKD-induced dysbiosis drives hepatic synthesis and systemic accumulation of indoxyl sulfate, which is proposed to promote carcinogenesis via Aryl Hydrocarbon Receptor (AhR) and Akt signaling, ultimately upregulating c-Myc and EGFR. We propose a two-compartment model: while systemic indoxyl sulfate reflects the total gut indole pool (mainly from planktonic bacteria), adherent bacteria like Fusobacterium nucleatum may create high-concentration indole hotspots within the tumor microenvironment. Clinically, we advocate for protein-independent DNA methylation biomarkers (SEPT9, SDC2) to avoid renal confounding. Furthermore, we propose a novel diagnostic panel integrating serum indoxyl sulfate (systemic load) and urinary indoxyl sulfate (gut production) to guide therapy. Therapeutically, targeting upstream drivers (AhR/Akt) may bypass resistance to anti-EGFR therapies in KRAS-mutated tumors. We also discuss the repurposing of the oral adsorbent AST-120 and emerging bacteriophage therapies as strategies to disrupt this oncogenic axis. This review offers a comprehensive framework for stratified management of CKD-associated CRC. Full article

Antibiotic resistance is arguably one of the greatest threats to global health today. The worldwide emergence of multidrug-resistant and hypervirulent Klebsiella pneumoniae underscores the urgent need for alternative treatments. Bacteriophages (phages) are considered one of the most promising alternatives to address this crisis. In this review, we summarize current knowledge of phage–host interactions and highlight recent advances in phage therapy against K. pneumoniae, including phage cocktails, antibiotic combination therapy, and treatments based on phage-derived proteins. Despite their tremendous therapeutic potential, significant challenges remain. We therefore also discuss strategies to optimize phage research and recent innovations in the field. Full article

Antimicrobial resistance threatens a new “dark age” in medical practice. Chronic antibiotic overuse has driven the rise in antimicrobial resistance and promoted the emergence of multidrug-resistant organisms. To address this problem, researchers have developed new approaches. Antimicrobials derived from bacteriophage, which are viruses that target bacteria, are promising candidates. Amongst these candidates, bacteriophage enzymes used in the viral replication cycle are of significant interest. Specifically, endolysins are used by bacteriophage to lyse the bacterial cell wall, leading to structural collapse and cell lysis. Researchers are increasingly applying these proteins externally to multidrug-resistant organisms as a novel antimicrobial treatment. Following this increased interest, many studies have presented protein engineering methods to further enhance the effectiveness of endolysins as antimicrobials. These methods include attachment of membrane-permeabilizing peptides, domain-swapping, and catalytic-site modification. Recent advances in all three fields have seen the implementation of tools like novel in silico design pipelines and library-based screening methods. This review summarizes these recent advances in the rapidly developing field of endolysin engineering and discusses potential future directions in this field. Full article

Streptococcus thermophilus is a Gram-positive, homofermentative lactic acid bacterium classified within the Firmicutes phylum, recognized for its probiotic properties and significant role in promoting human health. This review consolidates existing understanding of its metabolic pathways, functional metabolites, and diverse applications, highlighting evidence-based insights to enhance scientific integrity. S. thermophilus predominantly ferments lactose through the Embden-Meyerhof-Parnas pathway, resulting in L(+)-lactic acid as the primary end-product, along with secondary metabolites including acetic acid, formic acid, and pyruvate derivatives. Exopolysaccharides (EPS) are composed of repeating units of glucose, galactose, rhamnose, and N-acetylgalactosamine. They display strain-specific molecular weights ranging from 10 to 2000 kDa and contribute to the viscosity of fermented products, while also providing antioxidant and immunomodulatory benefits. Aromatic compounds such as acetaldehyde and phenylacetic acid are products of amino acid catabolism and carbohydrate metabolism, playing a significant role in the sensory characteristics observed in dairy fermentations. Bacteriocins, such as thermophilins (e.g., Thermophilin 13, 110), exhibit extensive antimicrobial efficacy against pathogens including Listeria monocytogenes and Bacillus cereus. Their activity is modulated by quorum-sensing mechanisms that involve the blp gene cluster, and they possess significant stability under heat and pH variations, making them suitable for biopreservation applications. In food applications, S. thermophilus functions as a Generally Recognized as Safe (GRAS) starter culture in the production of yogurt and cheese, working in conjunction with Lactobacillus delbrueckii subsp. bulgaricus to enhance acidification and improve texture. Specific strains have been identified to mitigate lactose intolerance, antibiotic-related diarrhea, and inflammatory bowel diseases through the modulation of gut microbiota, the production of short-chain fatty acids, and the inhibition of Helicobacter pylori. The genome, characterized by a G + C content of approximately 37 mol%, facilitates advancements in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas technology and heterologous protein expression, with applications extending to non-dairy fermentations and the development of postbiotics. This review emphasizes the adaptability of S. thermophilus, showcasing the variability among strains and the necessity for thorough preclinical and clinical validation to fully utilize its potential in health, sustainable agriculture, and innovation. It also addresses challenges such as susceptibility to bacteriophages and limitations in proteolytic activity. Full article

The underlying structural features of the mismatch between catalytic and cytostatic properties in L-asparaginase from Rhodospirillum rubrum (RrA) and three of its mutants were investigated. The rationale for selecting the specific mutations (RrA_{A64V, E67K}; RrA_{R118H, G120R}; RrA_{E149R, V150P, F151T}) is to elucidate the role of inter-subunit interaction in RrA and its impact on catalytic efficiency and stability. Bioinformatic modeling revealed a predominantly negative surface charge on RrA with limited positive charge clusters in the vicinity of the interface region. Thus, some negatively charged groups were replaced with positively charged ones to enhance the electrostatic interactions and stabilize the enzyme quaternary structure. RrA_{A64V, E67K} and RrA_{R118H, G120R} additionally contained an N-terminal 17-amino acid capsid peptide derived from the bacteriophage T7 (MASMTGGQQMGRGSSRQ), which could potentially affect the conformational stability of theenzymes. Circular dichroism (CD) spectroscopy was applied to the kinetic parameters analysis of Asn hydrolysis and showed that native RrA displayed a V_max of 30 U/mg and a K_M of 4.5 ± 0.5 mM. RrA_{E149R, V150P, and F151T} exhibited a substantially increased V_max of 57 U/mg. The catalytic efficiency of V_max/K_M also improved compared to the native enzyme: the V_max/K_M increased from approximately 7 U/mg × mM⁻¹ (for the native enzyme) to 9 U/mg × mM⁻¹ for Mut3. Other mutants exhibited less pronounced changes. Thermo-denaturation studies allowed us to determine the phase transition parameters of the RrA variants in comparison with commercial reference sample EcA. RrA_{A64V, E67K} and RrA_{R118H, G120R} exhibited the most favorable phase transition parameters, with melting temperatures (T_m) of 60.3 °C and 59.4 °C, respectively, exceeding that of the wild-type RrA (54.6 °C) and RrA_{E149R, V150P, F151T} (52 °C). The EcA demonstrated a slightly superior thermal stability, with a T_m of 62 °C. The mutations showed a significant effect on protein stability during trypsinolysis. Therefore, RrA_{E149R, V150P, F151T} showed higher resistance (45% activity remaining after 30 min of trypsin exposure) compared to the native RrA retained 20% activity. EcA preparations exhibited lower stability to trypsinolysis (losing over 90% activity in 15 min). The cytostatic effects were evaluated using MTT assays against K562 (leukemic) and A549 (lung carcinoma) cell lines. The MTT assays with K562 cells revealed that RrA_{E149R, V150P, F151T} (IC₅₀ of 10 U/mL) and RrA_{R118H, G120R} (IC₅₀ of 11.5 U/mL) exhibited superior antiproliferative activity compared to native enzymes RrA (IC₅₀ of 15 U/mL) and EcA (24 U/mL). RrA_{E149R, V150P, F151T} showed the most significant improvement in cytostatic activity. The results obtained indicate that the substitutions in RrA_{E149R, V150P, F151T} resulted in the improvement of the enzyme biocatalytic properties and an increase in the resistance to aggregation and trypsinolysis. This highlights the role of electrostatic interactions in stabilizing the oligomeric structure of the enzyme, which eventually translates into an improvement in cytostatic efficiency and antiproliferative forces. Full article

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

The escalating global health crisis of antibiotic resistance, driven by the rapid emergence of multidrug-resistant (MDR) bacterial pathogens, necessitates urgent and innovative countermeasures. This review comprehensively examines the diverse mechanisms employed by bacteria to evade antibiotic action, including alterations in cell membrane permeability, efflux pump overexpression, biofilm formation, target site modifications, and the enzymatic degradation of antibiotics. Specific focus is given to membrane transport systems such as ATP-binding cassette (ABC) transporters, resistance–nodulation–division (RND) efflux pumps, major facilitator superfamily (MFS) transporters, multidrug and toxic compound extrusion (MATE) systems, small multidrug resistance (SMR) families, and proteobacterial antimicrobial compound efflux (PACE) families. Additionally, the review explores the global burden of MDR pathogens and evaluates emerging therapeutic strategies, including quorum quenching (QQ), probiotics, postbiotics, synbiotics, antimicrobial peptides (AMPs), stem cell applications, immunotherapy, antibacterial photodynamic therapy (aPDT), and bacteriophage. Furthermore, this review discusses novel antimicrobial agents, such as animal-venom-derived compounds and nanobiotics, as promising alternatives to conventional antibiotics. The interplay between clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) in bacterial adaptive immunity is analyzed, revealing opportunities for targeted genetic interventions. By synthesizing current advancements and emerging strategies, this review underscores the necessity of interdisciplinary collaboration among biomedical scientists, researchers, and the pharmaceutical industry to drive the development of novel antibacterial agents. Ultimately, this comprehensive analysis provides a roadmap for future research, emphasizing the urgent need for sustainable and cooperative approaches to combat antibiotic resistance and safeguard global health. Full article

Bacteriophages (or phages) remain the leading cause of failure in dairy fermentations. Thereby, phage-resistant Lactococcus lactis and Lactococcus cremoris dairy starters are in continuous demand. In this work, our goal was to identify phage defense mechanisms against ceduoviruses encoded by two wild isolates of dairy origin named L. lactis IPLA517 and IPLA1064. These strains were previously subjected to experimental evolution to select derivatives that are resistant to the bacteriocin Lcn972. It was observed that the Lcn972^R derivatives became sensitive to phage infection; however, the underlying mechanism was not defined. The long-read sequencing technologies applied in this work reveal that all of the Lcn972^R derivatives shared the loss of a 41 kb endogenous plasmid (p41) that harbors a putative exopolysaccharide (EPS) gene cluster with significant homology to one described in Lactococcus garvieae. Using a CRISPR-Cas9-based approach, p41 was selectively cured from L. lactis IPLA1064. Phage infection assays with three ceduoviruses demonstrated that curing p41 restored phage sensitivity at levels comparable to the Lcn972^R-IPLA1064 derivatives. Phage adsorption to Δp41 cells was also increased, consistent with the hypothesis of EPS production hindering access to the phage receptor protein Pip. Our results reinforce the role of EPSs in protecting Lactococcus against phage infection, a phenomenon that is rarely reported for ceduoviruses. Moreover, the results also exemplify the likely horizontal gene transfer that can occur between L. lactis and L. garvieae in a dairy environment. Full article

Electrostatic charge significantly influences microorganism–surface interactions, including viral adhesion and transmission. While bacterial surface charges are well characterized using electrophoretic mobility and X-ray photoelectron spectroscopy (XPS), similar studies for viruses are limited. This work bridges the gap by estimating the negative surface charge of the Phi6 bacteriophage using XPS data. A novel approach is applied, combining chemical functionalities derived from XPS with a system of equations to quantify surface polysaccharides, proteins, hydrocarbons, and negatively charged groups (RCOO⁻ and R₂PO₄⁻). The results indicate a predominance of proteins on the viral surface and a pH-dependent negative charge: phosphate groups dominate at low pH (1–3), while both groups contribute equally at pH 4–9. These findings provide a deeper understanding of virus–surface interactions and underscore the importance of pH in modulating viral surface charge. This method, which surpasses traditional electrophoretic mobility techniques, offers new perspectives for studying viral adhesion and developing improved antiviral materials and disinfection strategies. Full article

The recent SARS-CoV-2 (COVID-19) pandemic exemplifies how newly emerging and reemerging viruses can quickly overwhelm and cripple global infrastructures. Coupled with synergistic factors such as increasing population densities, the constant and massive mobility of people across geographical areas and substantial changes to ecosystems worldwide, these pathogens pose serious health concerns on a global scale. Vaccines form an indispensable defense, serving to control and mitigate the impact of devastating outbreaks and pandemics. Towards these efforts, we developed a tunable vaccine platform that can be engineered to simultaneously display multiple viral antigens. Here, we describe a second-generation version wherein chimeric proteins derived from SARS-CoV-2 and bacteriophage lambda are engineered and used to decorate phage-like particles with defined surface densities and retention of antigenicity. This streamlines the engineering of particle decoration, thus improving the overall manufacturing potential of the system. In a prime-boost regimen, mice immunized with particles containing as little as 42 copies of the chimeric protein on their surface develop potent neutralizing antibody responses, and immunization protects mice against virulent SARS-CoV-2 challenge. The platform is highly versatile, making it a promising strategy to rapidly develop vaccines against a potentially broad range of infectious diseases. Full article

Over the past few decades, dengue fever has emerged as a significant global health threat, affecting tropical and moderate climate regions. Current vaccines have practical limitations, there is a strong need for safer, more effective options. This study introduces novel vaccine candidates covering all four dengue virus (DENV) serotypes using virus-like particles (VLPs), a proven vaccine platform. The dengue virus envelope protein domain III (EDIII), the primary target of DENV-neutralizing antibodies, was either genetically fused or chemically coupled to bacteriophage-derived AP205-VLPs. To facilitate the incorporation of the large EDIII domain, AP205 monomers were dimerized, resulting in sterically optimized VLPs with 90 N- and C-termini. These vaccines induced high-affinity/avidity antibody titers in mice, and confirmed their protective potential by neutralizing different DENV serotypes in vitro. Administration of a tetravalent vaccine induced high neutralizing titers against all four serotypes without producing enhancing antibodies, at least not against DENV2. In conclusion, the vaccine candidates, especially when administered in a combined fashion, exhibit intriguing properties for potential use in the field, and exploring the possibility of conducting a preclinical challenge model to verify protection would be a logical next step. Full article

Healthcare faces a major problem with the increased emergence of antimicrobial resistance due to over-prescribing antibiotics. Bacteriophages may provide a solution to the treatment of bacterial infections given their specificity. Enzymes such as endolysins, exolysins, endopeptidases, endosialidases, and depolymerases produced by phages interact with bacterial surfaces, cell wall components, and exopolysaccharides, and may even destroy biofilms. Enzymatic cleavage of the host cell envelope components exposes specific receptors required for phage adhesion. Gram-positive bacteria are susceptible to phage infiltration through their peptidoglycan, cell wall teichoic acid (WTA), lipoteichoic acids (LTAs), and flagella. In Gram-negative bacteria, lipopolysaccharides (LPSs), pili, and capsules serve as targets. Defense mechanisms used by bacteria differ and include physical barriers (e.g., capsules) or endogenous mechanisms such as clustered regularly interspaced palindromic repeat (CRISPR)-associated protein (Cas) systems. Phage proteins stimulate immune responses against specific pathogens and improve antibiotic susceptibility. This review discusses the attachment of phages to bacterial cells, the penetration of bacterial cells, the use of phages in the treatment of bacterial infections, and the limitations of phage therapy. The therapeutic potential of phage-derived proteins and the impact that genomically engineered phages may have in the treatment of infections are summarized. Full article

Antimicrobial resistance (AMR) has emerged as a global health challenge, sparking worldwide interest in exploring the antimicrobial potential of natural compounds as an alternative to conventional antibiotics. In recent years, one area of focus has been the utilization of bacteriophages and their derivative proteins. Specifically, phage lytic proteins, or endolysins, are specialized enzymes that induce bacterial cell lysis and can be efficiently produced and purified following overexpression in bacteria. Nonetheless, a significant limitation of these proteins is their vulnerability to certain environmental conditions, which may impair their effectiveness. Encapsulating endolysins in vesicles could mitigate this issue by providing added protection to the proteins, enabling controlled release, and enhancing their stability, particularly at temperatures around 4 °C. In this work, the chimeric lytic protein CHAPSH3b was encapsulated within non-ionic surfactant-based vesicles (niosomes) created using the thin film hydrating method (TFH). These protein-loaded niosomes were then characterized, revealing sizes in the range of 30–80 nm, zeta potentials between 30 and 50 mV, and an encapsulation efficiency (EE) of 50–60%. Additionally, with the objective of exploring their potential application in the food industry, these endolysin-loaded niosomes were incorporated into gelatine films. This was carried out to evaluate their stability and antimicrobial efficacy against Staphylococcus aureus. Full article