Motohiro Matsuura

Biological activities of chemically synthesized analogues of the nonreducing sugar moiety of lipid A

Biological activities of five synthetic lipid A analogues (D‐glucosamine derivatives) were examined to elucidate the structure required for expression of the biological activities of endotoxin. Proclotting enzyme of horseshoe crab activation, interferon‐inducing and tumor necrosis factor‐inducing activities were significantly expressed by an analogue which possesses 4‐O‐phosphoryl, 3‐O‐tetradecanoyl and N‐tetradecanoyloxytetradecanoyl groups. The results obtained with different analogues show that the 4‐O‐phosphoryl and N‐tetradecanoyloxytetradecanoyl groups are important for expression of the above activities. The effect of 6‐O‐acylation in preventing the expression of these biological activities is also suggested. Pyrogenic activity was not detected in any of the compounds tested.

Structural modifications of bacterial lipopolysaccharide that facilitate Gram-negative bacteria evasion of host innate immunity

Bacterial lipopolysaccharide (LPS), a cell wall component characteristic of Gram-negative bacteria, is a representative pathogen-associated molecular pattern that allows mammalian cells to recognize bacterial invasion and trigger innate immune responses. The polysaccharide moiety of LPS primary plays protective roles for bacteria such as prevention from complement attacks or camouflage with common host carbohydrate residues. The lipid moiety, termed lipid A, is recognized by the Toll-like receptor 4 (TLR4)/MD-2 complex, which transduces signals for activation of host innate immunity. The basic structure of lipid A is a glucosamine disaccharide substituted by phosphate groups and acyl groups. Lipid A with six acyl groups (hexa-acylated form) has been indicated to be a strong stimulator of the TLR4/MD-2 complex. This type of lipid A is conserved among a wide variety of Gram-negative bacteria, and those bacteria are easily recognized by host cells for activation of defensive innate immune responses. Modifications of the lipid A structure to less-acylated forms have been observed in some bacterial species, and those forms are poor stimulators of the TLR4/MD-2 complex. Such modifications are thought to facilitate bacterial evasion of host innate immunity, thereby enhancing pathogenicity. This hypothesis is supported by studies of Yersinia pestis LPS, which contains hexa-acylated lipid A when the bacterium grows at 27°C (the temperature of the vector flea), and shifts to contain less-acylated forms when grown at the human body temperature of 37°C. This alteration of lipid A forms following transmission of Y. pestis from fleas to humans contributes predominantly to the virulence of this bacterium over other virulence factors. A similar role for less-acylated lipid A forms has been indicated in some other bacterial species, such as Francisella tularensis, Helicobacter pylori, and Porphyromonas gingivalis, and further studies to explore this concept are expected.

Listeria monocytogenes Strain-Specific Impairment of the TetR Regulator Underlies the Drastic Increase in Cyclic di-AMP Secretion and Beta Interferon-Inducing Ability

ABSTRACT Among a number of laboratory strains of Listeria monocytogenes used in experimental infection, strain LO28 is highly capable of inducing robust beta interferon (IFN-β) production in infected macrophages. In this study, we investigated the molecular mechanism of the IFN-β-inducing ability of LO28 by comparing it with that of strain EGD, a low-IFN-β-inducing strain. It was found that LO28 secretes a large amount of IFN-β-inducing factor, which turned out to be cyclic di-AMP. The secretion of cyclic di-AMP was dependent on MdrT, a multidrug resistance transporter, and LO28 exhibited a very high level of mdrT expression. The introduction of a null mutation into mdrT abolished the ability of LO28 to induce IFN-β production. Examination of genes responsible for the regulation of mdrT expression revealed a spontaneous 188-bp deletion in tetR of LO28. By constructing recombinant strains of LO28 and EGD in which tetR from each strain was replaced, it was confirmed that the distinct ability of LO28 is attributable mostly to tetR mutation. We concluded that the strong IFN-β-inducing ability of LO28 is due to a genetic defect in tetR resulting in the overexpression of mdrT and a concomitant increase in the secretion of cyclic di-AMP through MdrT.

Evasion of human innate immunity without antagonizing TLR4 by mutant Salmonella enterica serovar Typhimurium having penta-acylated lipid A

Modification of a lipid A moiety in Gram-negative bacterial LPS to a less acylated form is thought to facilitate bacterial evasion of host innate immunity, thereby enhancing pathogenicity. The contribution of less-acylated lipid A to interactions of whole bacterial cells with host cells (especially in humans) remains unclear. Mutant strains of Salmonella enterica serovar Typhimurium with fewer acylated groups were generated. The major lipid A form in wild-type (WT) and the mutant KCS237 strain is hexa-acylated; in mutant strains KCS311 and KCS324 it is penta-acylated; and in KCS369 it is tetra-acylated. WT and KCS237 formalin-killed and live bacteria, as well as their LPS, strongly stimulated production of pro-inflammatory cytokines in human U937 cells; this stimulation was suppressed by TLR4 suppressors. LPS of other mutants produced no agonistic activity, but strong antagonistic activity, while their formalin-killed and live bacteria preparations had weak agonistic and no antagonistic activity. Moreover, these less-acylated mutants had increased resistance to phagocytosis by U937 cells. Our results indicate that a decrease of one acyl group (from six to five) is enough to allow Salmonella to evade human innate immunity and that the antagonistic activity of less-acylated lipid A is not utilized for this evasion.

Effect of acyl substituents of synthetic lipid A-subunit analogues on their immunomodulating antiviral activity.

A chemically synthesized lipid A-subunit analogue, GLA-60, 2-deoxy-4-O-phosphono-2-[(3R)-3-hydroxytetradecanamido]-3-O-[(3R)- 3- tetradecanoyloxytetradecanoyl]-D-glucose, has many of the activities of endotoxin but has little toxicity. Then, compounds with various lengths of acyl side chain of the acyloxyacyl group at the 3-O position of GLA-60 were synthesized and evaluated for interferon (IFN)-inducing activity, natural killer (NK) cell activation and antiviral activity. The compounds with acyl side chains between C8 and C15 exhibited significant antiviral activity (inhibition of pox tail lesion formation in vaccinia virus-infected mice), serum IFN-inducing activity and NK cell activation. However, the compound carrying a C2 or a C16 acyl side chain did not exhibit these activities. The compounds with a C13 or C14 acyl side chain showed strong protective against herpes simplex virus type 1 in cyclophosphamide-immunosuppressed mice.

Enhancement of nonspecific resistance to microbial infections of synthetic lipid A-subunit analogues of GLA-27 modified at the C1 position of the glucosamine backbone

The C1 position of lipid A-subunit analogue GLA-27, 4-O-phosphono-D-glucosamine carrying N-3-tetradecanoyloxytetradecanoyl(C14-O-(C14)) and 3-O-tetradecanoyl (C14) groups, was S-acetylated, thiolated or phosphorylated. Enhancement of nonspecific resistance to Pseudomonas aeruginosa and vaccinia virus infections of these chemically modified compounds were investigated. Thiolation augmented the nonspecific resistance to P. aeruginosa infection. Protective activity against vaccinia virus infection was reduced by all the chemical modifications. NK cell activity was found not to be effected by S-acetylation, but to be decreased slightly by thiolation or phosphorylation. IFN-inducing activity was reduced remarkably by thiolation or S-acetylation, or completely diminished by phosphorylation, compared with that of GLA-27.

The role of cytokines in endotoxin activities

エンドトキシンは生体にとって有益な作用から障害的な作用にまで及ぶ,極めて多彩な活性を示す。その活性発現には,生体細胞より産生放出される種々の仲介物質の作用が介在していることが明らかになってきている。その仲介物質として,エンドトキシンの主要な標的細胞であるマクロファージ系細胞から産生放出される,TNFαやIL-1を始めIL-6,IL-8,IFNα/γ,CSFなどのサイトカイン類が大きな位置を占めていると考えられ,その役割の解明が進められている。これらサイトカインは各々が複数の活性を発現し,相互に関連し合うため,活性の発現が複雑多岐にわたり,また,活性が過剰に発現された場合には障害的な活性を発現することになると考えられている。サイトカインの産生や作用を抑制することによって,エンドトキシンショックの治療を目指す研究も進められている。また,エンドトキシン刺激によるサイトカイン産生のための細胞レベルでの刺激伝達機構に関する研究も急速に発展している。