Yasunori Isshiki

Oral Immunization with ATP-Dependent Protease-Deficient Mutants Protects Mice against Subsequent Oral Challenge with Virulent Salmonella enterica Serovar Typhimurium

ABSTRACT 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 × 108 CFU of the ClpXP- or Lon-deficient strain, bacteria were detected with 103 to 104 CFU in the spleen, mesenteric lymph nodes, Peyers 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, Peyers 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.

Isolation and characterisation of disodium (4-amino-4-deoxy-β-l-arabinopyranosyl)-(1⃗8)-(d-glycero-α-d-talo-oct-2-ulopyranosylonate)-(2⃗4)-(methyl 3-deoxy-d-manno-oct-2-ulopyranosid)onate from the lipopolysaccharide of Burkholderia cepacia

A trisaccharide was isolated from the core oligosaccharide in the lipopolysaccharide (LPS) of Burkholderia cepacia GIFU 645 (ATCC 25416, type strain) by methanolysis followed by HPLC and saponification. It was identified by MS, methylation analysis and 1H and 13C NMR spectroscopy as disodium (4-amino-4-deoxy-beta-L-arabinopyranosyl)-(1-->8)-(D-glycero- alpha-D-talo-oct-2-ulopyranosylonate)-(2-->4)-(methyl 3-deoxy-D-manno-oct-2-ulopyranosid)onate. In addition to the trisaccharide derivative, methanolysis gave dimethyl (D-glycero-alpha-D- talo-oct-2-ulopyranosylonate)-(2-->4)-(methyl 3-deoxy-D-manno-oct-2- ulopyranosid)onate in a relative proportion to the trisaccharide of 3:1, indicating a non-stoichiometric (approximately 25%) substitution of the octulosonic acid by 4-amino-4-deoxyarabinose in the LPS.

PROTECTION AGAINST SHIGA TOXIN 1 CHALLENGE BY IMMUNIZATION OF MICE WITH PURIFIED MUTANT SHIGA TOXIN 1

ABSTRACT 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.

Structural analysis of a new glycosphingolipid from the lipopolysaccharide-lacking bacterium Sphingomonas adhaesiva.

A new glycosphingolipid, GSL-4B, was isolated from Sphingomonas adhaesiva and found to share the ceramide moiety with GSL-1 and GSL-3 from Sphingomonas capsulata studied earlier [Kawahara, K.; Moll, H.; Knirel, Y. A.; Seydel, U.; Zähringer, U. Eur. J. Biochem. 2000, 267, 1837-1846]. It is heterogeneous with respect to the long-chain bases erythro-2-amino-1,3-octadecanediol (sphinganine), (13Z)-erythro-2-amino-13-eicosene-1,3-diol, and (13Z)-erythro-2-amino-13,14-methylene-1,3-eicosanediol which in GSL-4B are present in the ratios of 1.1:1.0:1.1, and all bearing amide-linked (S)-2-hydroxymyristic acid. Methylation analysis and MALDI-TOF-MS along with 1H and 13C NMR spectroscopy showed that the carbohydrate part of GSL-4B has the structure of alpha-D-Glcp-(1-->4)-alpha-D-Galp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-GlcpA-(1-->1)-Cer

Structural analysis of the carbohydrate backbone of Vibrioparahaemolyticus 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.

Lipopolysaccharide of Burkholderia cepacia and Its Unique Character To Stimulate Murine Macrophages with Relative Lack of Interleukin-1β-Inducing Ability

ABSTRACT Lipopolysaccharide (LPS) of Burkholderia cepacia was purified by the conventional phenol-water extraction method (preparation BcLPS-1), followed by enzymatic treatments with DNase, RNase, trypsin, and proteinase K (preparation BcLPS-2), and finally by deoxycholate-phenol-water extraction (preparation BcLPS-3). Cells of LPS-hyporesponsive C3H/HeJ mice were activated by both the BcLPS-1 and the BcLPS-2 preparations but barely activated by BcLPS-3. When LPS-responsive C3H/HeN mice were used as targets, endotoxic activities such as lethal toxicity to galactosamine-sensitized mice, mitogenicity to spleen cells, and activation of macrophages to induce tumor necrosis factor alpha and interleukin-6 (IL-6) were strongly exhibited even by highly purified BcLPS-3 at levels comparable to those of the highly active enterobacterial LPS of Salmonella enterica serovar Abortus-equi (SaeLPS), used as the control. The ability of BcLPS-3 to activate murine macrophages for induction of IL-1β was, however, much weaker than that of SaeLPS. Both accumulation of pro-IL-1β protein and expression of IL-1β mRNA in macrophages by stimulation with BcLPS-3 were much weaker than by stimulation with SaeLPS. These results indicate that LPS of B. cepacia has the potential to play a role as a pathogenic factor with strong activity comparable to that of usual enterobacterial LPS, but unlike the latter, this LPS has a relative lack of ability in the activation of murine macrophages to induce IL-1β. The lack of IL-1β-inducing ability appears to be caused by incomplete signal transduction somewhere in the upstream step(s) of IL-1β gene transcription.

Extraction and Characterization of the Smooth‐Type Lipopolysaccharide from Fusobacterium nucleatum JCM 8532 and Its Biological Activities

The lipopolysaccharide (LPS) of Fusobacterium nucleatum JCM 8532 was isolated by hot‐phenol water extraction. Most of the LPS was extracted in the phenolic phase and shown to be the smooth‐type, whereas the aqueous phase contained mainly rough‐type LPS. The chemical composition of the LPS was similar to that reported in other studies, but D‐quinovosamine, which may be a major component of O‐antigenic polysaccharide, and 3‐deoxy‐D‐manno‐2‐octulosonic acid (Kdo) were detected for the first time by gas chromatography‐mass spectrometry. The biological activities of smooth‐type LPS, including limulus activity, lethal toxicity, pyrogenicity, and B lymphocyte mitogenicity, were comparable to those of enterobacterial LPS. Smooth‐type LPS inhibited the cell growth and DNA synthesis of adult and fetal human gingival fibroblasts in a dose‐dependent manner, suggesting that LPS may play a role in the occurrence of human gingivitis.

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.

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

ABSTRACT 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-α) 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-1β (IL-1β)-inducing activity while activity to induce TNF-α and IL-6 has been shown to be as strong as that of the reference LPS. Remarkably, PmxB augmented the IL-1β-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-1β-inducing activity was also observed.

Enterohemorrhagic Escherichia coli O157:H7 possesses somatic (O) antigen identical with that of Salmonella O301.

The O antigen of enterohemorrhagicEscherichia coli O157:H7 is identical with that ofSalmonella O301 and is also related toSalmonella O301302 in an a-a, b type of relationship.