RESUMEN
Although Vibrio parahaemolyticus (V. parahaemolyticus) is a pathogen frequently found in seafood, there is a possibility of its presence in other foods, such as dairy products. The main virulence factors of V. parahaemolyticus are thermostable direct hemolysins (TDHs) which are lethal toxins, so it is necessary to establish qualitative and quantitative methods for determining TDHs. HPLC-ESI-TOF was employed to establish a method for identifying TDHs. The identification and quantification ions of TDHs were confirmed by HPLC-ESI-TOF. The method was developed for detecting TDHs in milk powder using HPLC-ESI-TOF in this paper, and limits of detection (were between 0.20 and 0.40 mg/kg, limits of quantitation were between 0.5 and 1.0 mg/kg and recoveries of all TDHs were between from 78% to 94% with relative standard deviation lower than 10%. This research will provide a reference for developing methods of HPLC-MS/MS to detect TDHs in food samples, which can provide a tool for the government to monitor TDHs contamination in foods.
Asunto(s)
Proteínas Hemolisinas , Límite de Detección , Leche , Espectrometría de Masa por Ionización de Electrospray , Cromatografía Líquida de Alta Presión/métodos , Leche/química , Leche/microbiología , Animales , Espectrometría de Masa por Ionización de Electrospray/métodos , Proteínas Hemolisinas/análisis , Proteínas Hemolisinas/química , Toxinas Bacterianas/análisis , Toxinas Bacterianas/química , Reproducibilidad de los Resultados , Modelos Lineales , Espectrometría de Masas en Tándem/métodos , Polvos/química , Contaminación de Alimentos/análisis , Vibrio parahaemolyticus/química , Vibrio parahaemolyticus/aislamiento & purificaciónRESUMEN
Siderophores are small-molecule iron chelators produced by many microorganisms that capture and uptake iron from the natural environment and host. Their biosynthesis in microorganisms is generally performed using non-ribosomal peptide synthetase (NRPS) or NRPS-independent siderophore (NIS) enzymes. Vibrio parahaemolyticus secretes its cognate siderophore vibrioferrin under iron-starvation conditions. Vibrioferrin is a dehydrated condensate composed of α-ketoglutarate, L-alanine, aminoethanol, and citrate, and pvsA (the gene encoding the ATP-grasp enzyme), pvsB (the gene encoding the NIS enzyme), pvsD (the gene encoding the NIS enzyme), and pvsE (the gene encoding decarboxylase) are engaged in its biosynthesis. Here, we elucidated the biosynthetic pathway of vibrioferrin through in vitro enzymatic reactions using recombinant PvsA, PvsB, PvsD, and PvsE proteins. We also found that PvsD condenses L-serine and citrate to generate O-citrylserine, and that PvsE decarboxylates O-citrylserine to form O-citrylaminoethanol. In addition, we showed that O-citrylaminoethanol is converted to alanyl-O-citrylaminoethanol by amidification with L-Ala by PvsA and that alanyl-O-citrylaminoethanol is then converted to vibrioferrin by amidification with α-ketoglutarate by PvsB.
Asunto(s)
Pirrolidinonas , Vibrio parahaemolyticus , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/química , Vibrio parahaemolyticus/metabolismo , Vías Biosintéticas , Ácidos Cetoglutáricos/metabolismo , Hierro/metabolismo , Sideróforos/química , Citratos/metabolismoRESUMEN
Thermostable direct hemolysin (TDH) is a ~19 kDa, hemolytic pore-forming toxin from the gram-negative marine bacterium Vibrio parahaemolyticus, one of the causative agents of seafood-borne acute gastroenteritis and septicemia. Previous studies have established that TDH exists as a tetrameric assembly in physiological state; however, there is limited knowledge regarding the molecular arrangement of its disordered N-terminal region (NTR)-the absence of which has been shown to compromise TDH's hemolytic and cytotoxic abilities. In our current study, we have employed single-particle cryo-electron microscopy to resolve the solution-state structures of wild-type TDH and a TDH construct with deletion of the NTR (NTD), in order to investigate structural aspects of NTR on the overall tetrameric architecture. We observed that both TDH and NTD electron density maps, resolved at global resolutions of 4.5 and 4.2 Å, respectively, showed good correlation in their respective oligomeric architecture. Additionally, we were able to locate extra densities near the pore opening of TDH which might correspond to the disordered NTR. Surprisingly, under cryogenic conditions, we were also able to observe novel supramolecular assemblies of TDH tetramers, which we were able to resolve to 4.3 Å. We further investigated the tetrameric and inter-tetrameric interaction interfaces to elaborate upon the key residues involved in both TDH tetramers and TDH super assemblies. Our current structural study will aid in understanding the mechanistic aspects of this pore-forming toxin and the role of its disordered NTR in membrane interaction.
Asunto(s)
Toxinas Bacterianas , Vibrio parahaemolyticus , Vibrio parahaemolyticus/química , Microscopía por Crioelectrón , Proteínas Hemolisinas/química , Proteínas Hemolisinas/toxicidad , Toxinas Bacterianas/químicaRESUMEN
Immunoglobulin-like (Ig-like) fold domains are abundant on the surface of bacteria, where they are required for cell-to-cell recognition, adhesion, biofilm formation, and conjugative transfer. Fibrillar adhesins are proteins with Ig-like fold(s) that have filamentous structures at the cell surface, being thinner and more flexible than pili. While the roles of fibrillar adhesins have been proposed in bacteria overall, their characterization in Vibrio parahaemolyticus has not been established and, therefore, understanding about fibrillar adhesins remain limited in V. parahaemolyticus. This in silico analysis can aid in the systematic identification of Ig-like-folded and fibrillar adhesin-like proteins in V. parahaemolyticus, opening new avenues for disease prevention by interfering in microbial interaction between V. parahaemolyticus and the host.
Asunto(s)
Adhesinas Bacterianas/química , Fimbrias Bacterianas/química , Inmunoglobulinas/química , Vibrio parahaemolyticus/química , Estructura MolecularRESUMEN
A dual-mode aptasensor using colorimetry and microfluidic chip (MC) together with stir bar sorptive extraction (SBSE) has been developed for firstly qualifying samples contaminated with Vibrio parahaemolyticus (V.P) and Salmonella typhimurium (S.T), then precisely determine both of them in positive samples. For this purpose, the aptamer-streptavidin encoded probes (Apt-SAEs) corresponding to different bacteria were prepared in advance. Then, a stir bar modified with 4-mercaptophenylboronic acid (MPBA) was made to extract bacteria together with Apt-SAE probes. The binding event of aptamer and target triggered the formation of two sandwich structures containing Apt-SAE, V.P or S.T. The concentration of bacteria could be enriched by 1000 times within 15 min to avoid long-time enrichment process. Finally, the stir bar was immersed in the 3,3',5,5'-Tetramethylbenzidine (TMB)-H2O2 solution for color development. The color could be observed by naked eyes to judge whether the analytes were present. The colorless samples were judged to be negative. For the positive samples, the adsorbed encoded probes corresponding to different bacteria would be eluted from the stir bar and rapidly analyzed by the MC. Under the optimized conditions, 100 CFU/mL of V.P or S.T or both of them could be observed by colorimetry and 35 CFU/mL of them could be detected (S/N = 3) by the MC. The assay has significant application value for on-site screening and multiple detection of food-borne pathogenic bacteria.
Asunto(s)
Mezclas Complejas/análisis , Oro/química , Nanopartículas del Metal/química , Salmonella typhimurium/química , Vibrio parahaemolyticus/química , Adsorción , Bencidinas/química , Técnicas Biosensibles , Ácidos Borónicos/química , Colorimetría , Peróxido de Hidrógeno/química , Límite de Detección , Microfluídica , Compuestos de Sulfhidrilo/químicaRESUMEN
VbrK and VbrR from the gastroenteritis-causing Vibrio parahaemolyticus are a histidine kinase and response regulator, respectively, that constitute a two-component regulatory system. VbrK responds to ß-lactam antibiotics or nitrate and activates VbrR via phosphorylation. Consequently, VbrR transcriptionally regulates the expression of ß-lactamase and ExsC and contributes to the survival or virulence of V. parahaemolyticus. Due to the unavailability of the VbrR structure, it remains unclear how VbrR is activated via its N-terminal receiver domain (RD) and recognizes dsDNA via its C-terminal DNA-binding domain (DBD). To reveal the mechanism underlying VbrR-mediated activation, we generated the phosphomimetic protein (VbrRRD-D51E) of the VbrR RD by replacing the D51 residue at the phosphorylation site with glutamate. VbrRRD-D51E exhibits a ß7α5 structure rather than the typical ß5α5 structure because it contains a unique two-stranded ß-sheet. The VbrRRD-D51E structure represents an active state in which the D51E residue interacts with the T78 residue. As a result, the Y97 residue adopts an inward conformation, allowing VbrRRD-D51E to dimerize using the α4-ß5-α5 face. These activation events are facilitated by a VbrR-specific residue, R52. Further structural study demonstrated that the VbrR DBD adopts a ß-strand-decorated three-helix structure. Based on a comparative structural study, we propose that VbrR recognizes dsDNA by inserting the α8 helix into the major groove of dsDNA and interacting with the minor groove of dsDNA via the ß11-ß12 region. Our findings will provide a new avenue for development of new antibacterial drugs for treating V. parahaemolyticus infections.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Vibrio parahaemolyticus/química , Proteínas Bacterianas/genética , Dominio Catalítico , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Genes Reguladores , Modelos Moleculares , Fosforilación , Dominios Proteicos , Vibrio parahaemolyticus/metabolismoRESUMEN
A type of grafted acrylate copolymer resins, containing 3-oxo-N-allyl-1,2-benzisothiazole-2(3H)-carboxamide monomer and heterocyclic monomers, was synthesized through the copolymeri- zation of methyl methacrylate (MMA) and butyl acrylate (BA) with functional monomers. The structures of the monomers and copolymers were validated by infrared (IR) and 1 H nuclear magnetic resonance (NMR) spectroscopies. The inhibitory activities of the copolymers on algae, bacteria, and barnacle larvae were measured, and the antifouling potencies against marine macrofouling organisms were investigated. The results showed that the grafted resin had significant inhibitory effects on the growth of three marine algae (Isochrysis galbana, Nannochloropsisoculata, and Chlorella pyrenoidosa), and three bacteria (Vibrio coralliilyticus, Staphylococcus aureus,and Vibrio parahaemolyticus). The target copolymers also showed excellent inhibition of the survival of barnacle larvae. Additionally, the release rate of the antifoulant and the results of the marine field tests indicated that the grafted copolymers had outstanding antifouling potency against the attachment of marine macrofouling organisms.
Asunto(s)
Resinas Acrílicas/química , Antibacterianos/química , Organismos Acuáticos/química , Materiales Biocompatibles Revestidos/química , Imidazoles/química , Acrilatos/química , Animales , Antibacterianos/farmacología , Incrustaciones Biológicas , Chlorella/efectos de los fármacos , Diseño de Fármacos , Imidazoles/metabolismo , Membranas Artificiales , Metacrilatos/química , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-Actividad , Tiazoles/química , Thoracica/química , Vibrio/efectos de los fármacos , Vibrio parahaemolyticus/químicaRESUMEN
Acute hepatopancreatic necrosis disease (AHPND) is a major debilitating disease that causes massive shrimp death resulting in substantial economic losses in shrimp aquaculture. The Pir toxin proteins secreted by a unique strain of Vibrio parahaemolyticus play an essential role in the pathogenesis of AHPND. At present, most studies on the effects of Pir toxin proteins in shrimp focus on digestive tissues or organs such as hepatopancreas, stomach, etc., with none on the immune organs. In the present study, two recombinant Pir toxin proteins (rPirA and rPirB) of V. parahaemolyticus were expressed with rPirB shown to enter shrimp hemocytes. Employing pull-down and LC-MS/MS analysis, GST-rPirB was found to interact with 13 proteins in hemocytes, including histone H3 and histone H4 and among which histone H4 had the highest protein score. Further analysis using GST pull-down and Far-Western blot analysis revealed that rPirB could interact with histone H4. In addition, using the purified nucleosome protein from Drosophila S2 cells, it was found that PirB protein could specifically bind to histones. When flow cytometry was applied, it was observed that the interaction between PirB and histones in shrimp hemocytes induces apoptosis, which results in the dephosphorylation of Serine 10 in histone H3. Collectively, the current study shows that in addition to its effect on the digestive tract of shrimp, the PirB toxin protein interacts with histones to affect the phosphorylation of histone H3-S10, thereby inducing apoptosis.
Asunto(s)
Apoptosis/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Hemocitos/patología , Histonas/metabolismo , Vibrio parahaemolyticus/química , Animales , Acuicultura , Drosophila/citología , Hepatopáncreas/patología , Penaeidae/citología , Penaeidae/microbiología , Fosforilación , Proteínas Recombinantes/genética , Vibrio parahaemolyticus/patogenicidadRESUMEN
A pair of composite probes based on aptamer modified polyhedral oligomeric silsesquioxane-perovskite quantum dots (POSS-PQDs-Apt) as signal probe and titanium carbide (Ti3C2) MXenes as quencher were prepared for the first time. They were employed to fabricate one turn-on-type aptasensor relying on fluorescence resonance energy transfer (FRET) for Vibrio parahaemolyticus (VP) determination. The POSS-PQDs-Apt can be adsorbed on the MXenes nanosheets, and its fluorescence was quenched due to the FRET. After the composite probes were incubated with VP for 50 min, the POSS-PQDs-Apt binding with VP can be released from the surface of MXenes, and the signal recovered due to its higher affinity to the VP than MXenes. The fluorescence intensity from 519 nm emission of the system was measured at 480 nm excitation. Under In optimized conditions, the assay can determine VP in the concentration range 102 - 106 cfu/mL, and the detection limit (LOD) was 30 cfu/mL using fluorescence detection. The LOD is still 100 cfu/mL by naked eye detection which is proper for on-line monitoring VP in aquaculture water. This method was also used to detect VP in actual samples of seawater, the recovery of spiked samples was between 93% and 106%, and relative standard deviation (RSD) was between 2.7% and 6.7%. The result is consistent with the plate count. Therefore, this assay could provide a candidate platform for screening VP in aquaculture industry.
Asunto(s)
Aptámeros de Nucleótidos/química , Compuestos de Organosilicio/química , Puntos Cuánticos/química , Titanio/química , Vibrio parahaemolyticus/aislamiento & purificación , Contaminantes del Agua/análisis , Carga Bacteriana/métodos , Técnicas Biosensibles/métodos , Compuestos de Calcio/química , Transferencia Resonante de Energía de Fluorescencia , Límite de Detección , Óxidos/química , Agua de Mar/análisis , Agua de Mar/microbiología , Vibrio parahaemolyticus/químicaRESUMEN
In this work, we study the interaction of graphdiyne oxide (GDYO)-, graphene oxide (GO)-, or black phosphorous (BP)-wrapped Janus micromotors using a model system relying on a fluorescence-labeled affinity peptide, which is released upon specific interaction with a target Cholera Toxin B. Such ON-OFF-ON system allows mimicking similar processes occurring at (bio)-interfaces and to study the related sorption and desorption kinetics. The distinct surface properties of each nanomaterial play a critical role in the loading/release capacity of the peptide, greatly influencing the release profiles. Sorption obeys a second-order kinetic model using the two-dimensional (2D) nanomaterials in connection with micromotors, indicating a strong influence of chemisorption process for BP micromotors. Yet, release kinetics are faster for GDYO and GO nanomaterials, indicating a contribution of π and hydrophobic interactions in the probe sorption (Cholera Toxin B affinity peptide) and target probe release (in the presence of Cholera Toxin B). Micromotor movement also plays a critical role in such processes, allowing for efficient operation in low raw sample volumes, where the high protein content can diminish probe loading/release, affecting the overall performance. The loading/release capacity and feasibility of the (bio)-sensing protocol are illustrated in Vibrio cholerae and Vibrio parahaemolyticus bacteria cultures as realistic domains. The new concept described here holds considerable promise to understand the interaction of micromotor with biological counterparts in a myriad of biomedical and other practical applications, including the design of novel micromotor-based sensors.
Asunto(s)
Técnicas Biosensibles , Toxina del Cólera/análisis , Nanoestructuras/química , Fluorescencia , Grafito/química , Tamaño de la Partícula , Péptidos/química , Fósforo/química , Propiedades de Superficie , Vibrio cholerae/química , Vibrio parahaemolyticus/químicaRESUMEN
Bacteria express ß-lactamase to counteract the bactericidal effects of ß-lactam antibiotics, which are the most widely employed antibacterial drugs. In gram-negative bacteria, the expression of ß-lactamase is generally regulated in response to the muropeptide that is generated from the peptidoglycan of the cell wall during ß-lactam antibiotic challenge. The direct regulation of ß-lactamase expression by ß-lactams was recently reported in Vibrio parahaemolyticus, and this regulation is mediated by a two-component regulatory system that consists of the histidine kinase VbrK and the response regulator VbrR. VbrK directly recognizes ß-lactam antibiotics using the periplasmic sensor domain (VbrKSD), a PF11884 Pfam family member, and it delivers the ß-lactam signal to VbrR to induce the transcription of the ß-lactamase gene. To determine the structural features of VbrKSD as the prototype of the PF11884 family and provide insights into the ß-lactam antibiotic-binding mode of VbrKSD, we determined the crystal structure of VbrKSD at 1.65 Å resolution. VbrKSD folds into a unique curved rod-like structure that has not been previously reported in other families. VbrKSD consists of two domains (D1 and D2). The D1 domain contains two helix-decorated ß-sheets, and the D2 domain adopts a helix-rich structure. VbrKSD features two terminal disulfide bonds, which would be the canonical property of the PF11884 family. In the VbrKSD structure, the L82 residue, which was previously shown to play a key role in ß-lactam antibiotic recognition, forms a pocket along with its neighboring hydrophobic or positively charged residues.
Asunto(s)
Antibacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Histidina Quinasa/metabolismo , Vibrio parahaemolyticus/metabolismo , beta-Lactamas/metabolismo , Proteínas Bacterianas/química , Cristalografía por Rayos X , Histidina Quinasa/química , Humanos , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Vibriosis/microbiología , Vibrio parahaemolyticus/químicaRESUMEN
Bacterial two-component regulatory systems (TCS) play important roles in sensing environmental stimuli and responding to them by regulating gene expression. VbrK/VbrR, a TCS in Vibrio parahaemolyticus, confers resistance to ß-lactam antibiotics through activating a ß-lactamase gene. Its periplasmic sensor domain was previously suggested to detect ß-lactam antibiotics by direct binding. Here, we report a crystal structure of the periplasmic sensing domain of VbrK (VbrKSD) using sulfur-based single-wavelength anomalous diffraction (S-SAD) phasing. Contrary to most bacterial sensor domains which form dimers, we show that VbrKSD is a monomer using size exclusion chromatography coupled with multi-angle light scattering. This observation is also supported by molecular dynamics simulations. To quantify the binding affinity of ß-lactam antibiotics to VbrKSD, we performed isothermal titration calorimetry and other biophysical analyses. Unexpectedly, VbrKSD did not show any significant binding to ß-lactam antibiotics. Therefore, we propose that the detection of ß-lactam antibiotics by VbrK is likely to be indirect via an as yet unidentified mechanism.
Asunto(s)
Antibacterianos/química , Histidina Quinasa/química , Periplasma/química , beta-Lactamas/química , Proteínas Bacterianas/química , Cristalografía por Rayos X/métodos , Unión Proteica , Vibrio parahaemolyticus/química , beta-Lactamasas/químicaRESUMEN
Fluctuations in osmolarity are one of the most prevalent stresses to which bacteria must adapt, both hypo- and hyperosmotic conditions. Most bacteria cope with high osmolarity by accumulating compatible solutes (osmolytes) in the cytoplasm to maintain the turgor pressure of the cell. Vibrio parahaemolyticus, a halophile, utilizes at least six compatible solute transporters for the uptake of osmolytes: two ABC family ProU transporters and four betaine-carnitine-choline transporter (BCCT) family transporters. The full range of compatible solutes transported by this species has yet to be determined. Using an osmolyte phenotypic microarray plate for growth analyses, we expanded the known osmolytes used by V. parahaemolyticus to include N,N-dimethylglycine (DMG), among others. Growth pattern analysis of four triple-bccT mutants, possessing only one functional BCCT, indicated that BccT1 (VP1456), BccT2 (VP1723), and BccT3 (VP1905) transported DMG. BccT1 was unusual in that it could take up both compounds with methylated head groups (glycine betaine [GB], choline, and DMG) and cyclic compounds (ectoine and proline). Bioinformatics analysis identified the four coordinating amino acid residues for GB in the BccT1 protein. In silico modeling analysis demonstrated that GB, DMG, and ectoine docked in the same binding pocket in BccT1. Using site-directed mutagenesis, we showed that a strain with all four residues mutated resulted in the loss of uptake of GB, DMG, and ectoine. We showed that three of the four residues were essential for ectoine uptake, whereas only one of the residues was important for GB uptake. Overall, we have demonstrated that DMG is a highly effective compatible solute for Vibrio species and have elucidated the amino acid residues in BccT1 that are important for the coordination of GB, DMG, and ectoine transport.IMPORTANCEVibrio parahaemolyticus possesses at least six osmolyte transporters, which allow the bacterium to adapt to high-salinity conditions. In this study, we identified several additional osmolytes that were utilized by V. parahaemolyticus We demonstrated that the compound DMG, which is present in the marine environment, was a highly effective osmolyte for Vibrio species. We determined that DMG is transported via BCCT family carriers, which have not been shown previously to take up this compound. BccT1 was a carrier for GB, DMG, and ectoine, and we identified the amino acid residues essential for the coordination of these compounds. The data suggest that for BccT1, GB is more easily accommodated than ectoine in the transporter binding pocket.
Asunto(s)
Aminoácidos Diaminos/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Sarcosina/análogos & derivados , Vibrio parahaemolyticus/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Betaína/metabolismo , Transporte Biológico , Carnitina/metabolismo , Colina/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Familia de Multigenes , Sarcosina/metabolismo , Vibrio parahaemolyticus/química , Vibrio parahaemolyticus/genéticaRESUMEN
Vibrio parahemolyticus is a halophilic bacterium which causes widespread seafood poisoning pathogenicity. Although the incidence of disease caused by V. parahemolyticus was stepwise increased, the pathogenic mechanism remained unclear. Herein, the difference of V. parahemolyticus's metabonomic which on blood agar and seawater beef extract peptone medium was detected via nuclear magnetic resonance and 55 metabolites were identified. Among them, 40 kinds of metabolites were upregulated in blood agar group, and 12 kinds were downregulated. Nine pathways were verified by enrichment analysis which were predicted involved in amino acids and protein synthesis, energy metabolism, DNA and RNA synthesis and DNA damage repair. We supposed that the metabolic pathway obtained from this study is related to V. parahemolyticus pathogenicity and our findings will aid in the identification of alternative targets or strategies to treat V. parahemolyticus-caused disease.
Asunto(s)
Medios de Cultivo/metabolismo , Vibrio parahaemolyticus/metabolismo , Agar/análisis , Medios de Cultivo/química , Humanos , Espectroscopía de Resonancia Magnética , Metabolómica , Vibriosis/microbiología , Vibrio parahaemolyticus/química , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/crecimiento & desarrolloRESUMEN
Thermostable direct hemolysin (TDH) of Vibrio parahemolyticus is a membrane-damaging pore-forming toxin with potent cytolytic/cytotoxic activity. TDH exists as a tetramer consisting of protomers with a core ß-sandwich domain, flanked by an 11-amino acid long N-terminal region (NTR). This NTR could not be modeled in the previously determined crystal structure of TDH. Moreover, the functional implication of NTR for the membrane-damaging action of TDH remains unknown. In the present study, we have explored the implications of NTR for the structure-function mechanism of TDH. Our data show that the presence of NTR modulates the physicochemical property of TDH in terms of augmenting the amyloidogenic propensity of the protein. Deletion of NTR compromises the binding of TDH toward target cell membranes and drastically affects the membrane-damaging cytolytic/cytotoxic activity of the toxin. Mutations of aromatic/hydrophobic residues within NTR also confer compromised cell-killing activity. Moreover, covalent trapping of NTR, via an engineered disulfide bond, against the core ß-sandwich domain also abrogates the cytolytic/cytotoxic activity of TDH. This observation suggests that an unrestrained configuration of NTR is crucial for the membrane-damaging action of TDH. On the basis of our study, we propose a model explaining the role of NTR in the membrane-damaging function of TDH.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/ultraestructura , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/ultraestructura , Proteínas Bacterianas/química , Toxinas Bacterianas/metabolismo , Fenómenos Bioquímicos/genética , Transporte Biológico/genética , Proteínas Hemolisinas/química , Proteínas Hemolisinas/fisiología , Hemólisis , Humanos , Mutación/genética , Subunidades de Proteína/metabolismo , Vibrio parahaemolyticus/química , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismoRESUMEN
Electrochemiluminescence immunoassays are usually carried out through "on-electrode" strategy, i.e., sandwich-type immunoassay format, the sensitivity of which is restricted by two key bottlenecks: (1) the number of signal labels is limited and (2) only a part of signal labels could participate in the electrode reaction. In this Perspective, we discuss the development of an "in-electrode" Faraday-cage-type concept-based immunocomplex immobilization strategy. The biggest difference from the traditional sandwich-type one is that the designed "in-electrode" Faraday-cage-type immunoassay uses a conductive two-dimensional (2-D) nanomaterial simultaneously coated with signal labels and a recognition component as the detection unit, which could directly overlap on the electrode surface. In such a case, electrons could flow freely from the electrode to the detection unit, the outer Helmholtz plane (OHP) of the electrode is extended, and thousands of signal labels coated on the 2-D nanomaterial are all electrochemically "effective." Thus, then, the above-mentioned bottlenecks obstructing the improvement of the sensitivity in sandwich-type immunoassay are eliminated, and as a result a much higher sensitivity of the Faraday-cage-type immunoassay can be obtained. And, the applications of the proposed versatile "in-electrode" Faraday-cage-type immunoassay have been explored in the detection of target polypeptide, protein, pathogen, and microRNA, with the detection sensitivity improved tens to hundreds of times. Finally, the outlook and challenges in the field are summarized. The rise of Faraday-cage-type electrochemiluminescence immunoassay (FCT-ECLIA)-based biosensing strategies opens new horizons for a wide range of early clinical identification and diagnostic applications.
Asunto(s)
Anticuerpos/química , Técnicas Biosensibles , Técnicas Electroquímicas , Inmunoensayo , Nanoestructuras/química , Electrodos , Óxido Ferrosoférrico/química , Oro/química , Grafito/química , Humanos , Límite de Detección , Luminiscencia , MicroARNs/análisis , Neurotensina/análisis , Vibrio parahaemolyticus/química , Vibrio parahaemolyticus/aislamiento & purificación , Vibrio vulnificus/química , Vibrio vulnificus/aislamiento & purificación , Factores de Transcripción p300-CBP/análisisRESUMEN
Swarmer cells of the Gram-negative uropathogenic bacteria Proteus mirabilis and Vibrio parahaemolyticus become long (>10 to 100 µm) and multinucleate during their growth and motility on polymer surfaces. We demonstrated that the increasing cell length is accompanied by a large increase in flexibility. Using a microfluidic assay to measure single-cell mechanics, we identified large differences in the swarmer cell stiffness (bending rigidity) of P. mirabilis (5.5 × 10-22 N m2) and V. parahaemolyticus (1.0 × 10-22 N m2) compared to vegetative cells (1.4 × 10-20 N m2 and 2.2 × 10-22 N m2, respectively). The reduction in bending rigidity (â¼2-fold to â¼26-fold) was accompanied by a decrease in the average polysaccharide strand length of the peptidoglycan layer of the cell wall from 28 to 30 disaccharides to 19 to 22 disaccharides. Atomic force microscopy revealed a reduction in P. mirabilis peptidoglycan thickness from 1.5 nm (vegetative cells) to 1.0 nm (swarmer cells), and electron cryotomography indicated changes in swarmer cell wall morphology. P. mirabilis and V. parahaemolyticus swarmer cells became increasingly sensitive to osmotic pressure and susceptible to cell wall-modifying antibiotics (compared to vegetative cells)-they were â¼30% more likely to die after 3 h of treatment with MICs of the ß-lactams cephalexin and penicillin G. The adaptive cost of "swarming" was offset by the increase in cell susceptibility to physical and chemical changes in their environment, thereby suggesting the development of new chemotherapies for bacteria that leverage swarming for the colonization of hosts and for survival.IMPORTANCEProteus mirabilis and Vibrio parahaemolyticus are bacteria that infect humans. To adapt to environmental changes, these bacteria alter their cell morphology and move collectively to access new sources of nutrients in a process referred to as "swarming." We found that changes in the composition and thickness of the peptidoglycan layer of the cell wall make swarmer cells of P. mirabilis and V. parahaemolyticus more flexible (i.e., reduce cell stiffness) and that they become more sensitive to osmotic pressure and cell wall-targeting antibiotics (e.g., ß-lactams). These results highlight the importance of assessing the extracellular environment in determining antibiotic doses and the use of ß-lactam antibiotics for treating infections caused by swarmer cells of P. mirabilis and V. parahaemolyticus.
Asunto(s)
Antibacterianos/farmacología , Locomoción , Fenómenos Mecánicos , Proteus mirabilis/efectos de los fármacos , Vibrio parahaemolyticus/efectos de los fármacos , beta-Lactamas/farmacología , Fenómenos Químicos , Viabilidad Microbiana , Microfluídica/métodos , Presión Osmótica , Peptidoglicano/química , Polisacáridos Bacterianos/análisis , Proteus mirabilis/química , Proteus mirabilis/fisiología , Análisis de la Célula Individual , Vibrio parahaemolyticus/química , Vibrio parahaemolyticus/fisiologíaRESUMEN
A method is described for single-step detection of V. parahaemolyticus in seafood via aptamer-based SERS. A gold-coated polydimethylsiloxane (PDMS) film was used for the enhancement of Raman scattering. The Raman reporter 4-mercaptobenzoic acid was linked to aptamer modified gold nanoparticles (AuNPs) served as a signalling probe. The negatively charged signalling probe was assembled onto the cysteamine-modified Au-PDMS film through electrostatic adsorption. On addition of V. parahaemolyticus, it will be bound by the aptamer as a biorecognition element, and this leads to the dissociation of the signalling probe from the Au-PDMS film. Hence, the Raman signal (at 1592 cm-1) decreases. The assay has a wide linear response that covers the 1.2 × 102 to 1.2 × 106 cfu·mL-1 V. parahaemolyticus concentration range. The detection limit is 12 cfu·mL-1. The method was successfully applied to the determination of V. parahaemolyticus in oyster and salmon samples. Graphical abstract Schematic presentation of a surface-enhanced Raman spectroscopic method for single step detection of Vibrio parahaemolyticus using gold coated polydimethylsiloxane as the active substrate and aptamer modified gold nanoparticles. This solid substrate simplified the analysis procedures and enhanced the sensitivity.
Asunto(s)
Aptámeros de Nucleótidos/química , Dimetilpolisiloxanos/química , Oro/química , Nanopartículas del Metal/química , Vibrio parahaemolyticus/química , Secuencia de Bases , Benzoatos/química , Técnicas Biosensibles/métodos , Cisteamina/química , Escherichia coli/química , Límite de Detección , Listeria monocytogenes/química , Salmonella typhimurium/química , Sensibilidad y Especificidad , Espectrometría Raman , Staphylococcus aureus/química , Compuestos de Sulfhidrilo/química , Vibrio parahaemolyticus/aislamiento & purificaciónRESUMEN
Vibrio parahaemolyticus is a major seafood-borne pathogen that causes life-threatening gastroenteric diseases in humans through the consumption of contaminated seafoods. V. parahaemolyticus produces different kinds of toxins, including thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH), and some effector proteins belonging to the Type 3 Secretion System, out of which TDH and TRH are considered to be the major factors for virulence. Although TRH is one of the major virulent proteins, there is a dearth of understanding about the structural and functional properties of this protein. This study therefore aimed to amplify the full length trh gene from V. parahaemolyticus and perform sequence-based analyses, followed by structural and functional analyses of the TRH protein using different bioinformatics tools. The TRH protein shares significant conservedness with the TDH protein. A multiple sequence alignment of TRH proteins from Vibrio and non-Vibrio species revealed that the TRH protein is highly conserved throughout evolution. The three dimensional (3D) structure of the TRH protein was constructed by comparative modelling and the quality of the predicted model was verified. Molecular dynamics simulations were performed to understand the dynamics, residual fluctuations, and the compactness of the protein. The structure of TRH was found to contain 19 pockets, of which one (pocket ID: 2) was predicted to be important from the view of drug design. Eleven residues (E138, Y140, C151, F158, C161, K162, S163, and Q164), which are reported to actively participate in the formation of the tetrameric structure, were present in this pocket. This study extends our understanding of the structural and functional dynamics of the TRH protein and as well as provides new insights for the treatment and prevention of V. parahaemolyticus infections.
Asunto(s)
Proteínas Hemolisinas/química , Simulación de Dinámica Molecular , Vibrio parahaemolyticus/química , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Proteínas Hemolisinas/genética , Hemolíticos/química , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Vibrio parahaemolyticus/genéticaRESUMEN
BACKGROUND: Vibrio parahaemolyticus causes not only various diseases in aquaculture animals but also seafood-borne illness in humans. Outer membrane proteins (OMPs) are species-specific proteins found in bilayer membranes of gram-negative bacteria. Egg yolk immunoglobulin (IgY) has been reported to serve as oral administration of antibodies against bacteria and virus. RESULTS: The present research extracted and identified OMPs from V. parahaemolyticus, and then the extracted OMPs were used to immunize hens to obtain specific IgY. The efficacy of IgY against V. parahaemolyticus were investigated in vitro and in vivo. The specific IgY effectively inhibited the growth of V. parahaemolyticus in liquid medium rather than Escherichia coli and Staphylococcus aureus. Specific IgY antibodies were incorporated into extruded food pellets and fed to bacteria-challenged white pacific shrimp to observe the anti-bacterial effect in vivo. The bacterial loads in muscles of V. parahaemolyticus infected shrimp fed with specific IgY-included diets were significantly fewer than those fed with non-specific IgY-included diets. The superoxide dismutase activities in muscles of infected shrimp fed with specific IgY-included diets were significantly higher than the control group. CONCLUSION: The results suggested that the specific IgY effectively inhibited the growth of V. parahaemolyticus and introduced passive immunity to shrimp. © 2018 Society of Chemical Industry.