Janus Micromotors Coated with 2D Nanomaterials as Dynamic Interfaces for (Bio)-Sensing.

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.

Investigations of Dimethylglycine, Glycine Betaine, and Ectoine Uptake by a Betaine-Carnitine-Choline Transporter Family Transporter with Diverse Substrate Specificity in Vibrio Species.

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.

Faraday-Cage-Type Electrochemiluminescence Immunoassay: A Rise of Advanced Biosensing Strategy.

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.

Surface-enhanced Raman spectroscopic single step detection of Vibrio parahaemolyticus using gold coated polydimethylsiloxane as the active substrate and aptamer modified gold nanoparticles.

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.

Conformational transitions of the sodium-dependent sugar transporter, vSGLT.

Sodium-dependent transporters couple the flow of Na+ ions down their electrochemical potential gradient to the uphill transport of various ligands. Many of these transporters share a common core structure composed of a five-helix inverted repeat and deliver their cargo utilizing an alternating-access mechanism. A detailed characterization of inward-facing conformations of the Na+-dependent sugar transporter from Vibrio parahaemolyticus (vSGLT) has previously been reported, but structural details on additional conformations and on how Na+ and ligand influence the equilibrium between other states remains unknown. Here, double electron-electron resonance spectroscopy, structural modeling, and molecular dynamics are utilized to deduce ligand-dependent equilibria shifts of vSGLT in micelles. In the absence and presence of saturating amounts of Na+, vSGLT favors an inward-facing conformation. Upon binding both Na+ and sugar, the equilibrium shifts toward either an outward-facing or occluded conformation. While Na+ alone does not stabilize the outward-facing state, gating charge calculations together with a kinetic model of transport suggest that the resting negative membrane potential of the cell, absent in detergent-solubilized samples, may stabilize vSGLT in an outward-open conformation where it is poised for binding external sugars. In total, these findings provide insights into ligand-induced conformational selection and delineate the transport cycle of vSGLT.

NMR-based metabolomics reveals the metabolite profiles of Vibrio parahaemolyticus under ferric iron stimulation.

Vibrio parahaemolyticus is a halophilic bacterium endemic to coastal areas, and its pathogenicity has caused widespread seafood poisoning. In our previous research, the protein expression of V. parahaemolyticus in Fe3+ medium was determined using isobaric tags for relative and absolute quantitation (iTRAQ). Here, nuclear magnetic resonance (NMR) was used to detect changes in the V. parahaemolyticus metabolome. NMR spectra were obtained using methanol-water extracts of intracellular metabolites from V. parahaemolyticus under various culture conditions, and 62 metabolites were identified, including serine, arginine, alanine, ornithine, tryptophan, glutamine, malate, NAD+, NADP+, oxypurinol, xanthosine, dCTP, uracil, thymine, hypoxanthine, and betaine. Among these, 21 metabolites were up-regulated after the stimulation of the cells by ferric iron, and 9 metabolites were down-regulated. These metabolites are involved in amino acid and protein synthesis, energy metabolism, DNA and RNA synthesis and osmolality. Based on these results, we conclude that Fe3+ influences the metabolite profiles of V. parahaemolyticus.

Characterization of Chemically-Induced Bacterial Ghosts (BGs) Using Sodium Hydroxide-Induced Vibrio parahaemolyticus Ghosts (VPGs).

Acellular bacterial ghosts (BGs) are empty non-living bacterial cell envelopes, commonly generated by controlled expression of the cloned lysis gene E of bacteriophage PhiX174. In this study, Vibrio parahaemolyticus ghosts (VPGs) were generated by chemically-induced lysis and the method is based on minimum inhibitory concentration (MIC) of sodium hydroxide (NaOH), acetic acid, boric acid, citric acid, maleic acid, hydrochloric acid, and sulfuric acid. The MIC values of the respective chemicals were 3.125, 6.25, <50.0, 25.0, 6.25, 1.56, and 0.781 mg/mL. Except for boric acid, the lysis efficiency reached more than 99.99% at 5 min after treatment of all chemicals. Among those chemicals, NaOH-induced VPGs appeared completely DNA-free, which was confirmed by quantitative real-time PCR. Besides, lipopolysaccharides (LPS) extracted from the NaOH-induced VPGs showed no distinctive band on SDS-PAGE gel after silver staining. On the other hand, LPS extracted from wild-type bacterial cells, as well as the organic acids-induced VPGs showed triple major bands and LPS extracted from the inorganic acids-induced VPGs showed double bands. It suggests that some surface structures in LPS of the NaOH-induced VPGs may be lost, weakened, or modified by the MIC of NaOH. Nevertheless, Limulus amoebocyte lysate assay revealed that there is no significant difference in endotoxic activity between the NaOH-induced VPGs and wild-type bacterial cells. Macrophages exposed to the NaOH-induced VPGs at 0.5 × 106 CFU/mL showed cell viability of 97.9%, however, the MIC of NaOH did not reduce the cytotoxic effect of wild-type bacterial cells. Like Escherichia coli LPS, the NaOH-induced VPGs are an excellent activator of pro-inflammatory cytokines (IL-1ß and iNOS), anti-inflammatory cytokine (IL-10), and dual activities (IL-6) in the stimulated macrophage cells. On the other hand, the induction of TNF-α mRNA was remarkable in the macrophages exposed with wild-type cells. Scanning electron microscopy showed the formation of trans-membrane lysis tunnel structures in the NaOH-induced VPGs. SDS-PAGE and agarose gel electrophoresis also confirmed that cytoplasmic proteins and genomic DNA released from the VPGs to culture medium through the lysis tunnel structures. Taken together, all these data indicate that the NaOH-induced VPGs show the potency of a safe, economical, and effective inactivated bacterial vaccine candidate.

Differential proteomics to explore the inhibitory effects of acidic, slightly acidic electrolysed water and sodium hypochlorite solution on Vibrio parahaemolyticus.

Slightly acidic electrolysed water (SlAEW) and acidic electrolysed water (AEW) have been demonstrated to effectively inactivate food-borne pathogens. However, the underlying mechanism of inactivation remains unknown. Therefore, in this study, a differential proteomic platform was used to investigate the bactericidal mechanism of SlAEW, AEW, and sodium hypochlorite (NaOCl) solutions against Vibrio parahaemolyticus. The upregulated proteins after SlAEW, AEW, and NaOCl treatments were identified as outer membrane proteins K and U. The downregulated proteins after the SlAEW, AEW, and NaOCl treatments were identified as adenylate kinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and enolase, all of which are responsible for energy metabolism. Protein synthesis-associated proteins were downregulated and identified as elongation factor Tu and GAPDH. The inhibitory effects of SlAEW and AEW solutions against V. parahaemolyticus may be attributed to the changes in cell membrane permeability, protein synthesis activity, and adenosine triphosphate (ATP) biosynthesis pathways such as glycolysis and ATP replenishment.

The sodium/iodide symporter: state of the art of its molecular characterization.

The sodium/iodide symporter (NIS or SLC5A5) is an intrinsic membrane protein implicated in iodide uptake into thyroid follicular cells. It plays a crucial role in iodine metabolism and thyroid regulation and its function is widely exploited in the diagnosis and treatment of benign and malignant thyroid diseases. A great effort is currently being made to develop a NIS-based gene therapy also allowing the radiotreatment of nonthyroidal tumors. NIS is also expressed in other tissues, such as salivary gland, stomach and mammary gland during lactation, where its physiological role remains unclear. The molecular identity of the thyroid iodide transporter was elucidated approximately fifteen years ago. It belongs to the superfamily of sodium/solute symporters, SSS (and to the human transporter family, SLC5), and is composed of 13 transmembrane helices and 643 amino acid residues in humans. Knowledge concerning NIS structure/function relationship has been obtained by taking advantage of the high resolution structure of one member of the SSS family, the Vibrio parahaemolyticus sodium/galactose symporter (vSGLT), and from studies of gene mutations leading to congenital iodine transport defects (ITD). This review will summarize current knowledge regarding the molecular characterization of NIS.

Probing adenylation: using a fluorescently labelled ATP probe to directly label and immunoprecipitate VopS substrates.

The bacterial effector VopS from Vibrio parahaemolyticus modifies host Rho GTPases to prevent downstream signalling, which leads to cell rounding and eventually apoptosis. While previous studies have used [α-(32)P] ATP for studying this enzyme, we sought to develop a non-radioactive chemical probe of VopS function. To guide these studies, the kinetic parameters were determined for a variety of nucleotides and the results indicated that the C6 position of adenosine was amenable to modification. Since Fl-ATP is a commercially available ATP analogue that is fluorescently tagged at the C6 position, we tested it as a VopS substrate, and the results show that VopS uses Fl-ATP to label Cdc42 in vitro and in MCF7 whole cell extracts. The utility of this probe was further demonstrated by immunoprecipitating Fl-ATP labeled Cdc42 as well as several novel substrate proteins. The proteins, which were identified by LC-MS/MS, include the small GTPases Rac1 and Cdc42 as well as several proteins that are potential VopS substrates and may be important for V. parahaemolyticus pathology. In total, these studies identify Fl-ATP as a valuable chemical probe of protein AMPylation.

Vibrioferrin, an unusual marine siderophore: iron binding, photochemistry, and biological implications.

Vibrioferrin (VF) is a member of the carboxylate class of siderophores originally isolated from Vibrio parahaemolyticus, an enteropathogenic estuarine bacterium often associated with seafood-borne gastroenteritis. Recently we have also isolated this siderophore from several species of Marinobacter, which are closely associated or "symbiotic" with toxic, bloom-forming dinoflagellates such as Gymnodinium catenatum. We have measured the overall metal-ligand binding constant for iron-vibrioferrin (FeVF) as 10(24.02(5)) making vibrioferrin one of the weakest iron chelators of any known marine siderophore. FeVF is also shown to be considerably more sensitive to photolysis under relatively low illumination conditions than other photoactive siderophores leading primarily to a monodecarboxylated photoproduct that has no significant affinity for Fe(III). The consequences that these features have on bacterial-algal interactions with potential importance to understanding the origin and sustenance of harmful algal blooms are discussed.

Bacterial FIC Proteins AMP Up Infection.

Proteins containing FIC (filamentation induced by cyclic adenosine monophosphate) domains are found in both prokaryotic and eukaryotic organisms, but their function has remained elusive. Recent studies indicate that bacterial FIC domain-containing proteins disrupt host cell processes after being delivered into eukaryotic host cells: The Vibrio parahaemolyticus VopS protein interferes with Rho guanine triphosphatase (GTPase) function, and the Legionella pneumophila AnkX protein disrupts the microtubule-dependent transport of vesicles. Analysis of the VopS protein revealed that the FIC domain covalently modifies Rac by transferring adenosine 5'-monophosphate (AMP) to a threonine residue in the switch 1 region of the protein. Thus, FIC domain-mediated AMPylation is involved in the posttranslational regulation of protein function, and this activity has been subverted by microbial pathogens to modulate cellular functions during infection.

Local conformational changes in the Vibrio Na+/galactose cotransporter.

Na(+) and sugar transport by cotransporters (symporters) is thought to occur as a series of ordered ligand-induced conformational changes. To localize these conformational changes in a bacterial Na(+)/galactose cotransporter, we have employed a combination of cysteine-scanning and fluorescence techniques. Single or pairs of cysteine residues were introduced into the external face of a cysteine-less Vibrio parahaemolyticus sodium/glucose cotransporter for expression in Escherichia coli, and each transporter was purified using affinity chromatography. All the mutant proteins retained transport activity in bacteria and proteoliposomes. Each mutant was exposed to two different fluorescence reagents, ThioGlo3 or pyrene maleimide, that are essentially nonfluorescent until they react with a thiol. Fluorescence was recorded as a function of time and ligand concentrations. The reagents specifically labeled six of the seven cysteine mutants, but only in Cysteine 423 was the fluorescence affected by ligands. The rate of labeling of Cys423 by ThioGlo3 or pyrene maleimide was reduced by D-galactose in Na(+) buffer. Furthermore, the fluorescence of Thioglo3-labeled Cys423 was quenched by D-galactose, but only in the presence of Na(+). This quench was not accompanied by a Stokes shift and was not produced by nontransported sugars, e.g., L-glucose. Reducing the sodium concentration from 200 to 10 mM decreased the apparent affinity for d-galactose without altering the maximum quench with saturating D-galactose. Reducing the galactose concentration from 20 to 0.5 mM reduced both the apparent affinity for Na(+) and the maximum quench at saturating Na(+). These results suggest an ordered reaction scheme with Na(+) binding first. The fluorescence results with ThioGlo3-labeled Cys423 indicate that conformational changes underlying Na(+)/galactose cotransport occur at or near the extracellular domain between transmembrane helices 10 and 11.

Proteomics on full-length membrane proteins using mass spectrometry.

A general technique has been developed that allows rapid mass spectrometric analysis of full-length membrane proteins [Whitelegge, J. P., le Coutre, J., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10695-10698]. Using in-line HPLC electrospray ionization mass spectrometry (LC-MS), different native and recombinant bacterial membrane proteins of up to 61 kDa are characterized. Mass spectrometric data of four entirely different membrane proteins from three bacterial organisms, two transporters, a channel, and a porin protein are presented. In addition to determination of the molecular mass with an accuracy of +/-0.01%, the technique monitors alkylation or oxidation of single Cys residues and errors in deduced amino acid sequences. Finally, using in-line LC-MS, unknown proteins can be identified from solubilized Escherichia coli membranes without prior purification.

Utilization of hemin and hemoglobin as iron sources by Vibrio parahaemolyticus and identification of an iron-repressible hemin-binding protein.

Several clinical isolates of Vibrio parahaemolyticus were examined for their ability to utilize either hemin or hemoglobin as a sole source of iron. Both compounds appeared to be equally good iron sources. Maximum growth was obtained at 5 microM hemin or 1.25 microM hemoglobin under the conditions tested. Using a hemin-agarose batch affinity method, the hemin-binding protein was isolated from crude total membranes of a hemin-utilizing strain, WP1, grown under iron-deficient but not under iron-sufficient conditions. This protein was identical to the 83 kDa outer membrane protein which was expressed in response to iron limitation. The protein was susceptible to proteinase K cleavage in whole cells, indicating its exposure at the cell surface. Hemin and hemoglobin, but not protoporphyrin IX, inhibited binding of the protein to hemin-agarose.

Structure and iron transport activity of vibrioferrin, a new siderophore of Vibrio parahaemolyticus.

The structure of vibrioferrin, a siderophore from Vibrio parahaemolyticus, was elucidated based on a combination of partial hydrolysis and spectroscopic techniques. HPLC of purified vibrioferrin showed two peaks with an area ratio of approximately 2:1. However, upon reinjection of each of those isolated compounds, the original chromatographic pattern was obtained, indicating an equilibrium between two compounds in aqueous solution. Consistent with this finding, most of the NMR signals of vibrioferrin were duplicated. The structure was determined as 1-(2-[2-(5-carboxy-5-hydroxy-2-oxo-1-pyrrolidinyl)propionamide]ethyl) citrate, which exists in two epimeric forms resulting from cyclization between an amidic nitrogen of the alanine residue and a keto group of the 2-ketoglutaric acid residue. Transport experiments with 55Fe-labeled vibrioferrin demonstrated the function of vibrioferrin as a siderophore in V. parahaemolyticus. Kinetic studies with mid-log phase cells revealed that the iron uptake system was receptor-mediated, with Km and Vmax values of 67 nM and 54 pmol Fe/mg cell protein/min, respectively. Moreover, iron uptake mediated by vibrioferrin was blocked both by uncouplers and by ATPase inhibitors.

Introduction of a series of alkyl thiomaltosides, useful new non-ionic detergents, to membrane biochemistry.

We synthesized a series of non-ionic detergents, alkyl thiomaltosides, and investigated their properties and usefulness. We solubilized membrane proteins of Vibrio parahaemolyticus using the detergents. With octyl thiomaltoside, nonyl thiomaltoside, decyl thiomaltoside, or undecyl thiomaltoside, we observed satisfactory solubilization of the membrane proteins. Alkyl thiomaltosides possessing longer alkyl chains showed better solubilization than ones possessing shorter chains. H(+)-translocating ATPase (F0F1), which is localized in the cytoplasmic membrane (inner membrane), was solubilized with the detergents, and the solubilized enzyme showed much higher specific activity than that solubilized with octyl glucoside or heptyl thioglucoside, other useful non-ionic detergents. 5'-Nucleotidase, which seems to be an outer membrane protein, was also efficiently solubilized with the alkyl thiomaltosides. Membrane proteins of Escherichia coli were also efficiently solubilized with the detergents. Octyl thiomaltoside and nonyl thiomaltoside were removed fairly rapidly on dialysis. Decyl thiomaltoside was removed slowly, and undecyl thiomaltoside and dodecyl thiomaltoside were difficult to remove by dialysis.