Kazuyoshi Kawahara

Recognition of bacterial glycosphingolipids by natural killer T cells

Natural killer T (NKT) cells constitute a highly conserved T lymphocyte subpopulation that has the potential to regulate many types of immune responses through the rapid secretion of cytokines. NKT cells recognize glycolipids presented by CD1d, a class I-like antigen-presenting molecule. They have an invariant T-cell antigen receptor (TCR) α-chain, but whether this invariant TCR recognizes microbial antigens is still controversial. Here we show that most mouse and human NKT cells recognize glycosphingolipids from Sphingomonas, Gram-negative bacteria that do not contain lipopolysaccharide. NKT cells are activated in vivo after exposure to these bacterial antigens or bacteria, and mice that lack NKT cells have a marked defect in the clearance of Sphingomonas from the liver. These data suggest that NKT cells are T lymphocytes that provide an innate-type immune response to certain microorganisms through recognition by their antigen receptor, and that they might be useful in providing protection from bacteria that cannot be detected by pattern recognition receptors such as Toll-like receptor 4.

Invariant natural killer T cells recognize glycolipids from pathogenic Gram-positive bacteria

Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell antigen receptor (TCR), but the forces that drive TCR conservation have remained uncertain. Here we show that NKT cells recognized diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells were required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is present in low concentrations in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR and, most notably, extend the range of microbes recognized by this conserved TCR to several clinically important bacteria.

Modification of the structure and activity of lipid A in Yersinia pestis lipopolysaccharide by growth temperature.

ABSTRACT Yersinia pestis strain Yreka was grown at 27 or 37°C, and the lipid A structures (lipid A-27°C and lipid A-37°C) of the respective lipopolysaccharides (LPS) were investigated by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Lipid A-27°C consisted of a mixture of tri-acyl, tetra-acyl, penta-acyl, and hexa-acyl lipid As, of which tetra-acyl lipid A was most abundant. Lipid A-37°C consisted predominantly of tri- and tetra-acylated molecules, with only small amounts of penta-acyl lipid A; no hexa-acyl lipid A was detected. Furthermore, the amount of 4-amino-arabinose was substantially higher in lipid A-27°C than in lipid A-37°C. By use of mouse and human macrophage cell lines, the biological activities of the LPS and lipid A preparations were measured via their abilities to induce production of tumor necrosis factor alpha (TNF-α). In both cell lines the LPS and the lipid A from bacteria grown at 27°C were stronger inducers of TNF-α than those from bacteria grown at 37°C. However, the difference in activity was more prominent in human macrophage cells. These results suggest that in order to reduce the activation of human macrophages, it may be more advantageous for Y. pestis to produce less-acylated lipid A at 37°C.

Invariant natural killer T cells recognize glycolipids from pathogenic Gram-positive bacteria.

Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell antigen receptor (TCR), but the forces that drive TCR conservation have remained uncertain. Here we show that NKT cells recognized diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells were required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is present in low concentrations in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR and, most notably, extend the range of microbes recognized by this conserved TCR to several clinically important bacteria.

Synergistic action of cholera toxin B subunit (and Escherichia coli heat-labile toxin B subunit) and a trace amount of cholera whole toxin as an adjuvant for nasal influenza vaccine

Cholera toxin B subunit (CTB) and Escherichia coli heat-labile toxin (LTB) (2 micrograms), each supplemented with a trace amount of cholera toxin (CT) (0.02-20 ng), were examined for the adjuvant effect on antibody (Ab) response against influenza inactivated HA (haemagglutinin) vaccine in Balb/c mice. Each mouse received a primary intranasal (i.n.) inoculation of the vaccine (1.5 micrograms) and the CT-containing CTB and in 4 weeks a second i.n. inoculation of the vaccine alone. The primary inoculation of the vaccine with CTB alone did not induce either anti-HA IgA or IgG Ab response, or haemagglutination-inhibition Ab responses in the serum. The vaccine with less than 2 ng of CT also failed to induce Ab response. On the other hand, the vaccine with CT-containing CTB induced a high Ab response, which increased depending on the CT dose. Moreover, the second vaccine induced a response more than ten times higher than the primary one and the response increased depending on the CT dose. Similar enhancement was found in the local anti-HA IgA Ab response in the nasal wash. Such synergistic effects were observed also between LTB and CT. The amount of Ab produced by the synergism was considered to be enough to protect against virus infection. These results suggest that CTB (or LTB) containing a trace amount of CT (about 0.1%) can be used practically as a potent adjuvant for nasal vaccination of humans against influenza.

Escherichia coli heat-labile enterotoxin B subunits supplemented with a trace amount of the holotoxin as an adjuvant for nasal influenza vaccine

Escherichia coli heat-labile enterotoxin B subunit (LTB) (2 micrograms), supplemented with a trace amount of the holotoxin (LT) (0.02-20 ng), was examined for the adjuvant effect on antibody (Ab) responses against influenza inactivated haemagglutinin (HA) vaccine in Balb/c mice. Each mouse received a primary intranasal (i.n.) inoculation with the vaccine (1.5 micrograms), prepared from PR8 (H1N1) virus, together with LT-containing LTB and in 4 weeks a second i.n. inoculation of the vaccine alone. The inoculation of the vaccine with the LT-containing LTB induced significantly high primary and secondary anti-HA IgA and IgG Ab responses in the nasal wash and the serum, while the vaccine with LTB or less than 2 ng of LT induced little response. The synergistic adjuvant effect was maximal in the concentration of LTB supplemented with 0.2-2 ng of LT. Under these conditions, the augmented IgA and IgG Ab responses, which are cross-protective to PR8 HA molecules, provided complete cross-protection against PR8 virus challenge in mice immunized with heterologous vaccine within the same subtype. These results suggest that LTB containing a trace amount of LT can be used as a potent adjuvant for nasal vaccination of humans against influenza.

Evidence of correlation between 50-kilobase plasmid of Salmonella choleraesuis and its virulence.

A plasmid of 50 kilobases (kb) was found in all six strains of Salmonella choleraesuis studied, originally isolated from septicemic swine or man. A derivative cured of the 50 kb plasmid was isolated after treatment with novobiocin, and its virulence compared to that of the parent by intraperitoneal injection into mice. The lethality of the parent strain was found higher than that of the cured strain. With a sublethal number of cells of the parent strain injected, the spleen, liver and lymph nodes in various sites were markedly enlarged, and the bacteria were isolated from the heart blood. In contrast to this, neither enlargement of the organs nor recovery of organisms was observed with the cured strain. The Tn1-tagged 50 kb plasmid was next introduced by transformation into the cured strain. The virulence of the plasmid-reintroduced strain was higher than that of the cured strain, and in fact identical to that of the parent. We conclude that the 50 kb plasmid of S. choleraesuis is closely related with the mouse virulence of the organism.

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.

Natural Sphingomonas Glycolipids Vary Greatly in Their Ability to Activate Natural Killer T Cells

Mouse natural killer T (NKT) cells expressing an invariant T cell antigen receptor (TCR) recognize glycosphingolipids (GSLs) from Sphingomonas bacteria. The synthetic antigens previously tested, however, were designed to closely resemble the potent synthetic agonist alpha-galactosyl ceramide (alphaGalCer), which contains a monosaccharide and a C18:0 sphingosine lipid. Some Sphingomonas bacteria, however, also have oligosaccharide-containing GSLs, and they normally synthesize several GSLs with different sphingosine chains including one with a cyclopropyl ring-containing C21:0 (C21cycl) sphingosine. Here we studied the stimulation of NKT cells with synthetic GSL antigens containing natural tetrasaccharide sugars, or the C21cycl sphingosine. Our results indicate that there is a great degree of variability in the antigenic potency of different natural Sphingomonas glycolipids, with the C21cycl sphingosine having intermediate potency and the oligosaccharide-containing antigens exhibiting limited or no stimulatory capacity.

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.