Tatsuya Funahashi

Identification and Characterization of Genes Required for Biosynthesis and Transport of the Siderophore Vibrioferrin in Vibrio parahaemolyticus

In response to low iron availability, Vibrio parahaemolyticus synthesizes and secretes a polyhydroxycarboxylate-type siderophore vibrioferrin which is composed of 1 mol each of 2-ketoglutaric acid, L-alanine, ethanolamine, and citric acid. We have previously reported the cloning and characterization of the pvuA gene, which encodes the 78-kDa outer membrane receptor protein for ferric vibrioferrin. In this study, nine genes involved in the biosynthesis and transport of vibrioferrin have been identified in the genomic regions surrounding the pvuA gene. The genes were sequenced, and gene disruptants were constructed by insertion mutation for phenotype analysis. Five of the genes, named pvsABCDE, constitute an operon that is expressed under iron-limiting conditions. Homology searches of their predicted protein products suggested that the four genes pvsABDE are implicated in the biosynthesis of the siderophore. Another gene in the same operon, pvsC, encodes a putative exporter that is homologous to members of the major facilitator superfamily of multidrug efflux pumps. The remaining four genes, named pvuBCDE, encode proteins strongly homologous to Escherichia coli FecBCDE, respectively, which are components of the ATP-binding cassette transporter system for ferric dicitrate. Reverse transcriptase PCR analysis revealed that these transport genes are transcribed as a single mRNA with the upstream genes, psuA and pvuA. Phenotypic comparison between the wild-type strain and its targeted gene disruptants supported the biological functions for the respective operons that were expected on the basis of the homology search.

Conversion of cannabidiol to Δ9-tetrahydrocannabinol and related cannabinoids in artificial gastric juice, and their pharmacological effects in mice

Cannabidiol (CBD), a nonpsychoactive cannabinoid, was found to be converted to 9α-hydroxyhexahydrocannabinol (9α-OH-HHC) and 8-hydroxy-iso-hexahydrocannabinol (8-OH-iso-HHC) together with Δ9-tetrahydrocannabinol (Δ9-THC), a psychoactive cannabinoid, and cannabinol in artificial gastric juice. These cannabinoids were identified by gas chromatography-mass spectrometry (GC-MS) by comparison with the spectral data of the authentic compounds. Pharmacological effects of 9α-OH-HHC and 8-OH-iso-HHC in mice were examined using catalepsy, hypothermia, pentobarbital-induced sleep prolongation, and antinociception against acetic acid-induced writhing as indices. The ED50 values (effective dose producing a 50% reduction of control; mg/kg, i.v.) of 9α-OH-HHC and 8-OH-iso-HHC for the cataleptogenic effect were 8.0 and 30.4, respectively. 8-OH-iso-HHC (10 mg/kg, i.v.) produced a significant hypothermia from 15 to 90 min after administration, although 9α-OH-HHC failed to induce such an effect at the same dose. However, both HHCs (10 mg/kg, i.v.) significantly prolonged pentobarbital-induced sleeping time by 1.8 to 8.0 times as compared with the control solution with 1% Tween 80-saline. The ED50 values (mg/kg, i.v.) of 9α-OH-HHC and 8-OH-iso-HHC for the antinociceptive effect were 14.1 and 39.4, respectively. The present study demonstrated that CBD can be converted to Δ9-THC and its related cannabinoids, 9α-OH-HHC and 8-OH-iso-HHC, in artificial gastric juice, and that these HHCs show Δ9-THC-like effects in mice, although their pharmacological effects were less potent than those of Δ9-THC.

Identification and Characterization of Two Contiguous Operons Required for Aerobactin Transport and Biosynthesis in Vibrio mimicus

In response to iron deprivation, Vibrio mimicus produces aerobactin as a major siderophore. Application of the Fur titration assay to a V. mimicus genomic DNA library followed by further cloning of the surrounding regions led to the identification of two adjacent, iron‐regulated operons. One contains three genes encoding homologs of the Escherichia coli FhuCDB and the other, five genes encoding homologs of the E. coli IucABCD IutA. Construction of the V. mimicus polar disruptants in the respective operons allowed us to confirm their functions. The genetic arrangement of the aerobactin‐mediated iron acquisition system in V. mimicus is unique in that the aerobactin operon (iucABCD iutA) is contiguous to the operon (matCDB) encoding components of an ATP‐binding cassette transport system for ferric aerobactin. This is the first report demonstrating that aerobactin transport and biosynthesis genes are present in a species outside the family Enterobacteriaceae.

Characterization of Vibrio parahaemolyticus Manganese-Resistant Mutants in Reference to the Function of the Ferric Uptake Regulatory Protein

In many bacteria, the ferric uptake regulatory protein (Fur) has a central role in the negative regulation of genes affected by iron limitation. In this study, Vibrio parahaemolyticus strains carrying mutations in the fur gene encoding Fur were isolated by the manganese selection method to assess the function of Fur in connection with alternations in the coordinate expression of the siderophore vibrioferrin (VF) and iron‐repressible outer membrane proteins (IROMPs). Ten out of 25 manganese‐resistant mutants constitutively produced VF and expressed at least two IROMPs irrespective of the iron concentration in the medium. PCR‐direct DNA sequencing of the fur genes in these mutants identified four different point mutations causing amino acid changes. Moreover, a fur overexpressing plasmid was constructed to prepare antiserum against V. parahaemolyticus Fur. Western blotting with this antiserum revealed that the intracellular abundance of the wild‐type Fur was not significantly affected by the iron concentrations in the growth medium, and that the Fur proteins of the mutant strains occurred at substantially smaller amounts and/or migrated more rapidly in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis than the wild‐type Fur. These data afford an additional insight into the structure‐function relationship of Fur and imply its involvement in the iron acquisition systems of V. parahaemolyticus, although it is yet unknown whether its action on the target genes is direct or indirect.

Identification and Characterization of the sodA Genes Encoding Manganese Superoxide Dismutases in Vibrio parahaemolyticus, Vibrio mimicus, and Vibrio vulnificus

Sequencing of Fur titration assay‐positive clones obtained from genomic DNA libraries of Vibrio parahaemolyticus, V. mimicus and V. vulnificus revealed open reading frames encoding proteins of 202, 205 and 202 amino acid residues, respectively. Each open reading frame was preceded by a predicted Fur box which overlaps a likely promoter with similarity to the −10 and −35 consensus sequence of Escherichia coli. The deduced amino acid sequences shared considerable homology with bacterial Mn‐containing superoxide dismutases (MnSODs). Consistent with this, these Vibrio strains produced proteins with SOD activity resistant to inhibition by H2O2 and KCN only when grown under iron‐limiting conditions. Primer extension analysis of the total RNA from these vibrios revealed iron‐repressible expression of the genes. Furthermore, when grown under iron‐limiting conditions, E. coli carrying a plasmid with each cloned gene over‐expressed protein with the same electrophoretic mobility and insensitivity of SOD activity to H2O2 and KCN. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis followed by N‐terminal amino acid sequencing revealed that proteins (MnSODs) having N‐terminal amino acid sequences consistent with those deduced from the corresponding genes were present in cell lysates of the vibrios grown under these iron‐limited conditions. These results demonstrate that the genes cloned in this study are sodA homologs encoding MnSODs, whose expression is regulated by the iron status of the growth medium. PCR using a primer set based on the V. parahaemolyticus sodA sequence revealed the presence of homologous genes in certain other Vibrio species.

Cloning and Sequencing of the Vibrio parahaemolyticus fur Gene

A ferric uptake regulatory gene (fur) was cloned from Vibrio parahaemolyticus WP1 by a polymerase chain reaction‐based technique followed by functional complementation of a fur mutation in Escherichia coli. A sequence analysis showed that, at the amino acid level, the V. parahaemolyticus Fur protein is 81% identical with the Fur protein from E. coli and over 90% identical with those of the Vibrio species.

Characterization of Vibrio parahaemolyticus genes encoding the systems for utilization of enterobactin as a xenosiderophore.

We determined the ability of Vibrio parahaemolyticus to utilize enterobactin (Ent) as a xenosiderophore. Homology searches of the V. parahaemolyticus genomic sequence revealed the presence of genes that are homologous to the V. cholerae ferric Ent utilization genes, which consist of the iron-repressible outer-membrane protein genes irgA and vctA, and the ATP-binding cassette transport system operon vctPDGC. Moreover, the irgB and vctR genes, which encode transcriptional regulators, were also found immediately upstream of irgA and vctA, respectively. Growth assays of V. parahaemolyticus indicated that both irgA and vctA mutants grew well in the presence of Ent under iron-limiting conditions, whereas both the irgA/vctA double mutant and the vctPDGC mutant barely grew under the same conditions. In addition, growth assays of three isogenic tonB mutants demonstrated that the TonB2 system, and to a lesser extent the TonB1 system, can provide energy for both IrgA and VctA to transport ferric Ent. SDS-PAGE analysis showed that expression of both IrgA and VctA was enhanced by the presence of Ent. Complementation of the irgB and vctR mutants with their respective genes resulted in the increased expression of IrgA and VctA, respectively. Finally, reverse transcriptase-quantitative PCR revealed that transcription of the Ent utilization system genes is iron-regulated, and that transcription of irgA and vctA under iron-limiting conditions is further activated by proteins encoded by irgB and vctR, respectively, together with Ent.

Characterization of a gene encoding the outer membrane receptor for ferric enterobactin in Aeromonas hydrophila ATCC 7966(T).

Aeromonas hydrophila ATCC 7966(T) produces a catecholate siderophore amonabactin in response to iron starvation. In this study, we determined that this strain utilizes exogenously supplied enterobactin (Ent) for growth under iron-limiting conditions. A homology search of the A. hydrophila ATCC 7966(T) genomic sequence revealed the existence of a candidate gene encoding a protein homologous to Vibrio parahaemolyticus IrgA that functions as the outer membrane receptor for ferric Ent. SDS-PAGE showed induction of IrgA under iron-limiting conditions. The growth of the double mutant of irgA and entA (one of the amonabactin biosynthetic genes) was restored when it was complemented with irgA in the presence of Ent. Moreover, a growth assay of three isogenic tonB mutants indicated that the tonB2 system exclusively provides energy for IrgA to transport ferric Ent. Finally, reverse transcriptase-quantitative PCR revealed that the transcription of irgA and the TonB2 system cluster genes is iron-regulated, consistently with the presence of a predicted Fur box in the promoter region.

8-Hydroxycannabinol: a new metabolite of cannabinol formed by human hepatic microsomes

Metabolism of cannabinol (CBN) was studied in vitro using hepatic microsomes from human livers. The metabolites formed were analyzed by thinlayer chromatography (TLC) and identified by gas chromatography-mass spectrometry as their trimethylsilyl derivatives. 11-Hydroxy-CBN, the major metabolite, was detected together with a smaller amount of another mono-hydroxylated metabolite. The minor metabolite was identified as 8-hydroxy-CBN, after comparing its Rf value by TLC, retention time by GC, and the mass spectrum with those of the authentic compound. 8-Hydroxy-CBN was confirmed to be a new metabolite of CBN formed by human hepatic microsomes.

The small RNA Spot 42 regulates the expression of the type III secretion system 1 (T3SS1) chaperone protein VP1682 in Vibrio parahaemolyticus.

The cytotoxicity of Vibrio parahaemolyticus has been related to the type III secretion system 1 effector protein VP1680, which is secreted and translocated into host cells with the help of the specific chaperone protein, VP1682. This study sought to confirm the in silico analysis, which predicted that a small regulatory RNA (Spot 42) could base pair with the region encompassing the ribosomal-binding site and initiation codon of the vp1682 mRNA. Electrophoresis mobility shift assays indicated that Spot 42 could bind to the vp1682 mRNA with the help of Hfq. Consistent with these results, the translation of the vp1682 mRNA was inhibited when both Hfq and Spot 42 were added to the in vitro translation reaction. The cytotoxic activity against infected Caco-2 cells was significantly increased in the Spot 42 deletion mutant (Δspf) at 4 h after infection as compared with the parental strain. Additionally, we observed that both VP1682 and VP1680 were more highly expressed in Δspf mutants than in the parental strain. These results indicate that Spot 42 post-transcriptionally regulates the expression of VP1682 in V. parahaemolyticus, which contributes to cytotoxicity in vivo.