Search Results (1,546)

Heat shock proteins (HSPs), particularly HSP70, play a vital role in fish immune defense against pathogens. The administration of DnaK (bacterial homolog of HSP70) may be a strategy to potentiate the immune response and survival of aquatic organisms. This study evaluates the effect of cells overexpressing DnaK on mortality and immune-related gene expression in gnotobiotic sea bass larvae challenged with Vibrio anguillarum. Larvae were subjected to different treatments: NB (no bacteria), YS0 (E. coli with no plasmid), YS1 (E. coli expressing truncated DnaK), and YS2 (E. coli expressing DnaK), and then infected with V. anguillarum at 7 days post-hatching (dph). Mortality was monitored, and RT-qPCR was used to evaluate immune gene expression at 0, 18, 24, 36, and 120 hpc. While no significant variations were recorded in the non-challenged larvae, constant and sustained mortality was observed in challenged larvae from 60 to 120 hpc. However, lower mortality was observed in the larvae treated with DnaK. DnaK treatment promoted the expression of antimicrobial (hepcidin, transferrin) and chemotaxis genes (ccl4), which was further enhanced after a challenge with V. anguillarum, in conjunction with the modulation of il1β and il-8 at 120 hpc. These findings suggest that DnaK induces a potent innate immune response, improving survival against V. anguillarum and supporting its potential use as a disease-preventive strategy in aquaculture. Full article

Foot-and-mouth disease virus (FMDV) continues to pose a significant threat to livestock health and the global agricultural economy, particularly in endemic regions of Asia, Africa, and the Middle East. Current vaccines based on chemically inactivated FMDV present several challenges, including biosafety risks, high production costs, and limited effectiveness against emerging viral variants. To overcome these limitations, we developed virus-like particle (VLP) vaccines targeting FMDV serotypes O, A, and Asia1 using a recombinant Escherichia coli expression system. The resulting VLPs self-assembled into 25–30 nm particles with native-like morphology and antigenic properties, as confirmed by transmission electron microscopy, SDS-PAGE, and Western blot analysis. Immunogenicity was evaluated in mice and pigs using ELISA and virus neutralization tests (VNT), and protective efficacy was assessed through viral challenge studies. All VLPs induced strong serotype-specific antibody responses, with ELISA PI values exceeding 50% and significantly increased VNT titers after booster immunization. In mice, PD₅₀ values were 73.5 (A-type), 32.0 (O-type), and 55.7 (Asia1-type); in pigs, PD₅₀ values reached 10.6 (O-type) and 22.6 (Asia1-type). Notably, the vaccines induced robust immune responses even at lower antigen doses, suggesting the feasibility of dose-sparing formulations. These findings demonstrate that FMDV VLPs produced in E. coli are highly immunogenic and capable of eliciting protective immunity, highlighting their promise as safe, scalable, and cost-effective alternatives to conventional inactivated FMD vaccines. Full article

Pathogenic bacteria of the genus Leptospira are the etiological agents of leptospirosis, a disease that affects humans and animals worldwide. Despite the increasing number of studies, the mechanisms of leptospiral pathogenesis remain poorly comprehended. In this study, we report various interactions of the LigA7’-13’ and LigB1’-7’ domains with host components. The LigA7’-13’ and LigB1’-7’ were cloned into the pET28a vector, and the recombinant proteins were expressed in E. coli C43 (DE3) and E. coli BL21 (DE3), respectively. Both recombinant protein domains were expressed in soluble form and purified using nickel-chelating chromatography. The rLigA7’-13’ and rLigB1’-7’ domains exhibited binding to several types of integrins, with most interactions occurring in a dose-dependent and saturable manner, consistent with the characteristics of typical receptor-ligand interactions. The recombinant domain LigA7’-13’ demonstrated affinity for the glycosaminoglycans (GAGs) chondroitin-4-sulfate, chondroitin sulfate, heparin, chondroitin sulfate B, and heparan sulfate, while no binding was detected for LigB1’-7’ with these molecules. Both rLigA7’-13’ and rLigB1’-7’ interacted with components of the terminal complement pathway and were capable of recruiting C9 from normal human serum (NHS). These interactions may inhibit the formation of polyC9, ultimately preventing the assembly of the membrane attack complex (MAC). Collectively, our data expand the repertoire of host components that interact with rLigA7’-13’ and rLigB1’-7’, opening new avenues for understanding leptospiral immune evasion and broadening the roles of these domains in bacterial virulence. Full article

Antimicrobial resistance (AMR) is a major global concern in both human and veterinary medicine. Among antimicrobial resistance (AMR) bacteria, Extended-Spectrum Beta-Lactamases (ESBLs) pose a serious health risk because infections can be difficult to treat. These Gram-negative bacteria can be frequently found in poultry and in Italy, where such protein production is established. ESBL-producing Escherichia coli, Salmonella and Klebsiella in chicken and turkey may pose a significant public health risk due to potential transmission between poultry and humans. This review aims to assess the prevalence of ESBL-producing E. coli, Salmonella and Klebsiella phenotypically and genotypically in Italian poultry, identifying the most common genes, detection methods and potential information gaps. An initial pool of 1462 studies found in scientific databases (Web of Sciences, PubMed, etc.) was screened and 29 were identified as eligible for our review. Of these studies, 79.3% investigated both phenotypic and genotypic ESBL expression while ${bla}_{CTX-M}$, ${bla}_{TEM}$ and ${bla}_{SHV}$ were considered as targeted gene families. Large differences in prevalence were reported (0–100%). The ${bla}_{CTX-M-1}$ and ${bla}_{TEM-1}$ genes were the most prevalent in Italian territory. ESBL-producing E. coli, Salmonella and Klebsiella were frequently detected in farms and slaughterhouses, posing a potential threat to humans through contact (direct and indirect) with birds through handling, inhalation of infected dust, drinking contaminated water, ingestion of meat and meat products and the environment. Considering the frequent occurrence of ESBL-producing bacteria in Italian poultry, it is advisable to further improve biosecurity and to introduce more systematic surveillance. Additionally, the focus should be on the wild birds as they are ESBL carriers. Full article

African swine fever (ASF) is a disease that affects both domestic and wild swine. It was recently reported in the Dominican Republic and Haiti (2021), representing a substantial risk to America. The goal of this study was to produce a truncated form of the ASF-p72 recombinant protein based on the ASF strain genotype II (Georgia 2017) as well as to develop and validate a sensitive and specific ASF indirect-ELISA (iELISA) for early detection of ASF. The truncated ASF-p72 recombinant protein was successfully expressed in E. coli BL21/DE3 cells using the pET-SUMO plasmid. Bioinformatics analysis showed 100% homology among the new isolates of ASFV from genotype II. The ASF-p72-truncated protein was used to develop an iELISA, which had a high sensitivity (88%) and strong specificity (97%); the concordance index kappa was K = 0.872, indicating nearly perfect agreement compared to the WOAH confirmatory immunoperoxidase test. The validation results utilizing the reference sera panel from the OIE-ASF Reference Laboratory show the excellent detection capabilities of ASF antibodies up to a 1:1000 serum dilution. The inter-assay coefficient of variation (CV 10.4%) and intra-assay CV (2.8%) data show that the assay is precise and reproducible. This biotechnology advancement can be used to conduct future epidemiological research for ASF surveillance in ASF-free American countries. Full article

The polyphagous aphid Aphis gossypii exhibits host-specific biotypes, notably the cotton (Hap1) and cucumber (Hap3) types. While both can adapt to new hosts via zucchini induction, the underlying molecular mechanisms remain unclear. Our investigation revealed that both Hap1 and Hap3 A. gossypii underwent significant body size enlargement following host transfer to zucchini. Transcriptomic analysis revealed that zucchini-mediated host adaptation in the A. gossypii biotypes (Hap1/Hap3) involves insulin metabolism and detoxification pathways, with 17 co-differentially expressed genes (e.g., Col-I (type I collagen), CYP450 6a13, peroxidase) potentially critical for adaptation. The findings provide new insights into the molecular mechanisms regulating A. gossypii phenotypic plasticity and contribute to the development of resistance management strategies. Full article

Escherichia coli and Klebsiella pneumoniae are major contributors to the global challenge of antimicrobial resistance, posing serious threats to public health. Among current preventive strategies, conjugate vaccines that utilize bacterial surface polysaccharides have emerged as a promising and effective approach to counter multidrug-resistant strains. In this study, both the Wzy/Wzx-dependent and ABC transporter-dependent biosynthetic pathways for antigenic polysaccharides were introduced into E. coli W3110 cells. This dual-pathway engineering enabled the simultaneous biosynthesis of two structurally distinct polysaccharides within a single host, offering a streamlined and potentially scalable strategy for vaccine development. Experimental findings confirmed that both polysaccharide types were successfully produced in the engineered strains, although co-expression levels were moderately reduced. A weak competitive interaction was noted during the initial phase of induction, which may be attributed to competition for membrane space or the shared use of activated monosaccharide precursors. Interestingly, despite a reduction in plasmid copy number and transcriptional activity of the biosynthetic gene clusters over time, the overall polysaccharide yield remained stable with prolonged induction. This suggests that extended induction does not adversely affect final product output. Additionally, two glycoproteins were efficiently generated through in vivo bioconjugation of the synthesized polysaccharides with carrier proteins, all within the same cellular environment. This one-cell production system simplifies the workflow and enhances the feasibility of generating complex glycoprotein vaccines. Whole-cell proteomic profiling followed by MFUZZ clustering and Gene Ontology analysis revealed that core biosynthetic genes were grouped into two functional clusters. These genes were predominantly localized to the cytoplasm and were enriched in pathways related to translation and protein binding. Such insights not only validate the engineered biosynthetic routes but also provide a molecular basis for optimizing future constructs. Collectively, this study presents a robust synthetic biology platform for the co-expression of multiple polysaccharides in a single bacterial host. The approach holds significant promise for the rational design and production of multivalent conjugate vaccines targeting drug-resistant pathogens. Full article

Considering the absence of a widely utilized EBV vaccine, we have developed an EBV gL-gH344-Ferritin nanoparticle vaccine utilizing ferritin as a carrier. The gL-gH344-Ferritin fusion gene was synthesized and inserted into the pET30a plasmid. The expression of the fusion protein in the recombinant plasmid was verified by Western blot. Then, the gL-gH344-Ferritin subunit nanoparticle vaccine was obtained by purification of the fusion protein. BALB/c mice were immunized using a two-dose protocol. The titers of EBV specific antibodies were determined using enzyme-linked immunosorbent assay at 4, 8, and 12 weeks after the initial immunization. Moreover, the levels of EBV gL-gH344 specific splenocytes secreting interferon (IFN)-γ and interleukin (IL)-6 were determined using an enzyme-linked immunospot assay. The pET30a-gL-gH344-Ferritin prokaryotic expression plasmid was successfully constructed. gL-gH344-Ferritin was efficiently expressed in E. coli. Following immunization with gL-gH344-Ferritin, the mice sera demonstrated elevated titers of EBV specific immunoglobulin G. Moreover, after stimulating with EBV gL-gH344 specific peptides, the splenocytes of the immunized mice showed a marked tendency to secrete large amounts of IFN-γ and IL-6. The gL-gH344-Ferritin nanoparticle vaccine carrying the EBV gL-gH344 fusion protein induced robust and sustained specific humoral and cellular immune responses in mice. Full article

Phenylalanine butyramide (FBA) is a novel butyric acid derivative with favorable sensory properties, which has broad prospects in medicine and feed processing. However, there is currently limited research on the enzymatic synthesis of FBA. As is well known, lipase plays a crucial role in amide bond synthesis, but it typically requires completely anhydrous conditions. The lipase from Sphingomonas sp. HXN-200 (SpL) is the only intracellular lipase identified to date, capable of catalyzing the ammonolysis of esters or acids in an aqueous phase. In this study, we developed a method for the synthesis of FBA catalyzed by SpL in a biphasic reaction system of water and n-hexane. SpL was successfully expressed in E. coli BL21, and the optimal induction conditions were 0.4 mM IPTG and 18 h. It was ascertained that the n-hexane system containing 2% water was conducive to the reaction. Under optimized reaction conditions, 0.89 mg/mL of FBA was obtained within 15 h at 30 °C. Additionally, we found that SpL also has the ability to hydrolyze amides in the reaction of SpL catalyzing the formation of amides, so we further analyzed its catalytic mechanism. Full article

Industrial biotechnology offers a potential ecological solution for PET recycling under relatively mild reaction conditions via enzymatic degradation, particularly using the leaf branch compost cutinase (LCC) quadruple mutant ICCG. To improve the efficient downstream processing of this biocatalyst after heterologous gene expression with a suitable production host, protein crystallization can serve as an effective purification/capture step. Enhancing protein crystallization was achieved in recent studies by introducing electrostatic (and aromatic) interactions in two homologous alcohol dehydrogenases (Lb/LkADH) and an ene reductase (NspER1-L1,5) produced with Escherichia coli. In this study, ICCG, which is difficult to crystallize, was utilized for the application of crystal contact engineering strategies, resulting in ICCG mutant L50Y (ICCGY). Previously focused on the Lys-Glu interaction for the introduction of electrostatic interactions at crystal contacts, the applicability of the engineering strategy was extended here to an Arg-Glu interaction to increase crystallizability, as shown for ICCGY T110E. Furthermore, the rationale of the engineering approach is demonstrated by introducing Lys and Glu at non-crystal contacts or sites without potential interaction partners as negative controls. These resulting mutants crystallized comparably but not superior to the wild-type protein. As demonstrated by this study, crystal contact engineering emerges as a promising approach for rationally enhancing protein crystallization. This advancement could significantly streamline biotechnological downstream processing, offering a more efficient pathway for research and industry. Full article

Gossypol is a polyphenolic toxic compound present in cotton plants. To determine whether the candidate cytochrome P450BM3 enzymes could reduce gossypol in vitro, functional recombinant cytochrome P450BM3 enzymes were successfully expressed in E. coli. Site-directed mutagenesis generated mutants (R162H, R162K, Q129H, Q129N) to explore structural determinants of catalytic efficiency. Both wild-type P450BM3 and mutants exhibited significant ability to reduce gossypol levels, with R162H and R162K showing 33.4% and 24.2% reduced catalytic efficiency compared with the wild-type enzyme, respectively. Q129H and Q129N mutants maintained comparable catalytic efficiency to the wild type. Metabolomic profiling revealed two distinct reducing pathways catalyzed by wild-type P450BM3 and its mutants (R162H/Q129H), involving decarboxylation, hydroxylation, and C-C bond cleavage. This study demonstrated the feasibility of P450BM3 as a highly efficient biocatalyst for reducing gossypol levels, speculated that Arg162 might be a critical active residue, and hypothesized the potential pathways by which P450BM3 catalyzes the reduction of gossypol content, thereby providing a theoretical foundation for the enzymatic reduction of gossypol. Full article

Background: Enterohemorrhagic Escherichia coli (EHEC) is a considerable public health concern associated with several foodborne outbreaks of bloody diarrhea (BD) and the potentially lethal hemolytic uremic syndrome (HUS), the pathophysiology of which is attributable to the Shiga toxin (Stx) produced by this bacterium. In most patients, supportive treatment will be sufficient; however, in some cases, antibiotic treatment may be necessary. Most antibiotics are not recommended for EHEC infection treatment, particularly those that kill the bacteria, since this triggers the release of Stx in the body, inducing or worsening HUS. Azithromycin, which prevents the release of Stx and is a weaker inducer of the SOS system, has been successfully used to reduce EHEC shedding. It is necessary to identify compounds that eliminate EHEC without inducing Stx release. The use of natural compounds such as curcumin (CUR), a polyphenol derived from turmeric, has been highlighted as an alternative bactericidal treatment approach. Objective: The objective of this study was to establish the effect of CUR and its interactions with selected antibiotics on resistant EHEC O157/H7/EDL933. Methods: Bacterial cultures were exposed to CUR at three different concentrations (110, 220, and 330 µg/mL) and 1.2% DMSO, and the antimicrobial activity of CUR was assessed by measuring the optical density at 600 nm (OD600). The synergy of CUR and the antibiotics was determined with the FIC method. RT-PCR was performed to determine the expression levels of the bla_CTX-M-15, catA1, acrAB-tolC stx2A, and stx2B genes. Results: Our data indicate that CUR did not affect the growth of EHEC, but when combined with the antibiotics, it acted as a bacterial resistance breaker. Synergistic combinations of CUR and cefotaxime or chloramphenicol significantly reduced colony counts. Conclusions: Our findings support the potential of CUR as a sensitizer or in combination therapy against EHEC. Full article

The intestinal health and functionality of animals play pivotal roles in nutrient digestion and absorption, as well as in maintaining defense against pathogenic invasions. These biological processes are modulated by various determinants, including husbandry conditions, dietary composition, and gut microbial ecology. The excessive use of anthropogenic antibiotics may disrupt intestinal microbiota composition, potentially leading to dysbiosis that directly compromises host homeostasis. While Lactobacillus species are recognized for their immunomodulatory properties, their precise mechanisms in regulating host anti-inflammatory gene expression and influencing mucosal layer maturation, particularly regarding E. coli colonization resistance, require further elucidation. To investigate the regulatory mechanisms of Lactobacillus in relation to intestinal architecture and function during E. coli infection, we established a colonic infection model using Bal b/c mice, conducting systematic analyses of intestinal morphology, inflammatory mediator profiles, and microbial community dynamics. Our results demonstrate that Lactobacillus supplementation (Pediococcus acidilactici) effectively mitigated E. coli O78-induced enteritis, with co-administration during infection facilitating the restoration of physiological parameters, including body mass, intestinal histoarchitecture, and microbial metabolic functions. Microbiome profiling revealed that the Lactobacillus intervention significantly elevated Lactococcus abundance while reducing Weissella populations (p < 0.05), concurrently enhancing metabolic pathways related to nutrient assimilation and environmental signal processing (including translation mechanisms, ribosomal biogenesis, amino acid transport metabolism, and energy transduction systems; p < 0.05). Mechanistically, Lactobacillus administration attenuated E. coli-induced intestinal pathology through multiple pathways: downregulating pro-inflammatory cytokine expression (IL-1β, IL-1α, and TNF-α), upregulating epithelial junctional complexes (Occludin, Claudin-1, and ZO-1), and stimulating mucin biosynthesis (MUC1 and MUC2; p < 0.05). These modifications collectively enhanced mucosal barrier integrity and promoted epithelial maturation. This investigation advances our comprehension of microbiota–host crosstalk during enteropathogenic infections under probiotic intervention, offering valuable insights for developing novel nutritional strategies and microbial management protocols in animal husbandry. Full article

Photothermal therapy combines the effects of near-infrared laser (NIR laser) and strong light-absorbing materials to combat pathogens and unwanted biofilms. Graphene derivatives have a negative effect on microorganisms, and the combination of NIR laser irradiation and carbon nanomaterials (CNMs) can enhance their antibacterial effect. This investigation is devoted to the determination of the expression level of bacterial stress response genes (soxS and rpoS) under graphene oxide (GO), reduced graphene oxide (rGO), and NIR laser irradiation (1270 nm). GO, rGO and NIR laser irradiation separately and irradiation in the presence of graphene derivatives cause an increase in the expression level of rpoS associated with the general stress response of bacteria. GO and rGO do not change the expression level of soxS associated with the cell response to oxidative stress, and decrease it in the presence of a strong oxidizing agent paraquat (PQ). The expression of soxS increases under laser irradiation, but decreases under NIR laser irradiation in combination with graphene derivatives. The effect of GO, rGO, and NIR laser irradiation on the formation and eradication of E. coli biofilms was studied. NIR laser with GO and rGO suppresses the metabolic rate and decreases the intracellular ATP content by 94 and 99.6%, respectively. CNMs are shown to reduce biofilm biomass and the content of extracellular polymeric substances (EPSs), both exopolysaccharides and protein in the biofilm matrix. Graphene derivatives in combination with NIR laser irradiation may be an effective means of combating emerging and mature biofilms of Gram-negative bacteria. Full article

Campylobacter spp. is one of the most prevalent causes of zoonotic foodborne infections associated with diarrhea in humans. The growing threat of antibiotic resistance calls for innovative approaches. The antimicrobial lipopeptide brevibacillin produced by Brevibacillus laterosporus and its synthetic analog brevibacillin Thr1 showed promising activity against Salmonella and E. coli. The latter is a 1602.13 Da positively charged (+3) synthetic peptide of 13 residues that showed reduced cytotoxicity (IC₅₀ of 32.2 µg/mL against Caco-2 cells) and hemolytic activity (1.2% hemolysis at 128 µg/mL) compared to the native peptide. It contains an N-terminal L-isoleucic fatty acid chain and four non-proteinogenic amino acids and ends with valinol at its C-terminus. One key structural modification is the substitution of α,β-dehydrobutyric acid with threonine. We investigated the antimicrobial potential of the synthetic brevibacillin Thr1 analog against a collection of 44 clinical Campylobacter spp. that were obtained from two reference laboratories. Susceptibility testing revealed marked resistance to ciprofloxacin, tetracycline, and ampicillin among the strains, with more than half expressing a multidrug-resistant phenotype. The genomes of the 44 strains were sequenced to study the genes responsible for their antimicrobial resistance. Tetracycline resistance was associated with tet(O), ciprofloxacin resistance with mutations in gyrA and regulatory sequences modulating the expression of an efflux system, and aminoglycoside resistance with genes of the aph family. The brevibacillin Thr1 analog was produced by chemical synthesis, and evaluation of its activity against a subset of clinical strains by microdilution revealed minimum inhibitory concentration and minimum bactericidal concentration ranging from 8 µg/mL to 64 µg/mL. The peptide was active against multidrug-resistant isolates with a bactericidal effect. Of note, despite numerous attempts, it proved impossible to select Campylobacter spp. for resistance to the brevibacillin Thr1 analog. These results underline the potential of lipopeptides, notably brevibacillin, as antimicrobial alternatives against antibiotic-resistant Campylobacter bacterial infections. Full article