ABSTRACT
BACKGROUND: Antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), pose a significant threat to public health. Existing detection methods, like cultivation-based techniques, demand significant time and labor, while molecular diagnostic techniques, such as PCR, necessitate sophisticated instrumentation and skilled personnel. Although previous multiplex loop-mediated isothermal amplification assays based on fluorescent dyes (mfLAMP) offer simplicity and cost-effectiveness, they are prone to false-positive results. Therefore, developing a rapid and efficient multiplex assay for high-sensitivity MRSA is imperative to create a practical diagnostic tool for point-of-care testing. RESULTS: Here, we developed a mfLAMP combined with a lateral flow assay (mfLAMP-LFA) for the visual and simultaneous detection of the mecA (PBP2a-specific marker) and nuc (S. aureus-specific marker) genes in MRSA. We optimized mfLAMP-LFA using graphene oxide (GO)-based purification and specific DNA probes and evaluated its sensitivity, specificity, and stability. Utilizing GO to mitigate false-positive results by acting as a trap for free DNA probes, the mfLAMP-LFA method successfully identified mecAf and nucf-probes, exhibiting distinct red, green, and yellow fluorescence signals. The detection sensitivity of the developed mfLAMP-LFA method (1 CFU mL-1 in phosphate-buffered saline (PBS)) was comparable to other highly sensitive MRSA detection methods (1 CFU mL-1 in PBS). Furthermore, the method demonstrated specificity for MRSA, detecting it in irrigation water samples within the desired range and achieving reliable recovery rates from spiked samples. SIGNIFICANCE: This novel strategy is the first to incorporate GO into mfLAMP-LFA, enabling specific and sensitive MRSA detection and advancing rapid bacterial detection. This assay facilitates MRSA diagnostics, contributing to improved public health and food safety by delivering rapid, cost-effective point-of-care results. It enables the simultaneous detection of multiple bacteria, even in irrigation water samples artificially inoculated with MRSA, which contain aerobic bacteria at 2.7 × 102 CFU mL-1.
Subject(s)
Bacterial Proteins , Methicillin-Resistant Staphylococcus aureus , Micrococcal Nuclease , Nucleic Acid Amplification Techniques , Penicillin-Binding Proteins , Methicillin-Resistant Staphylococcus aureus/isolation & purification , Methicillin-Resistant Staphylococcus aureus/genetics , Penicillin-Binding Proteins/genetics , Nucleic Acid Amplification Techniques/methods , Micrococcal Nuclease/genetics , Bacterial Proteins/genetics , Fluorescence , Molecular Diagnostic Techniques/methods , Fluorescent Dyes/chemistry , GraphiteABSTRACT
In this study, a molecular beacon (MB) was designed for colorimetric loop-mediated isothermal amplification (cLAMP). The length of complementary bases on the MB, guanine and cytosine content (GC content), and hybridization sites of complementary bases were investigated as key factors affecting the design of the MB. We designed MBs consisting of 10, 15, and 20 complementary bases located at both ends of the HRPzyme. In the case of the long dumbbell DNA structure amplified from the hlyA gene of Listeria monocytogenes, possessing a flat region (F1c-B1) of 61 base pairs (bp), an MB was designed to intercalate into the flat region between the F1c and B1 regions of the LAMP amplicons. In the case of the short dumbbell DNA structure amplified from the bcfD gene of Salmonella species possessing a flat region (F1c-B1) length of 6 bp, another MB was designed to intercalate into the LoopF or LoopB regions of the LAMP amplicons. The results revealed that the hybridization site of the MB on the LAMP amplicons was not crucial in designing the MB, but the GC content was an important factor. The highest hybridization efficiencies for LAMP amplicons were obtained from hlyA gene-specific and bcfD gene-specific MBs containing 20- and 15-base complementary sequences, respectively, which exhibited the highest GC content. Therefore, designing MBs with a high GC content is an effective solution to overcome the low hybridization efficiency of cLAMP assays. The results obtained can be used as primary data for designing MBs to improve cLAMP accessibility.
Subject(s)
Colorimetry , Listeria monocytogenes , Nucleic Acid Amplification Techniques , Nucleic Acid Amplification Techniques/methods , Colorimetry/methods , Listeria monocytogenes/genetics , Listeria monocytogenes/isolation & purification , DNA, Bacterial/genetics , DNA, Bacterial/analysis , Salmonella/genetics , Salmonella/isolation & purification , Nucleic Acid Hybridization/methods , Molecular Diagnostic TechniquesABSTRACT
Eukaryotes have evolved sophisticated post-translational modifications to regulate protein function and numerous biological processes, including ubiquitination controlled by the coordinated action of ubiquitin-conjugating enzymes and deubiquitinating enzymes (Dubs). However, the function of deubiquitination in pathogenic fungi is largely unknown. Here, the distribution of Dubs in the fungal kingdom was surveyed and their functions were systematically characterized using the phytopathogen Fusarium graminearum as the model species, which causes devastating diseases of all cereal species world-wide. Our findings demonstrate that Dubs are critical for fungal development and virulence, especially the ubiquitin-specific protease 15 (Ubp15). Global ubiquitome analysis and subsequent experiments identified three important substrates of Ubp15, including the autophagy-related protein Atg8, the mitogen-activated protein kinase Gpmk1, and the mycotoxin deoxynivalenol (DON) biosynthetic protein Tri4. Ubp15 regulates the deubiquitination of the Atg8, thereby impacting its subcellular localization and the autophagy process. Moreover, Ubp15 also modulates the deubiquitination of Gpmk1 and Tri4. This modulation subsequently influences their protein stabilities and further affects the formation of penetration structures and the biosynthetic process of DON, respectively. Collectively, our findings reveal a previously unknown regulatory pathway of a deubiquitinating enzyme for fungal virulence and highlight the potential of Ubp15 as a target for combating fungal diseases.
Subject(s)
Fusarium , Mycotoxins , Virulence , Fungal Proteins/metabolism , Mycotoxins/metabolism , Deubiquitinating Enzymes/metabolism , Plant Diseases/microbiologyABSTRACT
The Med1 transcriptional coactivator is a crucial component of the Mediator middle complex, which regulates the expression of specific genes involved in cell development, differentiation, reproduction, and homeostasis. The Med1 LxxLL motif, a five-amino-acid peptide sequence, is essential for Med1-mediated gene expression. Our previous study revealed that the disruption of the Med1 subunit leads to a significant increase in fumonisin B1 (FB1) production in the maize pathogen Fusarium verticillioides. However, our understanding of how Med1 regulates FB1 biosynthesis in F. verticillioides, particularly through the Med1 LxxLL motifs, remains limited. To characterize the role of LxxLL motifs, we generated a series of Med1 LxxLL deletion and amino acid substitution mutants. These mutants exhibited impaired mycelial growth and conidia germination while demonstrating enhanced conidia production and virulence. Similar to the Med1 deletion mutant, Med1 LxxLL motif mutants also exhibited increased FB1 biosynthesis in F. verticillioides. Proteomic profiling revealed that the Med1 LxxLL motif regulated the biosynthesis of several key substances that affected FB1 production, including starch and carotenoid. Subsequent studies demonstrated that the production of amylopectin, which is strongly linked to FB1 biosynthesis, was significantly increased in Med1 LxxLL motif mutants. In addition, the disruption of carotenoid metabolic genes decreased carotenoid content, thus stimulating FB1 biosynthesis in F. verticillioides. Taken together, our results provide valuable insights into how the Med1 LxxLL motif regulates FB1 biosynthesis in the mycotoxigenic fungus F. verticillioides.
Subject(s)
Fumonisins , Fusarium , Fumonisins/metabolism , Proteomics , Fusarium/metabolism , Carotenoids/metabolism , Zea mays/microbiologyABSTRACT
Processed foods containing pork fat tissue to improve flavor and gain economic benefit may cause severe issues for Muslims, Jews, and vegetarians. This study aimed to develop an indirect enzyme-linked immunosorbent assay (iELISA) based on a monoclonal antibody specific to thermal stable-soluble protein in pork fat tissue and apply it to detect pork fat tissue in heat-processed (autoclave, steam, roast, and fry) beef meatballs. To develop a sensitive iELISA, the optimal sample pre-cooking time, coating conditions, primary and secondary dilution time, and various buffer systems were tested. The change in the iELISA sensitivity with different 96-well microtiter microplates was confirmed. The detection limit of iELISA performed with an appropriate microplate was 0.015% (w/w) pork fat in raw and heat-treated beef. No cross-reactions to other meats or fats were shown. These results mean that the iELISA can be used as an analytical method to detect trace amounts of pork fat mixed in beef.
ABSTRACT
In this study, the influence of microenvironments on antibody production of hybridoma cells was analyzed using six types of functionalized parylene films, parylene-N and parylene-C (before and after UV radiation), parylene-AM, and parylene-H, and using polystyrene as a negative control. Hybridoma cells were cultured on modified parylene films that produced a monoclonal antibody against the well-known fungal toxin ochratoxin-A. Surface properties were analyzed for each parylene film, such as roughness, chemical functional groups, and hydrophilicity. The proliferation rate of the hybridoma cells was observed for each parylene film by counting the number of adherent cells, and the total amount of produced antibodies from different parylene films was estimated using indirect ELISA. In comparison with the polystyrene, the antibody-production by parylene-H and parylene-AM was estimated to be observed to be as high as 210-244% after the culture of 24 h. These results indicate that the chemical functional groups of the culture plate could influence antibody production. To analyze the influence of the microenvironments of the modified parylene films, we performed cell cycle analysis to estimate the ratio of the G0/G1, S, and G2/M phases of the hybridoma cells on each parylene film. From the normalized proportion of phases of the cell cycle, the difference in antibody production from different surfaces was considered to result from the difference in the proliferation rate of hybridoma cells, which occurred from the different physical and chemical properties of the parylene films. Finally, protein expression was analyzed using an mRNA array to determine the effect of parylene films on protein expression in hybridoma cells. The expression of three antibody production-related genes (CD40, Sox4, and RelB) was analyzed in hybridoma cells cultured on modified parylene films.
Subject(s)
Antibody Formation , Polystyrenes , Hybridomas , Antibodies, MonoclonalABSTRACT
Plant pathogens are challenged by host-derived iron starvation or excess during infection, but the mechanism through which pathogens counteract iron stress is unclear. Here, we found that Fusarium graminearum encounters iron excess during the colonization of wheat heads. Deletion of heme activator protein X (FgHapX), siderophore transcription factor A (FgSreA) or both attenuated virulence. Further, we found that FgHapX activates iron storage under iron excess by promoting histone H2B deubiquitination (H2B deub1) at the promoter of the responsible gene. Meanwhile, FgSreA is shown to inhibit genes mediating iron acquisition during iron excess by facilitating the deposition of histone variant H2A.Z and histone 3 lysine 27 trimethylation (H3K27 me3) at the first nucleosome after the transcription start site. In addition, the monothiol glutaredoxin FgGrx4 is responsible for iron sensing and control of the transcriptional activity of FgHapX and FgSreA via modulation of their enrichment at target genes and recruitment of epigenetic regulators, respectively. Taken together, our findings elucidated the molecular mechanisms for adaptation to iron excess mediated by FgHapX and FgSreA during infection in F. graminearum and provide novel insights into regulation of iron homeostasis at the chromatin level in eukaryotes.
Subject(s)
Fusarium , Histones , Iron , Chromatin , Histones/genetics , Histones/metabolism , Iron/metabolism , Nucleosomes , Siderophores/genetics , Fusarium/metabolismABSTRACT
Food allergy represents a severe problem for many societies, including sensitive populations, academies, health authorities, and the food industry. Peanut allergy occupies a special place in the food allergy spectrum. To prevent consumption by consumers suffering from a peanut allergy, a rapid and sensitive detection method is essential to identify unintended peanut adulteration in processed foods. In this study, we produced four monoclonal antibodies (MAbs; RO 3A1-12, PB 4C12-10, PB 5F9-23, and PB 6G4-30) specific to thermo-stable and soluble proteins (TSSPs) of peanut and developed an enzyme-linked immunosorbent assay (ELISA) based on the MAbs. Among them, PB 5F9-23 MAb was firmly bound to Ara h 1, and other MAbs strongly reacted to Ara h 3 in the Western blot analysis. An antibody cocktail solution of the MAbs was used to enhance the sensitivity of an indirect ELISA, and the limit of detection of the indirect ELISA based on the antibody cocktail solution was 1 ng/ml and improved compared to the indirect ELISA based on the single MAb (11 ng/ml). The cross-reaction analysis revealed the high specificity of developed MAbs to peanut TSSPs without cross-reaction to other food allergens, including nuts. Subsequently, analyzing processed foods by indirect ELISA, all foods labeled as containing peanuts in the product description were confirmed to be positive. The results indicate that the developed antibodies exhibit high specificity and sensitivity to peanuts and can be used as bio-receptors in immunoassays or biosensors to detect intentional or unintentional adulteration of peanuts in processed foods, particularly heat-processed foods.
Subject(s)
Food Hypersensitivity , Peanut Hypersensitivity , Antibodies, Monoclonal , Arachis , Plant Proteins , Peanut Hypersensitivity/diagnosis , Enzyme-Linked Immunosorbent Assay/methods , AllergensABSTRACT
Contamination by Escherichia coli O157:H7 is considered a threat in the livestock and food industries. Therefore, it is necessary to develop methods for the convenient and rapid detection of Shiga-toxin-producing E. coli O157:H7. This study aimed to develop a colorimetric loop-mediated isothermal amplification (cLAMP) assay using a molecular beacon to rapidly detect E. coli O157:H7. Primers and a molecular beacon were designed for targeting the Shiga-toxin-producing virulence genes (stx1 and stx2) as molecular markers. Additionally, Bst polymerase concentration and amplification conditions for bacterial detection were optimized. The sensitivity and specificity of the assay were also investigated and validated on artificially tainted (100-104 CFU/g) Korean beef samples. The cLAMP assay could detect 1 × 101 CFU/g at 65 °C for both genes, and the assay was confirmed to be specific for E. coli O157:H7. The cLAMP takes about an hour and does not require expensive devices (e.g., thermal cycler and detector). Hence, the cLAMP assay proposed herein can be used in the meat industry as a fast and simple way to detect E. coli O157:H7.
Subject(s)
Escherichia coli O157 , Animals , Cattle , Escherichia coli O157/genetics , Colorimetry , Nucleic Acid Amplification Techniques/methods , Molecular Diagnostic Techniques/methods , Food MicrobiologyABSTRACT
Buckwheat is considered a severe food allergen, and its adulteration and mislabeling cause serious health risks. For protecting consumers suffering from buckwheat allergy, a high-sensitivity detection method is necessary to accurately identify intentional or unintentional adulteration of buckwheat in processed foods. The study revealed that buckwheat contains a significant amount of thermally stable-soluble proteins (TSSPs), which keep antigenicity even after heat treatment. Therefore, we used TSSPs to produce three monoclonal antibodies (MAbs) specific to buckwheat. A MAbs cocktail solution was subjected to enhance the sensitivity of an indirect enzyme-linked immunosorbent assay (iELISA), and the LOD was 1 ng/mL. The MAbs cocktail solution based-iELISA can successfully detect buckwheat adulterated in processed foods. The results suggested that the TSSPs in buckwheat can be used as suitable immunogens, and MAbs produced can be used as bioreceptor to develop immunoassays and biosensors for detecting buckwheat in food facilities and processed foods.
Subject(s)
Biological Products , Fagopyrum , Food Hypersensitivity , Antibodies, Monoclonal , Enzyme-Linked Immunosorbent Assay/methods , Immunoassay , AllergensABSTRACT
Fumonisin contamination of corn caused by Fusarium verticillioides is a major concern worldwide. While key genes involved in fumonisin biosynthesis are known, the location within the fungal cell where this process occurs has yet to be fully characterized. In this study, three key enzymes, i.e., Fum1, Fum8, and Fum6, associated with early steps of fumonisin biosynthesis pathway, were tagged with GFP, and we examined their cellular localization. Results showed that these three proteins co-localized with the vacuole. To further understand the role of the vacuole in fumonisin B1 (FB1) biosynthesis, we disrupted two predicted vacuole associated proteins, FvRab7 and FvVam7, resulting in a significant reduction of FB1 biosynthesis and a lack of Fum1-GFP fluorescence signal. Furthermore, we used the microtubule-targeting drug carbendazim to show that proper microtubule assembly is critical for proper Fum1 protein localization and FB1 biosynthesis. Additionally, we found that α1 tubulin is a negative regulator in FB1 biosynthesis. We concluded that vacuole proteins with optimized microtubule assembly play a crucial role in proper Fum1 protein localization and fumonisin production in F. verticillioides.
ABSTRACT
A one-step immunoassay based on filtration was presented, which used microbeads for target analyte detection and filters with appropriate pore sizes to distinguish the complexity of target analyte and microbeads. For effective bacterial detection, the microbead size and the filter's pore size must be optimized. The optimal concentrations of the enzyme (urease) and antibody were determined at the maximum absorbance change, that is, the maximum pH change. The pH change was measured using a field-effect transistor (FET). The correlation between pH change and threshold voltage was estimated to be 21.7 mV/pH, and the correlation between pH change and the source-drain current was estimated to be -379 nA/pH. For the one-step immunoassay, antibodies against target bacteria were isolated from horse serum by filtration, and these antibodies were estimated to have a sufficiently high specificity to overcome cross-reactivity among five types of food poisoning-related bacteria: Escherichia coli O157, Salmonella typhimurium, Listeria monocytogenes, Bacillus cereus, and Staphylococcus aureus. Finally, the FET-based one-step immunoassay was demonstrated for five types of food poisoning-related bacteria in human serum.
Subject(s)
Food Microbiology , Foodborne Diseases , Humans , Immunoassay , Salmonella typhimurium , Bacteria , Foodborne Diseases/diagnosis , Antibodies , Colony Count, Microbial , Food Contamination/analysisABSTRACT
This study investigated the antimicrobial and antibiofilm efficacy of separate and combined treatments of Lactobacillus curvatus B67-produced postbiotic and the polyphenolic flavanol quercetin against Listeria monocytogenes and Salmonella enterica ser. Typhimurium. The antimicrobial potentiality of the postbiotic was chiefly associated with organic acids (e.g., lactic and acetic acids). At sub-minimum inhibitory concentration (1/2 MIC), the postbiotic and quercetin effectively reduced the pathogenic biofilm cells on processed pork sausage and meat-processing surfaces (e.g., stainless-steel and rubber). Moreover, the postbiotic exhibited strong residual antimicrobial efficacy over diverse pH and temperature ranges. In addition, the combination of postbiotic with quercetin increased the leakage of pathogenic intracellular metabolites (e.g., nucleic acids and protein) and inhibited pathogenic biofilm formation on both biotic and abiotic surfaces. Therefore, this study confirmed that lactic acid bacteria-derived postbiotic and plant-derived quercetin could be used as potential alternative bioprotective agents in the meat processing industry.
Subject(s)
Listeria monocytogenes , Salmonella enterica , Lactobacillus , Quercetin/pharmacology , Food Preservation , Meat , Food MicrobiologyABSTRACT
Fusarium verticillioides is a key maize pathogen and produces fumonisins, a group of mycotoxins detrimental to humans and animals. Unfortunately, our understanding on how this fungus interacts with maize to trigger mycotoxin biosynthesis is limited. We performed a systematic computational network-based analysis of large-scale F. verticillioides RNA-seq datasets to identify gene subnetwork modules associated with virulence and fumonisin regulation. F. verticillioides was inoculated on two different maize lines, moderately resistant line hybrid 33K44 and highly susceptible line maize inbred line B73, to generate time-course RNA-Seq data. Among the highly discriminative subnetwork modules, we identified a putative hub gene FvLCP1, which encodes a putative a type-D fungal LysM protein with a signal peptide, three LysM domains, and two chitin binding domains. FvLcp1 is a unique protein that harbors these domains amongst five representative Fusarium species. FvLcp1 is a secreted protein important for fumonisin production with the LysM domain playing a critical role. The chitin-binding domain was essential for in vitro chitin binding. Using Magnaporthe oryzae, we learned that FvLcp1 accumulates in appressoria, suggesting that FvLcp1 is involved in host recognition and infection. Full length FvLcp1 suppressed BAX-triggered plant cell death in Nicotiana benthamiana. This unique type-D LysM secreted protein with a chitin-binding domain in F. verticillioides was shown to be potentially involved in suppressing host cell death and promoting fumonisin biosynthesis while the pathogen colonizes maize kernels.
Subject(s)
Fumonisins , Fusarium , Mycotoxins , Chitin/metabolism , Fumonisins/analysis , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression Profiling , Humans , Mycotoxins/metabolism , Plant Diseases/microbiology , Protein Sorting Signals/genetics , Zea mays/microbiology , bcl-2-Associated X Protein/geneticsABSTRACT
Noroviruses (NoVs) are the most common causes of epidemic gastroenteritis, responsible for at least 50% of all gastroenteritis outbreaks worldwide and significant causes of foodborne illness. In the USA, approximately 21 million illnesses attributable to NoVs have annually occurred. Therefore, there is a great demand to develop a rapid, low-cost, and accurate detection method for NoVs. This study first reported colorimetric helicase-dependent amplification (HDA) methods based on specific primers integrated with HRPzyme for the rapid and sensitive detection of NoV GI and GII. The colorimetric HDA methods exhibited a detection limit of 10 copies mL-1 of each NoV GI and GII and were confirmed to be specific to each NoV GI and GII. The period required to complete the HDA method was 2 h, including a step of RNA extraction and cDNA synthesis without expensive instruments such as a thermal cycler and detector. The cutoff value of the method for the oyster artificially inoculated with a known amount of NoV was all 102 copies g-1 for NoV GI and GII. Therefore, the HDA method developed in this study can be useful tool for the on-site detection of NoVs in food samples.
Subject(s)
Caliciviridae Infections , Gastroenteritis , Norovirus , Caliciviridae Infections/diagnosis , Caliciviridae Infections/epidemiology , Colorimetry , DNA Primers/genetics , Gastroenteritis/epidemiology , Genotype , Humans , Norovirus/genetics , Phylogeny , RNA, Viral/geneticsABSTRACT
One-step homogeneous immunoassay was developed for detecting influenza viruses A and B (Inf-A and Inf-B) using the switching peptide H2. As the fluorescence-labeled switching peptide dissociated from the binding pocket of detection antibodies, the fluorescence signal could be directly generated by the binding of Inf-A and Inf-B without washing (i.e., one-step immunoassay). For the one-step homogeneous immunoassay with detection antibodies in solution, graphene was labeled with the antibodies as a fluorescence quencher. To test the feasibility of the homogeneous one-step immunoassay, the stability of the antibody complex with the switching peptide was evaluated under different pH and salt conditions. The one-step homogeneous immunoassay with switching peptide was conducted using influenza virus antigens in phosphate-buffered saline and real samples with inactivated Inf-A and Inf-B spiked in serum. Finally, the one-step homogeneous immunoassay results were compared with those of commercially available lateral flow immunoassays.
ABSTRACT
In this study, a homogeneous one-step immunoassay based on switching peptides is presented for the detection of influenza viruses A and B (Inf-A and Inf-B, respectively). The one-step immunoassay represents an immunoassay method that does not involve any washing steps, only treatment of the sample. In this method, fluorescence-labeled switching peptides quantitatively dissociate from the antigen-binding site of immunoglobulin G (IgG). In particular, the one-step immunoassay based on soluble detection antibodies with switching peptides is called a homogeneous one-step immunoassay. The immunoassay developed uses switching peptides labeled with two types of fluorescence dyes (FAM and TAMRA) and detection antibodies labeled with two types of fluorescence quenchers (TQ2 for FAM and TQ3 for TAMRA). The optimal switching peptides for the detection of Inf-A and Inf-B have been selected as L1-peptide and H2-peptide. The interactions between the four kinds of switching peptides and IgG have been analyzed using computational docking simulation and SPR biosensor. The location of labeling for the fluorescence quenchers has been determined based on the distance between the fluorescence dyes of the switching peptides and the fluorescence quenchers, calculated on the basis of the efficiency of fluorescence quenching, using the Förster equation. To demonstrate the feasibility of the one-step immunoassay, binding constants (KD) have been calculated for detection antibodies against Inf-A and Inf-B with target antigens (Inf-A and Inf-B) and switching peptides (L1- and H2-peptides), using an isotherm model. The immunoassay has been demonstrated to be feasible using antigens as well as real samples of Inf-A and Inf-B with a critical cycle number (Ct). The immunoassay has also been compared to other commercially available rapid test kits for Inf-A and Inf-B and found to be far more sensitive for detection of Inf-A and Inf-B over the entire detection range.
Subject(s)
Orthomyxoviridae , Antigens , Fluorescent Dyes/chemistry , Immunoassay/methods , Immunoglobulin G , Peptides/chemistryABSTRACT
Foodborne pathogen-mediated biofilms in food processing environments are severe threats to human lives. In the interest of human and environmental safety, natural substances with antimicrobial properties and generally regarded as safe (GRAS) status are the futuristic disinfectants of the food industry. In this study, the efficacy of bioactive, soluble products (metabolic by-products) from lactic acid bacteria (LAB) and plant-derived essential oils (EO) were investigated as biocidal agents. The postbiotic produced by kimchi-derived Leuconostoc mesenteroides J.27 isolate was analyzed for its metabolic components to reveal its antimicrobial potential against three pathogenic microorganisms (Vibrio parahaemolyticus, Pseudomonas aeruginosa, and Escherichia coli). Additionally, the efficacy of food-grade EO (eugenol and thymol, respectively) was also assessed in our study. Determination of the minimum inhibitory concentration (MIC) of postbiotic and EO against three tested pathogens revealed that the sub-MIC (0.5 MIC) of postbiotic and EO could efficiently inhibit the biofilm formation on both seafood (squid) and seafood-processing surfaces (rubber and low-density polyethylene plastic). Moreover, the polymerase chain reaction (PCR) analysis confirmed that the LAB J.27 isolate possesses bacteriocin- and enzyme-coding genes. The residual antibacterial activity of the produced postbiotic was maintained over a diverse pH range (pH 1-6) but was entirely abolished at neutral or higher pH values. However, the activity was unaffected by exposure to high temperatures (100 and 121 °C) and storage (30 days). Notably, the leakage of intracellular metabolites, damage to DNA, and the down-regulation of biofilm-associated gene expression in the pathogens increased significantly (p > 0.05) following the combination treatment of postbiotic with thymol compared to postbiotic with eugenol. Nonetheless, all in vitro results indicated the prospective use of combining Leu. mesenteroides J.27-derived postbiotic with both EO as a "green preservative" in the seafood industry to inhibit the formation of pathogenic microbial biofilms.
Subject(s)
Anti-Infective Agents , Leuconostoc mesenteroides , Oils, Volatile , Vibrio parahaemolyticus , Anti-Bacterial Agents/pharmacology , Anti-Infective Agents/pharmacology , Biofilms , Escherichia coli , Eugenol , Humans , Oils, Volatile/pharmacology , Plant Oils/pharmacology , Prospective Studies , Pseudomonas aeruginosa , Seafood , Thymol/pharmacologyABSTRACT
Mediator is a nucleus-localized, multisubunit protein complex highly conserved across eukaryotes. It interacts with RNA polymerase II transcription machinery as well as various transcription factors to regulate gene expression. However, systematic characterization of the Mediator complex has not been performed in filamentous fungi. In our study, the goal was to investigate key biological functions of Mediator subunits in a mycotoxigenic plant pathogen Fusarium verticillioides. Although there is some level of divergence in the constituent subunits, the overall structure was conserved between Saccharomyces cerevisiae and F. verticillioides. We generated 11 Mediator subunit deletion mutants and characterized vegetative growth, conidia formation, environmental stress response, carbon and fatty acid use, virulence, and fumonisin B1 (FB1) biosynthesis. Each Mediator subunit deletion mutant showed deficiencies in at least three of the phenotypes tested, suggesting that each subunit has different principal functions in F. verticillioides development, metabolism, and virulence. The deletion of FvMed1 led to increased FB1 production, and we confirmed that FvMed1 is transported from the nucleus to the cytoplasm under fumonisin-producing conditions. Taken together, our study characterized various important functional roles for Mediator subunits in F. verticillioides and suggests that select subunits can perform unique cytoplasmic functions independent of the core Mediator in fungal nucleus.
Subject(s)
Fumonisins , Fusarium , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Plant Diseases/microbiology , Secondary Metabolism , Zea mays/microbiologyABSTRACT
The highly sensitive detection of peanut allergens (PAs) using silicon-based electrolyte-gated transistors (Si-EGTs) was demonstrated. The Si-EGT was made using a top-down technique. The fabricated Si-EGT showed excellent intrinsic electrical characteristics, including a low threshold voltage of 0.7 V, low subthreshold swing of <70 mV/dec, and low gate leakage of <10 pA. Surface functionalization and immobilization of antibodies were performed for the selective detection of PAs. The voltage-related sensitivity (SV) showed a constant behavior from the subthreshold regime to the linear regime. The current-related sensitivity (SI) was high in the subthreshold regime and then significantly decreased as the drain current increased. The limit of detection (LOD) was calculated to be as low as 25 pg/mL based on SI characteristics, which is the lowest value reported to date in the literature for various sensor methodologies. The Si-EGT showed selective detection of PA through a non-specific control test. These results confirm that Si-EGT is a high-sensitivity and low-power biosensor for PA detection.