Effects of Oakmoss Components on Extra- and Intracellular Legionella pneumophila and Its Host Acanthamoeba castellanii.

Acanthamoeba castellanii is a ubiquitous organism found in environmental water. The amoeba is pathogenic to toward humans and is also a reservoir of bacteria of the genus Legionella, a causative agent of legionellosis. Oakmoss, a source of natural fragrance ingredients, and its components are antibacterial agents that are specifically active against the genus Legionella. In the present study, oakmoss and its components were investigated for their inhibitory effects on total (extra- and intracellular) Legionella pneumophila within A. castellanii and on L. pneumophila within A. castellanii. Among the oakmoss components, 3-hydroxy-5-methylphenyl 2,4-dihydroxy-6-methylbenzoate (1), 3-methoxy-5-methylphenyl 2,4-dihydroxy-6-methylbenzoate (2), and 8-(2,4-dihydroxy-6-(2-oxoheptyl)phenoxy)-6-hydroxy-3-pentyl-1H-isochromen-1-one (8) reduced the number of total bacteria (extra- and intracellular) in a test culture and also exhibited high amoebicidal activity against L. pneumophila within A. castellanii at concentrations lower than their IC50 values for A. castellanii. In contrast, 6,8-dihydroxy-3-pentyl-1H-isochromen-1-one (5) reduced the total number of L. pneumophila and, also that of total bacteria after 24 h of treatment (P < 0.05), whereas the compound did not exhibit amoebicidal activity against L. pneumophila within A. castellanii at concentrations lower than its IC50 value against A. castellanii. Thus, it is suggested that these oakmoss components could be good candidates for disinfectants to protect from Legionella infection.

Evaluation of Antibacterial Activity Expression of the Hinokitiol/Cyclodextrin Complex Against Bacteria.

The purpose of this study was to assess the antimicrobial activity of a solid dispersion prepared by mixing and grinding hinokitiol (HT) with α-cyclodextrin (αCD), ß-cyclodextrin (ßCD), or γ-cyclodextrin (γCD). Antimicrobial activity was evaluated by calculating the minimum inhibitory concentration (MIC) and evaluating the change in the number of bacteria over time. The test microbes used were two Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus), two Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa), and two fungi (Candida albicans and Aspergillus brasiliensis). Calculation of the MIC value of HT using the agar dilution method revealed that the MIC of HT/CD inclusion complexes was lower than that of HT alone. HT irreversibly inhibited the growth of microorganisms in a short amount of time. HT/CD complexes retained the antimicrobial activity of HT as a result of including HT in a CD complex. These results suggest that inclusion of HT, an antimicrobial component, using CDs could lead to appropriate control of the drug release rate and efficient display of antimicrobial activity.

Effects of Fragrance Ingredients on the Uptake of Legionella pneumophila into Acanthamoeba castellanii.

Acanthamoeba castellanii, a ubiquitous organism in water environments, is pathogenic toward humans and also is a host for bacteria of the genus Legionella, a causative agent of legionellosis. Fragrance ingredients were investigated for their antibacterial activity against planktonic Legionella pneumophila, amoebicidal activity against A. castellanii, and inhibitory effect against L. pneumophila uptake into A. castellanii. Helional® exhibited relatively high antibacterial activity [minimum inhibitory concentration (MIC) , 32.0 µg/mL] . Anis aldehyde, canthoxal, helional® and vanillin exhibited amoebicidal activity (IC50 values, 58.4±2.0, 71.2±14.7, 66.8±8.3 and 49.1±2.5µg/mL, respectively) . L. pneumophila pretreatment with sub-MICs (0.25×MIC) of anis aldehyde, canthoxal, cortex aldehyde® 50 percent or vanillin evidently reduced L. pneumophila uptake into A. castellanii (p < 0.01) . Thus, fragrance ingredients were good candidates for disinfectant against L. pneumophila and A. castellanii.

Effects of oakmoss and its components on Acanthamoeba castellanii ATCC 30234 and the uptake of Legionella pneumophila JCM 7571 (ATCC 33152) into A. castellanii.

Acanthamoeba castellanii, a ubiquitous organism in water environments, is pathogenic toward humans and also is a host for bacteria of the genus Legionella, a causative agent of legionellosis. Oakmoss, a natural fragrance ingredient, and its components are antibacterial agents specifically against the genus Legionella. In the present study, oakmoss and its components were investigated for their amoebicidal activity against A. castellanii ATCC 30234 and the inhibitory effect on the uptake of L. pneumophila JCM 7571 (ATCC 33152) into A. castellanii. The oakmoss and its components 3-hydroxy-5-methylphenyl 2,4-dihydroxy-6-methylbenzoate(5), and 6,8-dihydroxy-3-pentyl-1H-isochromen-1-one (12) exhibited high amoebicidal activity (IC50 values; 10.5 ± 2.3, 16.3 ± 4.0 and 17.5 ± 2.8 µg/mL, respectively) after 48 h of treatment, which were equivalent to that of the reference compound, chlorhexidine gluconate. Pretreatment of L. pneumophila with sub-minimal inhibitory concentration of oakmoss, compound 5, 3-hydroxy-5-methylphenyl 2-hydroxy-4-methoxy-6-methylbenzoate (10) and 8-(2,4-dihydroxy-6-pentylphenoxy)-6-hydroxy-3-pentyl-1H-isochromen-1-one (14) obviously reduced the uptake of L. pneumophila into A.castellanii (p < 0.05).The inhibitory effect of compound 5 on the uptake of L. pneumophila was almost equivalent to that of ampicillin used as a reference. Thus, the oakmoss and its components were considered to be good candidates for disinfectants against not only genus Legionella but also A. castellanii.

Evaluation of the live vaccine efficacy of virulence plasmid-cured, and phoP- or aroA-deficient Salmonella enterica serovar Typhimurium in mice.

We evaluated the protective efficacy of 94-kb virulence plasmid-cured, and phoP- or aroA-deficient strains of Salmonella enterica serovar Typhimurium (ΔphoP or ΔaroA S. Typhimurium) as oral vaccine candidates in BALB/c mice. Two weeks after the completion of 3 oral immunizations with 1 × 10(8) colony-forming units (CFU) of virulence plasmid-cured, and ΔphoP or ΔaroA S. Typhimurium at 10-day intervals, S. Typhimurium lipopolysaccharide (LPS)-specific mucosal secretory immunoglobulin A (s-IgA) antibody titers were detected in the cecal homogenate, bile and lung lavage fluid, but not in the intestinal lavage fluid. In addition, the increases in S. Typhimurium LPS-specific immunoglobulin G (IgG) and IgA antibody titers in the serum were also observed 2 weeks after completing 3 oral immunizations with virulence plasmid-cured, and ΔphoP or ΔaroA S. Typhimurium. The series of 3 oral immunizations protected the mice against an oral challenge with 5 × 10(8) CFU of the virulent strain of S. Typhimurium, suggesting that both the virulence plasmid-cured, and ΔphoP and ΔaroA S. Typhimurium strains are promising candidates for safe and effective live S. Typhimurium vaccines.

Effects of oakmoss and its components on biofilm formation of Legionella pneumophila.

Oakmoss and its components are known as antibacterial agents, specifically against Legionella pneumophila. In the present study, we investigated the effects of oakmoss and its components (phenol, didepside and isochromen derivatives) on L. pneumophila biofilm formation, with particular reference to the bactericidal activity (minimum bactericidal concentration; MBC) of these components against the bacterial cells in the biofilm. Of the 20 compounds tested, two didepside derivatives and four phenol derivatives reduced biofilm formation by more than 50% of that observed for the control at their respective minimum inhibitory concentrations (1/2×MIC). The inhibitory activities of these compounds were either equivalent to or greater than that of the clarithromycin reference. Isochromen derivatives had no effect on biofilm formation. Analysis of bactericidal activity of didepside and isochromen derivatives revealed that three of four didepside derivatives and one of four isochromen derivatives exhibited high bactericidal activity (MBC: 32.0-74.7 µg/mL) against the L. pneumophila in the biofilm after 24 h or 48 h of co-incubation; the antibacterial activities of these compounds were almost equivalent to clarithromycin and chlorhexidine gluconate (MBC: 42.7-64.0 µg/mL) that were used as references. Thus, based on their anti-biofilm forming and bactericidal activities, didepside derivatives are considered to be good candidates for disinfectants against L. pneumophila.

The antibacterial activity of compounds isolated from oakmoss against Legionella pneumophila and other Legionella spp.

Oakmoss is a natural fragrance ingredient exhibiting highly specific, potent antibacterial activity against Legionella pneumophila, a causative agent of severe water-bone pneumonia. In the present study, the antibacterial activity of individual compounds isolated from oakmoss was investigated against L. pneumophila and other Legionella spp. A total of 18 known compounds and two minor novel compounds (i.e., 3-methoxy-5-methylphenyl-2,4-dihydroxy-6-methylbenzoate (compound 9) and 8-(2,4-dihydroxy-6-(2-oxoheptyl)-phenoxy)-6-hydroxy-3-pentyl-1H-isochromen-1-one (compound 20)) were purified from oakmoss. The minimum inhibitory concentrations (MICs) against clinical and environmental isolates of L. pneumophila, L. bozemanii, L. micdadei, L. longbeachae, and L. dumoffii for 11 of the 20 compounds were less than 100 µg/mL (range 0.8-64.0 µg/mL). Novel compounds 9 and 20 exhibited potent antibacterial activity against L. pneumophila strains (MIC ranges of 1.3-8.0 µg/mL and 3.3-13.3 µg/mL, respectively) and also against four other Legionella species (MIC ranges of 0.8-8.0 µg/mL and 3.3-21.3 µg/mL, respectively). Time-kill assays indicated that compounds 9 and 20 kill bacteria at a concentration equivalent to 2×MIC after 1 h and 6 h co-incubations, respectively. While oakmoss and the purified components exhibited antibacterial activity against Legionella spp., they were not active against other Gram-negative and -positive bacteria such as Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Staphylococcus aureus.

Characterization of the carbohydrate backbone of Vibrio parahaemolyticus O6 lipopolysaccharides.

Structural characterization studies have been carried out on the carbohydrate backbone of Vibrio parahaemolyticus serotype O6 lipopolysaccharides (LPS). The carbohydrate backbone isolated from O6 LPS by sequential derivatization, that is, dephosphorylation, O-deacylation, pyridylamination, N-deacylation and N-acetylation, is a nonasaccharide consisting of 3 mol of D-glucosamine (GlcN) (of which one is pyridylaminated), 2 mol of L-glycero-D-manno-heptose (Hep), and 1 mol each of D-galactose (Gal), D-glucose (Glc), D-glucuronic acid (GlcA) and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). Structural analyses by nuclear magnetic resonance spectroscopy and fast-atom bombardment mass spectrometry demonstrated that the carbohydrate backbone is ß-Galp-(1→2)-α-Hepp-(1→3)-α-Hepp-(1→5)-α-Kdop-(2→6)-ß-GlcpNAc-(1→6)-GlcNAc-PA, in which the 3-substituted α-Hepp is further substituted by ß-GlcpNAc-(1→4)-ß-Glcp at position 4 and by ß-GlcpA at position 2. In native O6 LPS, an additional 1 mol of D-galacturonic acid, which is liberated by dephosphorylation in hydrofluoric acid, is present at an unknown position. A previous study by the present authors reported that, of 13 O-serotype LPS of V. parahaemolyticus, the only LPS from which Kdo was detected was from O6 LPS after mild acid hydrolysis. In the present study, we have demonstrated that only 1 mol of Kdo is present at the lipid A proximal position, a component which is common to the LPS in all serotypes of the bacterium, and that there is no additional Kdo in the carbohydrate backbone of O6 LPS. ELISA and ELISA inhibition analysis using antisera against O6 and Salmonella enterica Minnesota R595 and LPS of both strains further revealed that Kdo is not involved as an antigenic determinant of O6 LPS.

The antibacterial activity of fragrance ingredients against Legionella pneumophila.

In the current study we investigated the antibacterial activity of fragrance ingredients against Legionella pneumophila, a causative agent of severe pneumonia. Among the 41 different fragrance ingredients tested, we found that the natural fragrance ingredients oakmoss (OM) and birch tar oil (BT), which contain many components, exhibit potent antibacterial activity. The minimum inhibitory concentration (MIC, % (v/v)) of OM and BT were 0.0020 and 0.0024, respectively and were lower than that of cinnamic aldehyde (0.0078), which has been previously shown to possess high antimicrobial activity. In a time-kill assay of OM and BT at MIC and two times MIC, the colony forming units (CFU) of the microbe were reduced to between 10(-3) to 10(-4) of the original CFU after 1 h co-incubation. After this time, the CFU gradually decreased in number, but remained above detection levels even after a 48-h co-incubation, except for BT at two times MIC. In contrast, at a concentration of 0.1% OM and BT (approximately 50 times MIC), CFU were not detected after co-incubation for 1 h. Another 18 fragrance ingredients including ketone, aldehyde, lactone, acid, phenol derivative, aliphatic alcohol and quinoline also exhibited a lesser degree of antibacterial activity against L. pneumophila at a MIC of less than 0.10.

Reactions of 26-iodopseudodiosgenin and 26-iodopseudodiosgenone with various nucleophiles and pharmacological activities of the products.

26-Iodopseudodiosgenin (8) and 26-iodopseudodiosgenone (9) were reacted with various nucleophiles (KSCN, KOCN, NaCN, NaN(3) and various amines) to give pseudodiosgenin derivatives (4, 12, 16-20, 26) and pseudodiosgenone derivatives (5, 13, 21-25, 27), respectively. The reactions of 8 and 9 with KOCN gave the elimination products (10) and (11), respectively. The reaction of 9 with NaCN gave 5alpha,26- (14) and 5beta,26-dicyanocholestan-3-one (15). The reaction of 8 with NaN3 gave triazepine derivative (30), while that of 9 gave 26-azidopseudodiosgenone (31). Compound 31 was converted into triazepine derivative (32) by heating at 120 degrees C. The cytotoxicity of the pseudodiosgenins and pseudodiosgenones on P-gp-underexpressing HCT 116 cells and P-gp-overexpressing Hep G2 cells was examined by MTT assay. Pseudodiosgenins 2, 4, 12 and 30 showed strong cytotoxic activity (IC50 values: 2.6+/-0.3-6.7+/-1.4 microM), as did pseudodiosgenones 3, 5, 11, 13, 21-25 and 27 (IC50 values: 1.3+/-0.3-6.4+/-0.3 microM) toward HCT 116 cells. Pseudodiosgenins 12, 16 and 30 (IC50 values: 1.2+/-0.7-2.2+/-0.6 microM) and pseudodiosgenones 22, 23, 25 and 27 (IC50 values: 0.6+/-0.1-2.5+/-0.3 microM) were highly cytotoxic to Hep G2 cells. Compounds 3 and 27 showed efficient antibacterial activity (MIC: 15.6, 10.4 microg/ml) and (MIC: 7.8, 15.6 microg/ml) against Bacillus subtilis and Staphylococcus aureus, respectively.

Azithromycin inhibits the formation of flagellar filaments without suppressing flagellin synthesis in Salmonella enterica serovar typhimurium.

The present study shows that a sub-MIC of the macrolide antibiotic azithromycin (AZM) diminishes the virulence function of Salmonella enterica serovar Typhimurium. We first constructed an AZM-resistant strain (MS248) by introducing ermBC, an erythromycin ribosome methylase gene, into serovar Typhimurium. The MIC of AZM for MS248 exceeded 100 microg/ml. Second, we managed to determine the efficacy with which a sub-MIC of AZM reduced the virulence of MS248 in vitro. On the one hand, AZM (10 microg/ml) in the culture medium was unable to inhibit the total protein synthesis, growth rate, or survival within macrophages of MS248. On the other hand, AZM (10 microg/ml) reduced MS248's swarming and swimming motilities in addition to its invasive activity in Henle-407 cells. Electron micrographs revealed no flagellar filaments on the surface of MS248 after overnight growth in L broth supplemented with AZM (10 microg/ml). However, immunoblotting analysis showed that flagellin (FliC) was fully synthesized within the bacterial cells in the presence of AZM (10 microg/ml). In contrast, the same concentration of AZM reduced the export of FliC to the culture medium. These results indicate that a sub-MIC of AZM was able to affect the formation of flagellar filaments, specifically by reducing the amount of flagellin exported from bacterial cells, but it was not involved in suppressing the synthesis of flagellin. Unfortunately, AZM treatment was ineffective against murine salmonellosis caused by MS248.

Structure of the core-oligosaccharide with a characteristic D-glycero-alpha-D-talo-oct-2-ulosylonate-(2-->4)-3-deoxy-D-manno-oct-2-ulosonate [alpha-Ko-(2-->4)-Kdo] disaccharide in the lipopolysaccharide from Burkholderia cepacia.

The core oligosaccharide in the lipopolysaccharide (LPS) of Burkholderia cepacia GIFU 645(T) was investigated. After mild acid hydrolysis of the LPS, a heptasaccharide was isolated and identified by chemical analyses, GLC-MS, FABMS, and NMR spectroscopy as follows: [carbohydrate structure: see text] where L-alpha-D-Hep stands for L-glycero-alpha-D-manno-heptose, Ko for D-glycero-D-talo-oct-2-ulosonic acid, and Kdo for 3-deoxy-D-manno-oct-2-ulosonic acid.

Structural characterization of the carbohydrate backbone of the lipopolysaccharide of Vibrio parahaemolyticus O-untypeable strain KX-V212 isolated from a patient.

Vibrio parahaemolyticus strain KX-V212 of a novel serotype, which does not belong to any of the known 13 O-serotypes of this vibrio, was isolated from a patient. Its O-antigen harbors a unique strain-specific O-antigenic factor(s), in addition to that shared by the O-antigen of V. parahaemolyticus serotype O2. A carbohydrate backbone nonasaccharide was isolated from the lipopolysaccharide (LPS) of strain KX-V212 by dephosphorylation, reduction and deacylation and found to consist of one residue each of D-glucose, D-galactose, D-GlcN, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and 5-acetamido-7-(N-acetyl-D-alanyl)amino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (Non5Ac7Ala), and two residues each of D-GlcA and L-glycero-D-manno-heptose (LD-Hep). Analysis of the isolated and deacylated lipid A showed that this oligosaccharide was an artifact resulting from a loss of one GlcN residue from the lipid A backbone. Therefore, the carbohydrate backbone of the LPS is a decasaccharide having the structure shown below. The initial LPS contains also D-GalA and phosphoethanolamine at unknown positions. Both similarity and differences are observed between the LPS of V. parahaemolyticus serotype O2 and strain KX-V212. [carbohydrate structure: see text]

Unusual interaction of a lipopolysaccharide isolated from Burkholderia cepacia with polymyxin B.

We have demonstrated that lipopolysaccharide (LPS) obtained from Burkholderia cepacia, an important opportunistic pathogen, has unique characteristics in both structure and activity. One of the structural characteristics is that the B. cepacia LPS has 4-amino-4-deoxy-L-arabinose (Ara4N) in its inner core region. Polymyxin B (PmxB) is known to act as an LPS antagonist, but LPS with Ara4N is suggested to be PmxB resistant by decreasing the binding capability of PmxB. Interaction of B. cepacia LPS with PmxB was investigated and compared with that of a reference LPS of Salmonella enterica serovar Abortusequi, referred to hereafter as the reference LPS. B. cepacia LPS suffered no suppressive effect of PmxB in its activity to stimulate murine peritoneal macrophages for induction of tumor necrosis factor alpha (TNF-alpha) and IL-6 even when the activity of the reference LPS was completely suppressed. A characteristic of B. cepacia LPS is that it has selectively weak interleukin-1 beta (IL-1 beta)-inducing activity while activity to induce TNF-alpha and IL-6 has been shown to be as strong as that of the reference LPS. Remarkably, PmxB augmented the IL-1 beta-inducing activity of B. cepacia LPS to the level of that of the reference LPS and, in contrast, completely suppressed the strong activity of the reference LPS. Using PmxB-immobilized beads, the adsorbances of these LPSs to the beads were compared, and it was found that B. cepacia LPS bound to PmxB with a high affinity similar to that of the reference LPS. These results indicate an unusual interaction of B. cepacia LPS with PmxB whereby B. cepacia LPS not only allows the binding of PmxB with high affinity, even though it contains Ara4N, but also suffers no suppressive effect of PmxB on its activity. Moreover, a remarkable increase in its IL-1 beta-inducing activity was also observed.

Protection against Shiga toxin 1 challenge by immunization of mice with purified mutant Shiga toxin 1.

Shiga toxin 1 (Stx1) of enterohemorrhagic Escherichia coli O157:H7 was cloned, and four mutant Stx1s were constructed by site-directed mutagenesis with PCR. The wild-type and mutant Stx1s with amino acid replacements at positions 167 and 170 of the A subunit were purified by one-step affinity chromatography with commercially available Globotriose Fractogel, and the mutant Stxs were used for the immunization of mice. The mutant toxins were nontoxic to Vero cells in vitro and to mice in vivo and induced the immunoglobulin G antibody against the wild-type Stx1, which neutralized the cytotoxicity of Stx1. The induced antibody titers depended on the mutation at position 170 of the A subunit. The mice immunized with the mutant Stx1s were protected against a challenge of approximately 100 times the 50% lethal dose of the wild-type Stx1, suggesting that the mutant toxins are good candidates for toxoid vaccines for infection by Stx1-producing E. coli.

Structure and serological characterization of 5,7-diamino-3,5,7,9-tetradeoxy-non-2-ulosonic acid isolated from lipopolysaccharides of Vibrio parahaemolyticus O2 and O-untypable strain KX-V212.

Lipopolysaccharides (LPS) of Vibrio parahaemolyticus O2 and O-untypable (OUT) strain (KX-V212) isolated from an individual patient were shown to contain 5,7-diamino-3,5,7,9-tetradeoxy-non-2-ulosonic acid (NonlA), which was readily released from LPS by mild acid hydrolysis. In the present study, we investigated the chemical and serological properties of NonlA isolated from LPS of V. parahaemolyticus O2 and OUT KX-V212. GC-MS and NMR analysis identified the NonlA from LPS of O2 to be 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (5NAc7NAcNonlA) and that from LPS of KX-V212 to be 5-acetamido-7-(N-acetyl-D-alanyl)amido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (5NAc7NAlaNAcNonlA). In ELISA inhibition analysis, 5NAc7NAcNonlA inhibited the O2 LPS/anti-O2 antiserum system, whereas, 5NAc7NAlaNAcNonlA did not show any inhibitory activity. However, after N-deacylation of 5NAc7NAlaNAcNonlA followed by N-acetylation, the product (5NAc7NAcNonlA) inhibited the O2 LPS/anti-O2 antiserum system to the same extent as that of 5NAc7NAcNonlA obtained from O2 LPS. These results suggest that 5NAc7NAcNonlA might be related to the serological specificity of O2 LPS as one of main epitope(s) involved in O2 LPS.

Structural analysis of the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides.

A structural investigation has been carried out on the carbohydrate backbone of Vibrio parahaemolyticus O2 lipopolysaccharides (LPS) isolated by dephosphorylation, O-deacylation and N-deacylation. The carbohydrate backbone is a short-chain saccharide consisting of nine monosaccharide units i.e., 1 mol each of D-galactose (Gal), D-glucose (Glc), D-glucuronic acid (GlcA), L-glycero-D-manno-heptose (L,D-Hep), D-glycero-D-manno-heptose (D,D-Hep), 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (NonlA), and 2 mol of 2-amino-2-deoxy-D-glucose (D-glucosamine, GlcN). Based on the data obtained by NMR spectroscopy, fast-atom bombardment mass spectrometry (FABMS) and methylation analysis, a structure was elucidated for the carbohydrate backbone of O2 LPS. In the native O2 LPS, the 2-amino-2-deoxy-D-glucitol (GlcN-ol) at the reducing end of the nonasaccharide is present as GlcN. The lipid A backbone is a beta-D-GlcN-(1-->6)-D-GlcN disaccharide as is the case for many Gram-negative bacterial LPS. The lipid A proximal Kdo is substituted by the distal part of the carbohydrate chain at position-5. In the native O2 LPS, D-galacturonic acid, which is liberated from LPS by mild acid treatment or by dephosphorylation in hydrofluoric acid, is present although its binding position is unknown at present.

Serum antibodies of periodontitis patients compared to the lipopolysaccharides of Porphyromonas gingivalis and Fusobacterium nucleatum.

Serum antibody titers against the lipopolysaccharides (LPSs) of Porphyromonas gingivalis and Fusobacterium nucleatum were compared between 9 periodontitis patients and 24 healthy persons. The IgG titers against the LPSs of P. gingivalis ATCC 33277(T) and W50 were clearly higher in the patients than in the healthy persons. However, IgM titers against the LPSs of P. gingivalis strains were relatively low, and no significant difference was observed between the patients and healthy persons. On the other hand, IgG and IgM titers against the LPS of Fusobacterium nucleatum JCM 8532(T) in some patients were significantly higher than those in the healthy persons, although the difference in IgG titers was not large compared to that of the LPS of P. gingivalis. These results suggest that the antibody measurement of patients' sera against the LPS of periodontal bacteria can be applied for the diagnosis of periodontitis.

Oral immunization with ATP-dependent protease-deficient mutants protects mice against subsequent oral challenge with virulent Salmonella enterica serovar typhimurium.

We evaluated the efficacy of mutants with a deletion of the stress response protease gene as candidates for live oral vaccine strains against Salmonella infection through infection studies with mice by using a Salmonella enterica serovar Typhimurium mutant with a disruption of the ClpXP or Lon protease. In vitro, the ClpXP protease regulates flagellum synthesis and the ClpXP-deficient mutant strain exhibits hyperflagellated bacterial cells (T. Tomoyasu et al., J. Bacteriol. 184:645-653, 2002). On the other hand, the Lon protease negatively regulates the efficacy of invading epithelial cells and the expression of invasion genes (A. Takaya et al., J. Bacteriol. 184:224-232, 2002). When 5-week-old BALB/c mice were orally administered 5 x 10(8) CFU of the ClpXP- or Lon-deficient strain, bacteria were detected with 10(3) to 10(4) CFU in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum 1 week after inoculation and the bacteria then decreased gradually in each tissue. Significant increases of lipopolysaccharide-specific immunoglobulin G (IgG) and secretory IgA were detected at week 4 and maintained until at least week 12 after inoculation in serum and bile, respectively. Immunization with the ClpXP- or Lon-deficient strain protected mice against oral challenge with the serovar Typhimurium virulent strain. Both the challenged virulent and immunized avirulent salmonellae were completely cleared from the spleen, mesenteric lymph nodes, Peyer's patches, and even cecum 5 days after the challenge. These data indicate that Salmonella with a disruption of the ATP-dependent protease ClpXP or Lon can be useful in developing a live vaccine strain.

An immunochemical study of serological cross-reaction between lipopolysaccharides from Vibrio cholerae O22 and O139.

A comparative chemical and serological study of the LPS of Vibrio cholerae O139 and O22 was performed. Chemical analysis revealed that the sugar composition of the LPS of strain O22 was quite similar to that of O139 LPS. Each contained D-glucose, L-glycero-D-manno-heptose, colitose (3,6-dideoxy-L-galactose), D-fructose, D-glucosamine, D-quinovosamine and D-galacturonic acid. The O-antigenic relationship between the two strains was analysed by passive haemolysis (PH) and passive haemolysis inhibition (PHI) tests with the respective LPS being used as antigens to sensitize sheep red blood cells (SRBC) and, in the latter case, as inhibitors in a PH system that consisted of LPS-sensitized SRBC, guinea-pig complement and anti-O139 or anti-O22 antiserum, both unabsorbed and absorbed with the heterologous antigen. In the PH experiment, unabsorbed anti-O139 antiserum had haemolytic titres of 66,000 and 22,000 against O139 LPS- and O22 LPS-sensitized SRBC, respectively; unabsorbed anti-O22 antiserum had haemolytic titres of 900 and 13,000, respectively. Thus, the anti-O139 antiserum contained an antibody that reacted with a heterologous O22 antigen at a high titre (22,000) and this antibody was completely removed from anti-O139 antiserum with the O22 antigen. The anti-O22 antiserum contained an antibody that reacted with the heterologous O139 antigen at a low titre (900) and this antibody was completely removed from anti-O22 antiserum with the O139 antigen. In PHI tests O139 LPS and O22 LPS each strongly inhibited (the ID50 of LPS ranged from 0.03 to 0.14 microgram ml-1) the heterologous haemolytic systems of both O139 LPS-sensitized SRBC/anti-O22 antiserum and O22 LPS-sensitized SRBC/anti-O139 antiserm, which are substantially equivalent to the common antigen factor in the O139 LPS-sensitized SRBC/anti-O22 antiserum system and the common antigen factor in the O22 LPS-sensitized SRBC/anti-O139 antiserum system, respectively. The results indicated that the O antigen of O139 is closely related to that of O22 in an a,b-a,c type of relationship where a is common antigenic factor, b is an O139-specific antigenic factor and c is an O22-specific antigenic factor.