Monoclonal antibodies against pertussis toxin subunits

Abstract Twenty monoclonal antibodies (mAbs) reacting with cholera toxin (CT) of Vibrio cholerae strain 569B were characterized in cross-section and G_M1 ganglioside inhibition assays. MAbs were characterized by reaction with CT and Escherichia coli heat-labile porcine strain (LT_p) and human strain (LTh) enterotoxins, and by G_M1 ganglioside inhibition of mAb binding. Eight of 10 CT-A specific and 3 of 10 CT-B-specific mAbs cross-reacted with LTh and LTp. G_M1 ganglioside inhibited reactions of the CT-B cross-reacting antibodies. Results showed that these epitodes common to the B subunit of CT and LT are located in or near the G_M1 ganglioside binding region, and that the G_M1 ganglioside-binding region of LT differs from that of CT.     

Genome size and restriction fragment length polymorphism analysis of Vibrio cholerae strains belonging to different serovars and biotypes

Abstract The genome size of Vibrio cholerae has been determined by pulsed field gel electrophoresis following digestion of chromosomal DNA with endonucleases. The genome size of all the classical strains examined was about 3000 kb and that of El Tor biotype was 2500 kb. The Not I and S fi I digestion patterns of the genomes of several V. cholerae straimns belonging to different serovars and biotypes showed distinct restriction fragment length polymorphism (RFLP). RFLP analysis together with the genome size can be used to differentiate strains of different serovars and biotypes of V. cholerae .     

eIF3k inhibits NF-κB signaling by targeting MyD88 for ATG5-mediated autophagic degradation in teleost fish

Optimal activation of NF-κB signaling is crucial for the initiation of inflammatory responses and eliminating invading bacteria. Bacteria have likewise evolved the ability to evade immunity; however, mechanisms by which bacteria dysregulate host NF-κB signaling are unclear. In this study, we identify eukaryotic translation initiation factor eIF3k, a nonessential member of the eIF3 translation initiation complex, as a suppressor of the NF-κB pathway. Mechanistically, we show that eIF3k expression induced by Vibrio harveyi enhances E3 ligase Nrdp1-mediated K27-linked ubiquitination of MyD88, an upstream regulator of NF-κB pathway activation. Furthermore, we show that eIF3k acts as a bridge linking ubiquitin-tagged MyD88 and ATG5, an important mediator of autophagy. We demonstrate that the MyD88-eIF3k-ATG5 complex is transported to the autophagosome for degradation, and that innate immune signaling is subsequently terminated and does not attack invading V. harveyi. Therefore, our study identifies eIF3k as a specific inhibitor of the MyD88-dependent NF-κB pathway and suggests that eIF3k may act as a selective autophagic receptor that synergizes with ATG5 to promote the autophagic degradation of MyD88, which helps V. harveyi to evade innate immunity. We conclude that V. harveyi can manipulate a host''s autophagy process to evade immunity in fish and also provide a new perspective on mammalian resistance to bacterial invasion.     

Evaluation of mutagenic and antimutagenic properties of some bioactive xanthone derivatives using Vibrio harveyi test

Aims: Drug safety evaluation plays an important role in the early phase of drug development, especially in the preclinical identification of compounds’ biological activity. The Vibrio harveyi assay was used to assess mutagenic and antimutagenic activity of some aminoalkanolic derivatives of xanthone (1–5), which were synthesized and evaluated for their anticonvulsant and hemodynamic activities. Methods and Results: A novel V. harveyi assay was used to assess mutagenic and antimutagenic activity of derivatives of xanthone 1–5. Two V. harveyi strains were used: BB7 (natural isolate) and BB7M (BB7 derivative containing mucA and mucB genes on a plasmid pAB91273, products of these genes enhance error‐prone DNA repair). According to the results obtained, the most beneficial mutagenic and antimutagenic profiles were observed for compounds 2 and 3. A modification of the chemical structure of compound 2 by the replacement of the hydroxy group by a chloride improved considerably the antimutagenic activity of the compound. Thus, antimutagenic potency reached a maximum with the presence of tertiary amine and chloride atom in the side chain. Conclusions: Among the newly synthesized aminoalkanolic derivatives of xanthone with potential anticonvulsant properties, there are some compounds exhibiting in vitro antimutagenic activity. In addition, it appears that the V. harveyi assay can be applied for primary mutagenicity and antimutagenicity assessment of compounds. Significance and Impact of the Study: The obtained preliminary mutagenicity and antimutagenicity results encourage further search in the group of amino derivatives of xanthone as the potential antiepileptic drugs also presenting some antimutagenic potential. Furthermore, V. harveyi test may be a useful tool for compounds safety evaluation.     

Effect of Na+and K+Ions on the Luminescence of Intact Vibrio harveyiCells at Different pH Values

The bioluminescent activity of intact Vibrio harveyicells loaded with different concentrations of NaCl and KCl at different pH values was studied. In the pH range of 6.5–8.5, the effect of Na⁺was significantly higher than that of K⁺at all concentrations studied. Maximum luminescent activity was observed in cells loaded with 0.68 M NaCl. When Na⁺was nonuniformly distributed on the plasma membrane, the cell luminescence kinetics was nonstationary in the 20-min range: during incubation, the luminescence intensity increased at pH 6.5 and decreased at pH 8.5. The activation and damping rate constants depended on the Na⁺gradient value. The maximum of luminescent activity shifted during incubation from pH 8.5 to 6.5–7.0. The luminescence kinetics in the systems with KCl was stationary; the maximum level of luminescence was observed in the pH range of 7.0–7.5. Under Na⁺-controlled conditions, the cell respiration and luminescence changed in synchronism. The protonophore CCP at a concentration of 20 M completely inhibited luminescence at pH 6.5 and was ineffective at pH 8.5.     

Acidophilic granulocytes of the marine fish gilthead seabream (<Emphasis Type="Italic">Sparus aurata</Emphasis> L.) produce interleukin-1β following infection with<Emphasis Type="Italic"> Vibrio anguillarum</Emphasis>

The fish immune response to Gram-negative bacteria is poorly understood. In this study, we use a monoclonal antibody (mAb) specific to acidophilic granulocytes from the marine fish gilthead seabream (Sparus aurata L.), together with an antiserum specific to interleukin-1 (IL-1) from this species, in order to investigate whether these cells are involved in the immune response against the pathogenic bacterium Vibrio anguillarum and, in particular, in the production of the pro-inflammatory cytokine IL-1. We found that gilthead seabream head-kidney, peritoneal exudate and peripheral blood leukocytes accumulated proIL-1 intracellularly when challenged in vitro with V. anguillarum, whereas only peritoneal exudate and blood leukocytes were able to accumulate proIL-1 following infection. Importantly, the blood leukocytes from infected animals that accumulated proIL-1 were shown to be the acidophilic granulocytes. A rapid mobilization of such cells from the head-kidney to the site of inflammation following infection with V. anguillarum was also observed. This work was supported by the Spanish Ministry of Science and Technology (grants BIO2001-2324-C02-02 and AGL2002-03529, and fellowship to J. García-Castillo), Spanish Ministry of Education, Culture and Sport (fellowship to E. Chaves-Pozo) and Fundación Séneca, Coordination Centre for Research (grant PI-51/00782/FS/01 and fellowship to P. Pelegrín).E. Chaves-Pozo and P. Pelegrín contributed equally to this work.     

Significance of Na+ in the fish pathogen, Vibrio anguillarum, under energy depleted condition

Vibrio anguillarum kills various kinds of fish over salinities ranging from seawater to freshwater. In this study, we investigated the role of Na(+) in V. anguillarum, especially under energy-depleted conditions such as in natural seawater. V. angustum S14, which is a typical marine vibrio, was used for comparison. V. anguillarum only required Na(+) for starvation-survival, but in contrast, V. angustum S14 always required Na(+) for both growth and starvation-survival. In marine vibrios, Na(+) is used in the Na(+)-dependent respiratory chain that produces the sodium motive force (SMF) across the cell membrane. It has been considered that marine vibrios always need a SMF produced by Na(+), however in the case of V. anguillarum, the SMF is not required for growth, but becomes more important for starvation-survival.     

Kinetics of Light Emission and Oxygen Consumption by Bioluminescent Bacteria

Oxygen plays a key role in bacterial bioluminescence. The simultaneous and continuous kinetics of oxygen consumption and light emission during a complete exhaustion of the exogenous oxygen present in a closed system has been investigated. The kinetics are performed with Vibrio fischeri, V. harveyi, and Photobacterium phosphoreum incubated on respiratory substrates chosen for their different reducing power. The general patterns of the luminescence time courses are different among species but not among substrates. During steady-state conditions, substrates, which are less reduced than glycerol, have, paradoxally, a better luminescence efficiency. Oxygen consumption by luciferase has been evaluated to be 17% of the total respiration. Luciferase is a regulatory enzyme presenting a positive cooperative effect with oxygen and its affinity for this final electron acceptor is about 4–5 times higher than the one of cytochrome oxidase. The apparent Michaelis constant for luciferase has been evaluated to be in the range of 20 to 65 nM O₂. When O₂ concentrations are as low as 10 nM, luminescence can still be detected; this means that above this concentration, strict anaerobiosis does not exist. By n-butyl malonate titration, it was clearly shown that electrons enter the luciferase pathway only when the cytochrome pathway is saturated. It is suggested that, in bioluminescent bacteria, luciferase acts as a free-energy dissipating valve when anabolic processes (biomass production) are impaired.