Transcriptional regulation of the ferric aerobactin receptor gene by a GntR-like repressor IutR in Vibrio furnissii.

We found that Vibrio furnissii can utilize aerobactin (AERO) as a xenosiderophore. A homology search of its genome revealed that this bacterium possesses genes encoding an AERO-mediated iron acquisition system similar to that of V. vulnificus. The system consists of the ABC transporter gene vatCDB, the GntR-type transcriptional repressor gene iutR, and the outer membrane receptor gene iutA. The functions of the vatCDB operon and iutA in V. furnissii were confirmed by the inability of the corresponding deletion mutants to utilize AERO. Reverse transcription-quantitative PCR revealed that iutA transcription under iron-limiting conditions was extensively activated by the addition of AERO to the growth medium; therefore, we focused on elucidating this phenomenon. Electrophoretic mobility shift and DNase I footprinting assays revealed that glutathione S-transferase-fused IutR (GST-IutR) bound directly to a specific palindromic sequence in the iutA promoter region. However, GST-IutR did not bind to this sequence when either AERO or ferric AERO was present in the assay mixture. These in vitro findings suggest that, under iron-limiting conditions, iutA transcription in V. furnissii is artfully regulated both by IutR, acting as a direct repressor of iutA, and by AERO, acting as an effector for IutR, leading to the derepression of iutA transcription.

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.

Identification and characterization of Aeromonas hydrophila genes encoding the outer membrane receptor of ferrioxamine B and an AraC-type transcriptional regulator.

We found that, under iron-limiting conditions, Aeromonas hydrophila ATCC 7966(T) could utilize the xenosiderophore desferrioxamine B (DFOB) for growth by inducing the expression of its own outer membrane receptor. Two consecutive genes, desR and desA, were selected as candidates involved in DFOB utilization. The presence of the ferric-uptake regulator boxes in their promoters suggested that these genes are under iron-dependent regulation. Mutation of desA, a gene that encodes the outer membrane receptor of ferrioxamine B, disrupted the growth of the amonabactin-deficient mutant in the presence of DFOB. ß-Galactosidase reporter assays and reverse transcriptase-quantitative PCR demonstrated that desR, a gene that encodes an AraC-like regulator homolog is required for the induction of desA transcription in the presence of DFOB and under iron-limiting conditions. The functions of desA and desR were analyzed using complementation experiments. Our data provided evidence that DesA is powered primarily by the TonB2 system.

Regulation of the expression of the Vibrio parahaemolyticus peuA gene encoding an alternative ferric enterobactin receptor.

A pvsB-vctA-irgA triple deletion mutant of Vibrio parahaemolyticus can utilize enterobactin under iron-limiting conditions by inducing a previously undescribed receptor, PeuA (VPA0150), in response to extracellular alkaline pH and enterobactin. In silico analyses revealed the existence of a two-component regulatory system operon, peuRS, immediately upstream of peuA, which constitutes an operon with the TonB2 system genes. Both the peuRS and peuA-tonB2 operons were found to be upregulated under iron-limiting conditions in a ferric uptake regulator (Fur)-dependent manner. The involvement of peuA and peuRS in enterobactin utilization was analyzed by complementation experiments using deletion mutants. Primer extension analysis indicated that, under iron-limiting conditions, the transcription of peuA was initiated from the +1 site at pH 7.0 and from both the +1 and +39 sites at pH 8.0 in the presence of enterobactin. The +39 transcript was absent from the peuRS deletion mutant. Secondary structure prediction of their 5'-untranslated regions suggested that translation initiation is blocked in the +1 transcript, but not in the +39 transcript. Consistent with this, in vitro translation analysis demonstrated that production of PeuA was determined only by the +39 transcript. These studies establish a novel gene regulation mechanism in which the two-component regulatory system PeuRS enhances expression of the alternative +39 transcript that possesses non-inhibitory structure, allowing the peuA expression to be regulated at the translation stage.

Role of periplasmic binding proteins, FatB and VatD, in the vulnibactin utilization system of Vibrio vulnificus M2799.

Vibrio vulnificus, an opportunistic marine bacterium that causes a serious, often fatal, infection in humans, requires iron for its pathogenesis. This bacterium uses iron from the environment via the vulnibactin-mediated-iron-uptake system. In this study, we constructed the deletion mutants of the genes encoding the proteins involved in the vulnibactin-mediated-iron-uptake system, isochorismate synthase (ICS), vulnibactin utilization protein (VuuB), periplasmic ferric-vulnibactin binding protein (FatB), and ferric-vulnibactin receptor protein (VuuA). The Δics and ΔvuuA mutants were unable to grow under low-iron concentration conditions compared with the isogenic wild-type, indicating that the involvement of ICS in the vulnibactin biosynthesis pathway and uptake of ferric-vulnibactin through the VuuA receptor protein are essential for V. vulnificus M2799 growth under low-iron concentration conditions. Similar growth impairment was also observed in ΔfatB, with growth recovery of this mutant observed 6 h after the beginning of the culture. These results indicate that there must be other periplasmic ferric-vulnibactin binding proteins in V. vulnificus M2799 that complement the defective fatB gene. Complementary growth studies confirmed that VatD protein, which functions as a periplasmic ferric-aerobactin binding protein, was found to participate in the ferric-vulnibactin uptake system in the absence of FatB. Furthermore, the expression of ics, vuuB, fatB, vuuA, and vatD genes was found to be regulated by iron and the ferric uptake regulator.

Spermidine-analogous triamines suppressed the growth of Candida albicans.

We examined the antifungal activity of various synthetic triamines on several fungi. Among various triamines having a general structure H2N(CH2)aNH(CH2)bNH2 (a=2-5, b=3-8), some triamines (a=4 or 5) showed inhibitory effect on the growth of Candida albicans and C. tropicalis. Determination of the minimum inhibitory concentrations (MICs) of these triamines on C. albicans showed that triamine 4-8 (a=4, b=8) and triamine 5-8 had strong antifungal activity. Further analysis revealed that the antifungal effect of triamine 4-8 was fungistatic and the antifungal effect was diminished by the addition of spermidine, a physiological triamine, to the medium. These results suggested that triamine 4-8 is antagonistic to spermidine and the antifungal activity is due to the suppression of the action of intrinsic polyamines. On the agar medium, C. albicans formed microcolonies even in the presence of triamine 4-8 by long cultivation. We then observed the form of C. albicans using microscope and found that the cells cultivated with triamine 4-8 were round, similar to the yeast form, while most of the cells on the agar medium without triamine 4-8 were hyphal form. Subsequently, we investigated the synergistic effect of two compounds with triamine 4-8, cyclohexylamine and dl-α-difluoromethylornithine which are inhibitors of enzymes involving in the biosynthesis of physiological polyamines such as spermidine. The results showed that the antifungal activity of triamine 4-8 increased by the addition of these enzyme inhibitors.

The Vibrio parahaemolyticus small RNA RyhB promotes production of the siderophore vibrioferrin by stabilizing the polycistronic mRNA.

High-affinity iron acquisition in Vibrio parahaemolyticus is mediated by the cognate siderophore vibrioferrin. We have previously reported that the vibrioferrin biosynthesis operon (pvsOp) is regulated at the transcriptional level by the iron-responsive repressor Fur (T. Tanabe, T. Funahashi, H. Nakao, S. Miyoshi, S. Shinoda, and S. Yamamoto, J. Bacteriol. 185:6938-6949, 2003). In this study, we identified the Fur-regulated small RNA RyhB and the RNA chaperone Hfq protein as additional regulatory proteins of vibrioferrin biosynthesis. We found that vibrioferrin production was greatly impaired in both the ryhB and hfq deletion mutants, and a TargetRNA search (http://snowwhite.wellesley.edu/targetRNA/index2.html) revealed that the 5'-untranslated region of pvsOp mRNA (pvsOp 5'-UTR) contains a potential base-pairing region required for the formation of the RyhB-pvsOp 5'-UTR duplex. An electrophoresis mobility shift assay indicated that RyhB can directly bind to the pvsOp 5'-UTR with the aid of Hfq. Rifampin chase experiments indicated that the half-life of pvsOp mRNA in the ryhB and hfq mutants was approximately 3-fold shorter than that in the parental strain, suggesting that both RyhB and Hfq are engaged in the stabilization of pvsOp mRNA. Chrome azurol S assays followed by electrophoresis mobility shift assays and rifampin chase experiments carried out for mutant strains indicated that base pairing between RyhB and the pvsOp 5'-UTR results in an increase in the stability of pvsOp mRNA, thereby leading to the promotion of vibrioferrin production. It is unprecedented that RyhB confers increased stability on a polycistronic mRNA involved in siderophore biosynthesis as a direct target.

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.

Identification and characterization of a cluster of genes involved in biosynthesis and transport of acinetoferrin, a siderophore produced by Acinetobacter haemolyticus ATCC 17906T.

Acinetobacter haemolyticus ATCC 17906(T) is known to produce the siderophore acinetoferrin under iron-limiting conditions. Here, we show that an operon consisting of eight consecutive genes, named acbABCD and actBCAD, participates in the biosynthesis and transport of acinetoferrin, respectively. Transcription of the operon was found to be iron-regulated by a putative Fur box located in the promoter region of the first gene, acbA. Homology searches suggest that acbABCD and actA encode enzyme proteins involved in acinetoferrin biosynthesis and an outer-membrane receptor for ferric acinetoferrin, respectively. Mutants defective in acbA and actA were unable to produce acinetoferrin or to express the ferric acinetoferrin receptor under iron-limiting conditions. These abilities were rescued by complementation of the mutants with native acbA and actA genes. Secondary structure analysis predicted that the products of actC and actD may be inner-membrane proteins with 12 membrane-spanning helices that belong to the major facilitator superfamily proteins. ActC showed homology to Sinorhizobium meliloti RhtX, which has been characterized as an inner-membrane importer for ferric rhizobactin 1021 structurally similar to acinetoferrin. Compared to the parental ATCC 17906(T) strain, the actD mutant displayed about a 35 % reduction in secretion of acinetoferrin, which was restored by complementation with actD, suggesting that ActD acts as an exporter of the siderophore. Finally, the actB product was significantly similar to hypothetical proteins in certain bacteria, in which genes encoding ActBCA homologues are arranged in the same order as in A. haemolyticus ATCC 17906(T). However, the function of ActB remains to be clarified.

Identification and characterization of an outer membrane receptor gene in Acinetobacter baumannii required for utilization of desferricoprogen, rhodotorulic acid, and desferrioxamine B as xenosiderophores.

In this study, we found that Acinetobacter baumannii utilized exogenously supplied desferricoprogen, rhodotorulic acid, and desferrioxamine B for growth under iron-limiting conditions. The ferric uptake regulator (Fur) titration assay method was then successfully applied to select iron-regulated genes in A. baumannii genomic libraries. Part of the nucleotide sequence homologous to Escherichia coli, fhuE, obtained from one of the positive clones allowed us to clone the entire gene, which was named fhuE. The fhuE gene had an amino acid sequence consistent with the N-terminal amino acid sequence of the 76-kDa iron-repressible outer membrane proteins in A. baumannii. Reverse transcription-polymerase chain reaction analysis demonstrated that fhuE mRNA is transcribed under iron-limiting conditions, consistent with the presence of a sequence homologous to the consensus Fur box in the promoter region. Disruption of fhuE resulted in the loss of expression of the 76-kDa protein. In addition, the double disruptant of fhuE and basD, which encodes one of the biosynthetic genes for the cognate siderophore acinetobactin, was unable to grow in the presence of desferricoprogen, rhodotorulic acid or desferrioxamine B. However, growth of the double disruptant was restored by complementation with fhuE, demonstrating that A. baumannii FhuE functions as the receptor common to coprogen, ferric rhodotorulic acid and ferrioxamine B.

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.

NK1.1(+) cells regulate neutrophil migration in mice with Acinetobacter baumannii pneumonia.

Acinetobacter baumannii is a major cause of both community-associated and nosocomial infections worldwide. These infections are difficult to treat because the bacterium rapidly develops resistance to multiple antibiotics. However, little is known about the nature of the innate cellular response to A. baumannii infection. In the present study, we identified the cells infiltrating the lungs of mice with Acinetobacter pneumonia and analyzed their response to infection. Normal mice eradicated the A. baumannii infection within 3 days of inoculation. Neutrophils were rapidly recruited to the lungs, followed by macrophages and NK1.1(+) cells. Neutrophil-depleted mice showed acute and severe symptoms, and all of the mice died within 3 days of inoculation. The majority of macrophage-depleted mice responded in a similar manner to the control mice. These results indicate that neutrophils are essential for the elimination of A. baumannii. Half of NK1.1(+) cell-depleted mice died within 1 day of inoculation and the number of infiltrating neutrophils was lower than that in control mice up until 3 days post-inoculation. Moreover, the expression levels of keratinocyte chemoattractant protein (KC) decreased in NK1.1(+) cell-depleted mice. These results indicate that NK1.1(+) cells recruit neutrophils during the early phase of Acinetobacter infection by increasing KC expression.

The Vibrio parahaemolyticus pvuA1 gene (formerly termed psuA) encodes a second ferric vibrioferrin receptor that requires tonB2.

We previously reported that the Vibrio parahaemolyticus pvsABCDE and psuA-pvuABCDE operons are involved in the biosynthesis and transport of its own siderophore, vibrioferrin (VF). Of these, psuA and pvuA encode TonB-dependent outer-membrane proteins (OMPs). Although pvuA was characterized as the ferric vibrioferrin receptor gene, the role of the psuA product remains unknown. In this study, a growth assay of isogenic psuA, pvuA, and psuA-pvuA double-deletion mutants followed by complementation of the double-deletion mutant with psuA or pvuA was used to identify psuA as a gene encoding an OMP involved in the uptake of ferric VF. Thus, psuA and pvuA were renamed pvuA1 and pvuA2, respectively. Moreover, we clarified the TonB specificities of PvuA1 and PvuA2, because V. parahaemolyticus has three sets of the TonB systems. The triple deletion of pvuA1, tonB1, and tonB2, and the double deletion of pvuA2 and tonB2 resulted in the complete loss of growth promotion by VF. This finding indicates that the energy required for PvuA1 and PvuA2 to transport ferric VF across the outer membrane is provided by the TonB2 system and by both the TonB1 and TonB2 systems, respectively.

Identification of genes, desR and desA, required for utilization of desferrioxamine B as a xenosiderophore in Vibrio furnissii.

We found that Vibrio (V.) furnissii ATCC35016 can gain iron through a xenosiderophore desferrioxamine B (DFOB) for its growth under iron-limiting conditions, concurrent with the expression of the 79-kDa iron-repressible outer membrane protein (IROMP) in response to the presence of DFOB. Based on the sequence of the ferrioxamine B (an iron-bound form of DFOB) receptor gene in V. vulnificus, two V. furnissii genes, termed desA and desR, encoding the 79-kDa IROMP and AraC-type transcriptional regulator, respectively, were identified and cloned. Nucleotide sequences located in the promoter regions of both desR and desA predicted the presence of consensus ferric uptake regulation (Fur)-binding sequences. The transcription of both genes was negatively regulated by exogenous iron levels. Deletion of the desA gene abolished the ability of V. furnissii to utilize DFOB, and neither desA mRNA nor DesA was detected in the deletion mutant of desR regardless of the presence of DFOB. The functions of DesA and DesR as the ferrioxamine B receptor and transcriptional activator for desA, respectively, were confirmed by complementation of desA and desR deletion mutants.

Identification and characterization of a Vibrio mimicus gene encoding the heme/hemoglobin receptor.

The present authors have previously reported that Vibrio mimicus expresses 77-kDa and 80-kDa outer membrane proteins in response to iron-limited conditions, and that the 77-kDa protein serves as the receptor for ferriaerobactin. In this study, it was found that V. mimicus can use heme and hemoglobin as iron sources. FURTA was then applied to V. mimicus 7PT to obtain candidate gene fragments involved in utilization of heme and hemoglobin. One FURTA-positive clone was shown to contain a partial gene, whose predicted amino acid sequence correlated with the N-terminal amino acid sequence determined for the 80-kDa outer membrane protein and also shared homology with heme/hemoglobin receptors of Gram-negative bacteria. Based on this information, the entire gene (named mhuA), and a gene upstream of mhuA (named mhuB) encoding a LysR family of transcriptional activator, were cloned and analyzed. RNA analysis indicated that mhuA and mhuB are each transcribed from individual Fur-regulated promoters. Moreover, RNA analysis of an mhuB deletion mutant and a promoter reporter assay coupled with ß-galactosidase suggested that MhuB could function as an activator for mhuA transcription. Finally, the role of MhuA as the heme/hemoglobin receptor was confirmed by construction of an mhuA deletion mutant and its complemented strain followed by growth assay.

Identification and characterization of genes required for utilization of desferri-ferrichrome and aerobactin in Vibrio parahaemolyticus.

During the course of our investigation on the iron acquisition systems in Vibrio parahaemolyticus, a causative agent of seafood-related gastroenteritis, we found that this species utilizes desferri-ferrichrome for growth as a heterologous siderophore (a siderophore produced by other bacteria and fungi) under iron-limiting conditions. N-Terminal amino acid sequence analysis of the iron-repressible outer membrane proteins followed by searches of the reported genomic sequences of this species identified four relevant genes (called fhuACDB) forming an operon. Deletion analysis of the fhuA and fhuD genes indicated that the most upstream gene fhuA and the three downstream genes fhuCDB encode the ferrichrome receptor and the ATP-binding cassette transport components, respectively. Moreover, it was found that the fhuCDB genes are also required for transport of ferric aerobactin which restores growth of this species under iron-limiting conditions.

Proteomic analysis of Vibrio vulnificus M2799 grown under iron-repleted and iron-depleted conditions.

Vibrio vulnificus is an opportunistic marine bacterium that causes a serious, often fatal, infection in human. An important factor that determines the survival of V. vulnificus in the human body is the ability to acquire iron. The differential expression of proteins in whole-cell lysates of V. vulnificus M2799, a clinical isolate, was evaluated under iron-repleted and iron-depleted conditions during the early, mid and late logarithmic growth phases. A total of 32, 53 and 42 iron-regulated spots were detected by two-dimensional differential gel electrophoresis (2D-DIGE) in the early, mid and late logarithmic growth phases, respectively. Of these, 18 (early logarithmic growth phase), 31 (mid logarithmic growth phase) and 26 (late logarithmic growth phase) proteins were subsequently identified by matrix-assisted laser desorption/ionization-time of flight analysis. These proteins were classified into 10 functional categories, including inorganic ion transport and metabolism, carbohydrate transport and metabolism, and amino acid transport and metabolism. Based on this classification, the expression of proteins involved in the iron acquisition system increased from the early to the mid logarithmic growth phases, while that of proteins involved in other metabolic pathways increased from the mid to the late logarithmic growth phases. Furthermore, when the protein expression profile of the wild type bacterium was compared with that of the fur mutant grown under the iron-repleted condition, the expression of 18 proteins was found to be regulated by iron and Fur.

Role of aldose reductase in the peritoneal changes of patients undergoing peritoneal dialysis.

BACKGROUND/AIMS: The mesothelium of patients undergoing peritoneal dialysis (PD) is exposed to glucose in dialysate. Glucose metabolites 3-deoxyglucosone and advanced glycation endproducts (AGEs) in the PD fluid induce peritoneal damage. Circulating factors also affect the peritoneum in the uremic model and predialysis patients. Aldose reductase (AR) generates precursors of 3-deoxyglucosone. We have reported AR acceleration in uremic patients. Therefore, AR acceleration might affect the peritoneum. The purpose of this study was to evaluate the AR level in PD patients and to determine the factors that change the peritoneum of these patients. METHODS: We measured the PD effluent (eff-) concentration of cancer antigen 125 (CA125) as a marker of mesothelial viability in PD patients. Erythrocyte AR, eff-, and plasma (p-) concentrations of 3-deoxyglucosone, AGEs, and malondialdehyde were also studied in 30 PD patients, 18 patients undergoing hemodialysis, and 8 control subjects. RESULTS: In the PD group, AR, p-3-deoxyglucosone, p-AGEs, and p-malondialdehyde were higher than in the control group. The predictors for eff-CA125 were not only PD duration and eff-3-deoxyglucosone, but also AR. CONCLUSION: AR was upregulated in PD patients. AR acceleration may affect the peritoneum in these patients. Further studies are needed to clarify the role of AR in PD patients.

Vibrio vulnificus damages macrophages during the early phase of infection.

Vibrio vulnificus is an estuarine bacterium that can cause primary septicemia as well as serious wound infections. Generally, clinical isolates have a high lethal effect compared with environmental isolates. However, little is known about the mechanisms by which V. vulnificus causes disease. In this study, we compared the pathogenicity of a clinical isolate, strain M2799, with that of an environmental isolate, strain JCM3731. The clinical isolate showed 100 times higher lethality in mice than the environmental isolate. In strain M2799-inoculated mice, the number of macrophages decreased significantly, whereas there was no appreciable change in the number of macrophages in strain JCM3731-inoculated mice. The clinical isolate showed high cytotoxic activity, especially to macrophages, compared with the environmental isolate in vitro. The growth of the clinical isolate was almost completely inhibited in the presence of macrophages. Moreover, the survival rate of the clinical isolate-inoculated mice increased by recruitment of macrophages. These results indicate that V. vulnificus infection progresses by damage to macrophages during the early phase of infection.

Stereoselective hexobarbital 3'-hydroxylation by CYP2C19 expressed in yeast cells and the roles of amino acid residues at positions 300 and 476.

We examined the enzymatic function of recombinant CYP2C19 in enantiomeric hexobarbital (HB) 3'-hydroxylation, and searched the roles of amino acid residues, such as Phe-100, Phe-114, Asp-293, Glu-300, and Phe-476 of CYP2C19 in the stereoselective HB 3'-hydroxylation, using a yeast cell expression system and site-directed mutagenesis method. CYP2C19 wild-type exerted substrate enantioselectivity of (R)-HB>>(S)-HB and metabolite diastereoselectivity of 3'(R)<3'(S) in 3'-hydroxylation of HB enantiomers. The substitution of Asp-293 by alanine failed to yield an observable peak at 450 nm in its reduced carbon monoxide-difference spectrum. CYP2C19-E300A and CYP2C19-E300V with alanine and valine, respectively, in place of Glu-300 exerted total HB 3'-hydroxylation activities of 45 and 108%, respectively, that of the wild-type. Interestingly, these two mutants showed substrate enantioselectivity of (R)-HB<(S)-HB, which is opposite to that of the wild-type, while metabolite diasteroselectivity remained unchanged. The replacement of Phe-476 by alanine increased total HB 3'-hydroxylation activity to approximately 3-fold that of the wild-type. Particularly, 3'(S)-OH-(S)-HB-forming activity elevated to 7-fold that of the wild-type, resulting in the reversal of the substrate enantioselectivity. In contrast, the substitution of phenylalanine at positions 100 and 114 by alanine did not produce a remarkable change in the total activity or the substrate enantioselectivity. These results indicate that Glu-300 and Phe-476 are important in stereoselective oxidation of HB enantiomers by CYP2C19.