Specific proteomic adaptation to distinct environments in Vibrio parahaemolyticus includes significant fluctuations in expression of essential proteins.

Bacteria constantly experience changes to their external milieu and need to adapt accordingly to ensure their survival. Certain bacteria adapt by means of cellular differentiation, resulting in the development of a specific cell type that is specialized for life in a distinct environment. Furthermore, to understand how bacteria adapt, it is essential to appreciate the significant changes that occur at the proteomic level. By analysing the proteome of our model organism Vibrio parahaemolyticus from distinct environmental conditions and cellular differential states, we demonstrate that the proteomic expression profile is highly flexible, which likely allows it to adapt to life in different environmental conditions and habitats. We show that, even within the same swarm colony, there are specific zones of cells with distinct expression profiles. Furthermore, our data indicate that cell surface attachment and swarmer cell differentiation are distinct programmes that require specific proteomic expression profiles. This likely allows V. parahaemolyticus to adapt to life in different environmental conditions and habitats. Finally, our analyses reveal that the expression profile of the essential protein pool is highly fluid, with significant fluctuations that dependent on the specific life-style, environment and differentiation state of the bacterium.

A comparative genomics methodology reveals a widespread family of membrane-disrupting T6SS effectors.

Gram-negative bacteria deliver effectors via the type VI secretion system (T6SS) to outcompete their rivals. Each bacterial strain carries a different arsenal of effectors; the identities of many remain unknown. Here, we present an approach to identify T6SS effectors encoded in bacterial genomes of interest, without prior knowledge of the effectors' domain content or genetic neighborhood. Our pipeline comprises a comparative genomics analysis followed by screening using a surrogate T6SS+ strain. Using this approach, we identify an antibacterial effector belonging to the T6SS1 of Vibrio parahaemolyticus, representing a widespread family of T6SS effectors sharing a C-terminal domain that we name Tme (Type VI membrane-disrupting effector). Tme effectors function in the periplasm where they intoxicate bacteria by disrupting membrane integrity. We believe our approach can be scaled up to identify additional T6SS effectors in various bacterial genera.

Characteristics of viable-but-nonculturable Vibrio parahaemolyticus induced by nutrient-deficiency at cold temperature.

Vibrio parahaemolyticus has been consistently found to be involved in the food-borne disease outbreaks every year. Particularly, V. parahaemolyticus can be induced into a viable but nonculturable (VBNC) state under cold-starvation conditions. In this physiological state, V. parahaemolyticus losses its colony-forming ability and shows reduced metabolic activities. The subsequent failure of its detection may threaten public health-hygiene practices. Until now, evident information on physiological properties of VBNC V. parahaemolyticus and its underlying mechanism remains unclear and unorganized. Therefore, this review summarized survival behavior, persistence, and entry of pathogenic microorganisms into a VBNC state in response to various environmental conditions and discussed distinctive characteristics of VBNC cells. To survive under unfavorable environments, VBNC V. parahaemolyticus shows marked modifications in cell membrane composition, fatty acid synthesis, morphology, metabolism, gene expression, and capability of adhesion and virulence. These physiological modifications might be closely associated with an imbalance in maintaining essential biological processes within VBNC cells, thereby causing a decrease in cell membrane fluidity. To develop an efficient surveillance method and to prevent the recovery of VBNC cells in food, the induction of a VBNC state needs to be profoundly understood.

The release of a distinct cell type from swarm colonies facilitates dissemination of Vibrio parahaemolyticus in the environment.

Bacteria experience changes in their environment and have developed various strategies to respond accordingly. To accommodate environmental changes, certain bacteria differentiate between specialized cell types. Vibrio parahaemolyticus is a marine bacterium, a worldwide human pathogen and the leading agent of seafood-borne gastroenteritis. It exists as swimmer or swarmer cells, specialized for life in liquid and on solid environments, respectively. Swarmer cells are characteristically highly elongated-a morphology important for swarming behavior. When attached to surfaces it forms swarm colonies, however, it is not known how cells within swarming populations respond to changes in the external milieu and how its distinct life cycle influences its ecological dissemination. The worldwide distribution of V. parahaemolyticus accentuates the need for understanding the factors contributing to its dissemination. Here we determine the stage-wise development of swarm colonies and show how the swarm colony architecture fluctuates with changing environmental conditions. Swarm colonies act as a continuous source of cells that are released from the swarm colony into the environment. Surprisingly, the cell length distribution of released cells was very homogenous and almost no long cells were detected, indicating that swarmer cells are not released into the liquid environment but stay surface attached during flooding. Released cells comprise a distinct cell type that is morphologically optimized for swimming behavior and is capable of spreading in the liquid environment and attach to new surfaces. Release of this distinct cell type facilitates the dissemination of V. parahaemolyticus in the environment and likely influences the ecology of this bacterium.

Nuclear magnetic resonance biosensor for rapid detection of Vibrio parahaemolyticus.

BACKGROUND: Vibrio parahaemolyticus is a Gram-negative bacterium widely distributed in marine environments and a well-recognized invertebrate pathogen frequently isolated from seafood. V. parahaemolyticus may also spread into humans, via contaminated, raw, or undercooked seafood, causing gastroenteritis and diarrhea. METHODS: A Nuclear Magnetic Resonance (NMR)-based detection system was used to detect pathogenic levels of this microorganism (105 CFU/ml) with Molecular Mirroring using iron nanoparticles coated with target-specific biomarkers capable of binding to DNA of the target microorganism. The NMR system generates a signal (in milliseconds) by measuring NMR spin-spin relaxation time T2, which correlates with the amount of microorganism DNA. RESULTS: Compared with conventional microbiology techniques such as real-time PCR (qPCR), the NMR biosensor showed similar limits of detection (LOD) at different concentrations (105-108 CFU/ml) using two DNA extraction methods. In addition, the NMR biosensor system can detect a wide range of microorganism DNAs in different matrices within a short period of time. CONCLUSION: NMR biosensor represents a potential tool for diagnostic and quality control to ensure microbial pathogens such as V. parahaemolyticus are not the cause of infection. The "hybrid" technology (NMR and nanoparticle application) opens a new platform for detecting other microbial pathogens that have impacted human health, animal health and food safety.

A cell length-dependent transition in MinD-dynamics promotes a switch in division-site placement and preservation of proliferating elongated Vibrio parahaemolyticus swarmer cells.

Vibrio parahaemolyticus exists as swimmer and swarmer cells, specialized for growth in liquid and on solid environments respectively. Swarmer cells are characteristically highly elongated due to an inhibition of cell division, but still need to divide in order to proliferate and expand the colony. It is unknown how long swarmer cells divide without diminishing the population of long cells required for swarming behavior. Here we show that swarmer cells divide but the placement of the division site is cell length-dependent; short swarmers divide at mid-cell, while long swarmers switch to a specific non-mid-cell placement of the division site. Transition to non-mid-cell positioning of the Z-ring is promoted by a cell length-dependent switch in the localization-dynamics of the division regulator MinD from a pole-to-pole oscillation in short swarmers to a multi-node standing-wave oscillation in long swarmers. Regulation of FtsZ levels restricts the number of divisions to one and SlmA ensures sufficient free FtsZ to sustain Z-ring formation by preventing sequestration of FtsZ into division deficient clusters. By limiting the number of division-events to one per cell at a specific non-mid-cell position, V. parahaemolyticus promotes the preservation of long swarmer cells and permits swarmer cell division without the need for dedifferentiation.

Purification and antibacterial mechanism of fish-borne bacteriocin and its application in shrimp (Penaeus vannamei) for inhibiting Vibrio parahaemolyticus.

Vibrio parahaemolyticus: is recognized as the main cause of gastroenteritis associated with consumption of seafood. Bacteriocin-producing Lactobacillus plantarum FGC-12 isolated from golden carp intestine had strong antibacterial activity toward V. parahaemolyticus. The fish-borne bacteriocin was purified by a three-step procedure consisting of ethyl acetate extraction, gel filtration chromatography and high performance liquid chromatography. Its molecular weight was estimated at 4.1 kDa using SDS-PAGE. The fish-borne bacteriocin reached the maximum production at stationary phase after 20 h. It was heat-stable (30 min at 121 °C) and remained active at pH range from 3.0 to 5.5, but was sensitive to nutrasin, papain and pepsin. Its minimum inhibitory concentration for V. parahaemolyticus was 6.0 mg/ml. Scanning electron microscopy analysis showed that the fish-borne bacteriocin disrupted cell wall of V. parahaemolyticus. The antibacterial mechanism of the fish-borne bacteriocin against V. parahaemolyticus might be described as action on membrane integrity in terms of the leakage of electrolytes, the losses of Na+K+-ATPase, AKP and proteins. The addition of the fish-borne bacteriocin to shrimps leaded V. parahaemolyticus to reduce 1.3 log units at 4 °C storage for 6 day. Moreover, a marked decline in total volatile base nitrogen and total viable counts was observed in bacteriocin treated samples than the control. It is clear that this fish-borne bacteriocin has promising potential as biopreservation for the control of V. parahaemolyticus in aquatic products.

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells.

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ability to obtain single cells for fluorescence microscopy, which can be particularly challenging when imaging cells that exist within bacterial communities. For example, the human pathogen Vibrio parahaemolyticus exists as short rod-shaped swimmer cells in liquid conditions that upon surface contact differentiate into a subpopulation of highly elongated swarmer cells specialized for growth on solid surfaces. This paper presents a method to perform single cell fluorescence microscopy analysis of V. parahaemolyticus in its two differential states. This protocol very reproducibly induces differentiation of V. parahaemolyticus into a swarmer cell life-cycle and facilitates their proliferation over solid surfaces. The method produces flares of differentiated swarmer cells extending from the edge of the swarm-colony. Notably, at the very tip of the swarm-flares, swarmer cells exist in a single layer of cells, which allows for their easy transfer to a microscope slide and subsequent fluorescence microscopy imaging of single cells. Additionally, the workflow of image analysis for demographic representation of bacterial societies is presented. As a proof of principle, the analysis of the intracellular localization of chemotaxis signaling arrays in swimmer and swarmer cells of V. parahaemolyticus is described.

The bacterial effector VopL organizes actin into filament-like structures.

VopL is an effector protein from Vibrio parahaemolyticus that nucleates actin filaments. VopL consists of a VopL C-terminal domain (VCD) and an array of three WASP homology 2 (WH2) motifs. Here, we report the crystal structure of the VCD dimer bound to actin. The VCD organizes three actin monomers in a spatial arrangement close to that found in the canonical actin filament. In this arrangement, WH2 motifs can be modeled into the binding site of each actin without steric clashes. The data suggest a mechanism of nucleation wherein VopL creates filament-like structures, organized by the VCD with monomers delivered by the WH2 array, that can template addition of new subunits. Similarities with Arp2/3 complex and formin proteins suggest that organization of monomers into filament-like structures is a general and central feature of actin nucleation.

Association of a D-alanyl-D-alanine carboxypeptidase gene with the formation of aberrantly shaped cells during the induction of viable but nonculturable Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a halophilic Gram-negative bacterium that causes human gastroenteritis. When the viable but nonculturable (VBNC) state of this bacterium was induced by incubation at 4°C in Morita minimal salt solution containing 0.5% NaCl, the rod-shaped cells became coccoid, and various aberrantly shaped intermediates were formed in the initial stage. This study examined the factors that influence the formation of these aberrantly shaped cells. The proportion of aberrantly shaped cells was not affected in a medium containing D-cycloserine (50 µg/ml) but was lower in a medium containing cephalosporin C (10 µg/ml) than in the control medium without antibiotics. The proportion of aberrantly shaped cells was higher in a culture medium that contained 0.5% NaCl than in culture media containing 1.0 or 1.5% NaCl. The expression of 15 of 17 selected genes associated with cell wall synthesis was enhanced, and the expression of VP2468 (dacB), which encodes D-alanyl-D-alanine carboxypeptidase, was enhanced the most. The proportion of aberrantly shaped cells was significantly lower in the dacB mutant strain than in the parent strain, but the proportion was restored in the presence of the complementary dacB gene. This study suggests that disturbance of the dynamics of cell wall synthesis by enhanced expression of the VP2468 gene is associated with the formation of aberrantly shaped cells in the initial stage of induction of VBNC V. parahaemolyticus cells under specific conditions.

The crystal structure of the periplasmic domain of Vibrio parahaemolyticus CpxA.

The Cpx two-component system of Gram-negative bacteria senses extracytoplasmic stresses using the histidine kinase CpxA, a membrane-bound sensor, and controls the transcription of the genes involved in stress response by the cytosolic response regulator CpxR, which is activated by the phosphorelay from CpxA. CpxP, a CpxA-associated protein, also plays an important role in the regulation of the Cpx system by inhibiting the autophosphorylation of CpxA. Although the stress signals and physiological roles of the Cpx system have been extensively studied, the lack of structural information has limited the understanding of the detailed mechanism of ligand binding and regulation of CpxA. In this study, we solved the crystal structure of the periplasmic domain of Vibrio parahaemolyticus CpxA (VpCpxA-peri) to a resolution of 2.1 Å and investigated its interaction with CpxP. VpCpxA-peri has a globular Per-ARNT-SIM (PAS) domain and a protruded C-terminal tail, which may be required for ligand sensing and CpxP binding, respectively. The direct interaction of the PAS core of VpCpxA-peri with VpCpxP was not detected by NMR, suggesting that the C-terminal tail or other factors, such as the membrane environment, are necessary for the binding of CpxA to CpxP.

Morphological changes of Vibrio parahaemolyticus under cold and starvation stresses.

This study addresses morphological changes of Vibrio parahaemolyticus cells during the initial period of stress. Drastic changes occurred in cells under starvation with/without concomitant cold treatment for one to a few days. Under carbon starvation, rod-shaped cells became coccoid by reductive division, which was influenced by glucose/amino acid in the culture medium. Under concomitant cold and carbon starvation, cells entered the viable but nonculturable state and their shape changed from rod-like to coccoid with waning-shape and various aberrantly intermediates, such as club-shaped, budding, club-shaped with budding, dumbbell-shaped and rabbit-ear-like-shaped. Electron microscopy revealed characteristic features in the cells that were concomitantly stressed with cold and starved, such as densely stained peripheral part and lightly stained central part of cytoplasm, and a thick peptidoglycan cell wall in the temperature-upshifted cells. The expression of cytoskeleton genes, mreB, minE and ftsZ, was determined in a real-time quantitative reverse transcription-polymerase chain reaction, and results revealed a substantial decline in expression in cells under starvation (to 0.25 to 0.64 times that of the control) in the first 2 to 3 h, and a rapid upregulated expression in recovering cells. Alteration of the morphology of cells under such stresses may be associated with the expression of these cytoskeleton genes. This work demonstrates the uniqueness of concomitant cold and starvation treatment in V. parahaemolyticus. The details of the morphological changes may facilitate understanding of stress adaptation in bacteria.

Localization and expression of MreB in Vibrio parahaemolyticus under different stresses.

MreB, the homolog of eukaryotic actin, may play a vital role when prokaryotes cope with stress by altering their spatial organization, including their morphology, subcellular architecture, and localization of macromolecules. This study investigates the behavior of MreB in Vibrio parahaemolyticus under various stresses. The behavior of MreB was probed using a yellow fluorescent protein-MreB conjugate in merodiploid strain SC9. Under normal growth conditions, MreB formed helical filaments in exponential-phase cells. The shape of starved or stationary-phase cells changed from rods to small spheroids. The cells differentiated into the viable but nonculturable (VBNC) state with small spherical cells via a "swelling-waning" process. In all cases, drastic remodeling of the MreB cytoskeleton was observed. MreB helices typically were loosened and fragmented into short filaments, arcs, and spots in bacteria under these stresses. The disintegrated MreB exhibited a strong tendency to attach to the cytoplasmic membrane. The expression of mreB generally declined in bacteria in the stationary phase and under starvation but was upregulated during the initial periods of cold shock and VBNC state differentiation and decreased afterwards. Our findings demonstrated the behavior of MreB in the morphological changes of V. parahaemolyticus under intrinsic or extrinsic stresses and may have important implications for studying the cellular stress response and aging.

State transitions of Vibrio parahaemolyticus VBNC cells evaluated by flow cytometry.

BACKGROUND: Vibrio parahaemolyticus, in response to environmental conditions, may be present in a viable but nonculturable state (VBNC), which can still be responsible for cases of infectious diseases in humans. METHODS: The characterization of the cellular states of V. parahaemolyticus during entry into, persistence in, and resuscitation from the VBNC state, was assessed through plate culture method and epifluorescence microscope evaluation of actively respiring cells. Flow cytometry (FCM) in combination with SYBR Green I (SG) and propidium iodide allowed us to distinguish between viable, dead, and damaged-cells. Immunofluorescence labeling detected by FCM was used to study changes in antibody affinity. RESULTS: Two groups of bacteria, one with High Nucleic Acid (HNA) and one having Low Nucleic Acid (LNA) content, were differentiated using SG and FCM and each was correlated with cell viability. With the aging of the microcosm, the LNA bacteria population increased while the HNA population gradually disappeared. Cytofluorimetric immunofluorescence analyses showed that the bacterial cell levels dropped from 95% at day 0 to 40% at day 26 and by day 29, antibody affinity was virtually lost. FCM analyses of light scatter signals expressed by cell population highlighted morphological changes indicating a reduction in cell size, as also shown by scanning electron microscopy images and variations in cell structure. CONCLUSIONS: The methodology used has provided useful data in relation to the state transitions of V. parahaemolyticus regarding cell viability, antigenic surface components, and the quantification of morphological variations during its entry into the VBNC state.

Bactericidal effect of TiO2 on the selected Vibrio parahaemolyticus and optimization using response surface methodology.

In this work, the bactericidal effect of TiO2 on selected typical food pathogenic bacteria, Vibrio parahaemolyticus was studied. V parahaemolyticus is an important pathogen of humans and aqua-cultured animals. We established the response surface methodology (Box-Behnken Design) to investigate the effect of principal parameters on the cell sterilization such as TiO2 concentration, UV illumination time, temperature, and pH. The sterilization rate reached maximum value at the TiO2 concentration of 1.0 mg/ml. During irradiation under the time of 30 min with UV light with the 1g-TiO2/l, the sterilization rate was greater than 85%, and 99% or more cell lost their viability with 3 hours of irradiation. Sterilization rate of the cell increased with decrease in the pH and temperature.

Two type IV pili of Vibrio parahaemolyticus play different roles in biofilm formation.

Vibrio parahaemolyticus RIMD2210633 has two sets of type IV-A pilus genes. One set is similar to that found in other Gram-negative bacteria, such as Pseudomonas aeruginosa, Vibrio cholerae (chitin-regulated pilus; ChiRP), and Vibrio vulnificus. The other is homologous to the genes for the mannose-sensitive hemagglutinin (MSHA) pilus. In this study, we analyzed the effects of the deletions in the pilin genes for each type IV pilus (the ChiRP and the MSHA pilus) on biofilm formation. Although the MSHA pilin mutant formed aggregates, the number of bacteria that attached directly to the coverslip was reduced, suggesting that this pilus contributes to the bacterial attachment to the surface of the coverslip. In contrast, the ChiRP mutant attached to the surface of the coverslip, but did not form aggregates, suggesting that ChiRP plays a role in bacterial agglutination during biofilm formation. These results suggest that the two type IV pili of V. parahaemolyticus contribute to biofilm formation in different ways. Both mutants showed a lower fitness for adsorption onto chitin particles than that of the wild type. Collectively, these data suggest that the use of two type IV pili is a refined strategy of V. parahaemolyticus for survival in natural environments.

[Study on the antibacterial mechanisms of copper-bearing montmorillonite].

Vibrio parahaemolyticus ATCC 27519 was chosen as indicator of aquacultural pathogenic bacteria to determine the antibacterial activity and mechanism of copper-bearing montmorillonite (Cu-MMT) in vitro. The results indicated that montmorillonite (MMT) had no antibacterial activity. The minimum inhibitory concentration (MIC) and bactericidal concentration (MBC) of Cu-MMT on Vibrio parahaemolyticus were 75 and 300 mg/L, respectively. The activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) of bacteria were examined and the results showed treatment with Cu-MMT could lead to significant release of intracellular enzymes from the tested bacteria suggesting that the permeability of the cell membrane increased and bacteria suffered injury. Three typical inhibitors (malonic acid, iodine acetic acid and phosphate sodium) were used to further study the inhibitory pathways of respiratory metabolism. Cu-MMT effectively inhibited respiratory metabolism of Vibrio parahaemolyticus, with the respiratory inhibition percent (I(R)) of 31.8%. The respiratory superposing inhibition percent after addition of phosphate sodium, iodine acetic acid and malonic acid was 48.6%, 27.8% and 17.5%, respectively. These results indicated that the effect of malonic acid on superposing inhibition percent of Cu-MMT for bacteria is the lowest; thus, the synergic action between Cu-MMT and malonic acid is the weakest, indicating that they inhibited the same pathway of respiratory metabolism, i.e. the TCA pathway, which is the most important pathway of carbohydrate metabolism. The atomic force microscope image of Vibrio parahaemolyticus exposed to Cu-MMT showed that Cu-MMT could rupture the bacterial cell membrane.

Three new regulators of swarming in Vibrio parahaemolyticus.

Movement on surfaces, or swarming motility, is effectively mediated by the lateral flagellar (laf) system in Vibrio parahaemolyticus. Expression of laf is induced by conditions inhibiting rotation of the polar flagellum, which is used for swimming in liquid. However, not all V. parahaemolyticus isolates swarm proficiently. The organism undergoes phase variation between opaque (OP) and translucent (TR) cell types. The OP cell produces copious capsular polysaccharide and swarms poorly, whereas the TR type produces minimal capsule and swarms readily. OP<-->TR switching is often the result of genetic alterations in the opaR locus. Previously, OpaR, a Vibrio harveyi LuxR homolog, was shown to activate expression of the cpsA locus, encoding capsular polysaccharide biosynthetic genes. Here, we show that OpaR also regulates swarming by repressing laf gene expression. However, in the absence of OpaR, the swarming phenotype remains tightly surface regulated. To further investigate the genetic controls governing swarming, transposon mutagenesis of a TR (DeltaopaR1) strain was performed, and SwrT, a TetR-type regulator, was identified. Loss of swrT, a homolog of V. harveyi luxT, created a profound defect in swarming. This defect could be rescued upon isolation of suppressor mutations that restored swarming. One class of suppressors mapped in swrZ, encoding a GntR-type transcriptional regulator. Overexpression of swrZ repressed laf expression. Using reporter fusions and quantitative reverse transcription-PCR, SwrT was demonstrated to repress swrZ transcription. Thus, we have identified the regulatory link that inhibits swarming of OP strains and have begun to elucidate a regulatory circuit that modulates swarming in TR strains.

Separation of active and inactive fractions from starved culture of Vibrio parahaemolyticus by density dependent cell sorting.

The co-existence of physiologically different cells in bacterial cultures is a general phenomenon. We have examined the applicability of the density dependent cell sorting (DDCS) method to separate subpopulations from a long-term starvation culture of Vibrio parahaemolyticus. The cells were subjected to Percoll density gradient and separated into 12 fractions of different buoyant densities, followed by measuring the cell numbers, culturability, respiratory activity and leucine incorporation activity. While more than 78% of cells were in lighter fractions, about 95% of culturable cells were present in heavier fractions. The high-density subpopulations also had high proportion of cells capable of forming formazan granules. Although this was accompanied by the cell specific INT-reduction rate, both leucine incorporation rates and INT-reduction rates per cell had a peak at mid-density fraction. The present results indicated that DDCS could be used to separate subpopulations of different physiological conditions.