Bactericidal catechins damage the lipid bilayer.

The mode of antibacterial action of, the green tea (Camellia sinensis) extracts, (-)-epigallocatechin gallate (EGCg) and (-)-epicatechin (EC) was investigated. Strong bactericidal EGCg caused leakage of 5,6-carboxyfluorescein from phosphatidylcholine liposomes (PC), but EC with very weak bactericidal activity caused little damage to the membrane. Phosphatidylserine and dicetyl phosphate partially protected the membrane from EGCg-mediated damage when reconstituted into the liposome membrane with PC. EGCg, but not EC, caused strong aggregation and NPN-fluorescence quenching of PC-liposomes and these actions were markedly lowered in the presence of negatively charged lipids. These results show that bactericidal catechins primarily act on and damage bacterial membranes. The observation that Gram-negative bacteria are more resistant to bactericidal catechins than Gram-positive bacteria can be explained to some extent by the presence of negatively charged lipopolysaccharide.

Two forms of Vibrio cholerae O1 El Tor hemolysin derived from identical precursor protein.

Vibrio cholerae O1 grown in heart infusion broth produces two forms of El Tor hemolysin (ETH) monomers of 65 and 50 kDa. These monomers form several different sizes of mixed oligomers ranging from 180 to 280 kDa in the liposomal membranes. We found that the N-terminal amino acid sequences, NH2-Trp-Pro-Ala-Pro-Ala-Asn-Ser-Glu, of both the 65- and 50-kDa toxins were identical. We assumed, therefore, that the 65- and 50-kDa toxins were derivatives of the identical precursor protein and the 50-kDa protein was a truncated derivative of 65-kDa ETH. To substantiate this assumption, we treated the 260-kDa oligomer with trypsin and obtained a 190-kDa oligomer. This 190-kDa oligomer consisted of only the 50-kDa subunits. Both 260- and 190-kDa oligomers formed ion channels indistinguishable from each other in planar lipid bilayers. These results suggest that the essential part of the ETH in forming the membrane-damaging aggregate is a 50-kDa protein.

A cytocidal tissue kallikrein isolated from mouse submandibular glands.

A cytocidal factor against mouse thymocytes was purified from the submandibular glands of female BALB/c mice using Sephadex G-50 gel filtration chromatography and reverse-phase HPLC. SDS-PAGE and amino acid sequence analysis revealed that the cytocidal factor was mouse glandular kallikrein (mGK)-6. mGK-6 showed an optimal enzyme activity at pH 10 and a cytocidal activity against thymocytes in a dose-dependent manner.

Enhancement of lymphocyte proliferation by mouse glandular kallikrein.

Mouse glandular kallikrein (mGK) strongly enhanced the spontaneous and mitogen-induced proliferation of lymphocytes. Both blast formation and 3H-TdR incorporation were dose-dependently enhanced at the same time many cells were killed. The enhancing activity was independent of EGF, because EGF-binding proteins (mGK-9 in mGK-6,9 mixture and mGK-13), renal kallikrein (mGK-6) and human kallikrein all displayed the same enhancement. A serine proteinase inhibitor, diisopropyl fluorophosphate, could block the enhancement by mGK. The new function suggests that mGK is important in the immune system as a regulatory molecule.

A cytotoxic serine proteinase isolated from mouse submandibular gland.

We have isolated a novel cytotoxic factor from the submandibular glands of male BALB/c mice by Sephadex G-50 gel filtration chromatography and reverse-phase HPLC. The cytotoxic factor is a serine proteinase, which belongs to the mouse glandular kallikrein (mGK) family, with an Mr of approximately 27,000. The purified serine proteinase showed cytotoxic activity against mouse thymocytes in a dose-dependent manner, and a serine proteinase inhibitor, diisopropyl fluorophosphate, blocked its cytotoxic activity.

El Tor hemolysin of Vibrio cholerae O1 forms channels in planar lipid bilayer membranes.

We investigated the channel formation by El Tor hemolysin (molecular mass, 65 kDa) of Vibrio cholerae O1 biotype El Tor in planar lipid bilayers. The El Tor hemolysin channel exhibited asymmetric and hyperbolic membrane current with increasing membrane potential, meaning that the channel is voltage dependent. The zero-current membrane potential measured in KCI solution showed that permeability ratio PK+/PCl- was 0.16, indicating that the channel is 6-fold more anion selective over cation. The hemolysin channel frequently flickered in the presence of divalent cations, suggesting that the channel spontaneously opens and closes. These data imply that the El Tor hemolysin damages target cells by the formation of transmembrane channels and, consequently, is the cause of osmotic cytolysis.

[Antibacterial and anti-hemolysin activities of tea catechins and their structural relatives].

Among catechins tested, (-)epigallocatechin (EGC), (-)epicatechin gallate (ECg), (-) epigallocatechin gallate (EGCg) inhibited the growth of Staphylococcus aureus, Vibrio cholerae O1 classical Inaba 569B and El Tor Inaba V86. S. aureus was more sensitive than V. cholerae O1 to these compounds. EGCg showed also a bactericidal activity against V. cholerae O1 569B. Pyrogallol showed a stronger antibacterial activity against S. aureus and V. cholerae O1 than tannic and gallic acid. Rutin or caffein had no effect on them. ECg and EGCg showed the most potent anti-hemolysin activity against S. aureus alpha-toxin, Vibrio parahaemolyticus thermostable direct hemolysin (Vp-TDH) and cholera hemolysin. Among catechin relatives, only tannic acid had a potent anti-hemolysin activity against alpha-toxin. These results suggest that the catechol and pyrogallol groups are responsible for the antibacterial and bactericidal activities, while the conformation of catechins might play an important role in the anti-hemolysin activity.

[Relationship between the anti-hemolysin activity and the structure of catechins and theaflavins].

We examined the corresponding isomers of catechins and theaflavins for anti-hemolysin activities against Staphylococcus aureus alpha-toxin and Vibrio cholerae O1 hemolysin. Catechins and theaflavins showed anti-hemolysin activities in a dose-dependent manner. Among the catechins tested, (-)catechin gallate, (-)epicatechin gallate and (-)epigallocatechin gallate having galloyl groups in their molecules showed more potent anti-hemolysin activities against both toxins. On the other hand, free catechins, i. e. (-)catechin, (-)gallocatechin, (-) epicatechin and (-)epigallocatechin had low anti-hemolysin activities against alpha-toxin. Although (-)catechin or (-)gallocatechin had no effect on cholera hemolysin, (-) epicatechin and (-)epigallocatechin were slightly inhibitory. Among dextrocatechins, (+) epicatechin and (+)epigallocatechin proved to be more effective than (+)catechin and (+) gallocatechin. The anti-hemolysin activities of theaflavins against alpha-toxin and cholera hemolysin were dependent on the number of the galloyl group in their structure. These results suggest that the tertiary structure of the catechin or theaflavin and the active site of hemolysin, that affects the interaction between them, plays an important role in the anti-hemolysin activity.

Conformational alteration in alpha-toxin from Staphylococcus aureus concomitant with the transformation of the water-soluble monomer to the membrane oligomer.

The membrane-damaging alpha-toxin aggregate of Staphylococcus aureus was characterized physicochemically. The aggregate weight of the toxin formed by various methods appeared to be 6 times higher than the molecular weight of the monomer as determined by the laser light scattering technique, suggesting the presence of a hexamer in the membrane. The aggregates fluoresced 20 to 50% more than the monomer at 336 nm. Circular dichroism measurements revealed that both the monomer and the oligomer showed essentially beta-sheet structure with the maximum ellipticity about -8,400 deg.cm2.dmol-1 at 215 nm. Circular dichroism spectrum of the oligomers showed ellipticity difference of -6,600, -44 and +84 deg.cm2.dmol-1, at 200, 250 and 280 nm, respectively, compared with the monomer. All these results suggest that the conformational change in the toxin molecule occurs concomitant with the transformation of the water-soluble monomer to the membrane-embedded hexamer.

Interaction of the alpha-toxin of Staphylococcus aureus with the liposome membrane.

When the liposome membrane is exposed to the alpha-toxin of Staphylococcus aureus, fluorescence of the tryptophan residue(s) of the toxin molecule increases concomitantly with the degree of toxin-hexamer formation (Ikigai, H., and Nakae, T. (1985) Biochem. Biophys. Res. Commun. 130, 175-181). In the present study, the toxin-membrane interaction was distinguished from the hexamer formation by the fluorescence energy transfer from the tryptophan residue(s) of the toxin molecule to the dansylated phosphatidylethanolamine in phosphatidylcholine liposome. Measurement of these two parameters yielded the following results. The effect of the toxin concentration and phospholipid concentration on these two parameters showed first order kinetics. The effect of liposome size on the energy transfer and the fluorescence increment of the tryptophan residue(s) was only detectable in small liposomes. Under moderately acidic or basic conditions, the fluorescence energy transfer always preceded the fluorescence increment of the tryptophan residue(s). The fluorescence increment at 336 nm at temperatures below 20 degrees C showed a latent period, whereas the fluorescence energy transfer did not. These results were thought to indicate that when alpha-toxin damages the target membrane, the molecule interacts with the membrane first, and then undergoes oligomerization within the membrane.

Assembly of the alpha-toxin-hexamer of Staphylococcus aureus in the liposome membrane.

It has been shown that the access of the alpha-toxin of Staphylococcus aureus to the target membrane and assembly of the hexamer can be monitored independently by respectively measuring the fluorescence energy transfer from the tryptophan residue(s) of the toxin to the dansylated phosphatidylethanolamine in the liposome membrane and the fluorescence increment of the toxin at 336 nm (Ikigai, H., and Nakae, T., (1987) J. Biol. Chem. 262, 2150-2155). Measurement of these parameters under various conditions showed the following results: when phosphatidylcholine (PC) liposomes composed of saturated fatty acids were mixed with the toxin, the fluorescence energy transfer occurred below, at, and above the transition temperature of the lipid, but the change of fluorescence at 336 nm was never detectable; when PC-liposomes containing unsaturated fatty acids were used, both the fluorescence energy transfer and the fluorescence increment of 336 nm were observed. These results suggested that the toxin-membrane interaction occurs in PC-membranes containing saturated and/or unsaturated fatty acids and that the oligomerization occurs only in the presence of PC containing unsaturated fatty acid(s). This conclusion was supported by the results of quantitative determination of the toxin-hexamer assembly and leakage of carboxyfluorescein from PC-liposomes under conditions similar to the above.

Simplified method for preparation of concentrated exoproteins produced by Staphylococcus aureus grown on surface of cellophane bag containing liquid medium.

A simplified method for preparation of concentrated exoproteins including protein A and alpha-toxin produced by Staphylococcus aureus was successfully devised. The concentrated proteins were obtained by cultivating S. aureus organisms on the surface of a liquid medium-containing cellophane bag enclosed in a sterilized glass flask. With the same amount of medium, the total amount of proteins obtained by the method presented here was identical with that obtained by conventional liquid culture. The concentration of proteins obtained by the method, however, was high enough to observe their distinct bands stained on polyacrylamide gel electrophoresis. This method was considered quite useful not only for large-scale cultivation for the purification of staphylococcal proteins but also for small-scale study using the proteins. The precise description of the method was presented and its possible usefulness was discussed.

Luminol-dependent chemiluminescence in antibody-sensitized neutrophils stimulated with protein A-bearing staphylococci.

When mouse polymorphonuclear leukocytes (PMNs) sensitized with rabbit antibody to mouse Ehrlich ascites tumor cells were stimulated by Staphylococcus aureus Cowan I cells, a conspicuous luminol-dependent chemiluminescence was observed in the absence of opsonin. The profile of the chemiluminescence (CL) response evoked by staphylococcal cells from antibody-sensitized PMNs had two peaks. An initial peak, observed within 1 min after stimulation, was sharp and high and a second peak, observed about 5 min after stimulation, was low and extended. The CL response of antibody-sensitized PMNs stimulated by S. aureus Cowan I cells was dose-dependently blocked by preincubation with soluble SpA. Cells of a mutant derived from S. aureus Cowan I strain with trace amounts of cell-bound SpA failed to stimulate the antibody-sensitized PMNs to generate the CL response. The antibody-sensitized PMNs were found to phagocytize SpA-bearing S. aureus cells even in the absence of opsonic serum. These results suggest that the observation presented here might provide a useful tool for the investigation of CL response of PMNs.

The clinical significance of gastrointestinal decontamination in the occurrence of endogenous infections.

No significant difference was seen in the incidence of infections between subjects receiving complete, selective and no decontamination aimed at the intestinal microflora in studies evaluating the preventative potential against endogenous infections in the compromised host maintained under protective isolation. This finding is reported together with a report of Serratia marcescens septicemia in a patient with leukemia who was given antibiotics systemically and kept under protective isolation. The establishment of opportunistic infections in relation to these results is discussed in terms of the biological phenomena of the interaction between the intestinal flora and the host, and between the species comprising the intestinal flora.

The protective activity of tea against infection by Vibrio cholerae O1.

Extracts of black tea exhibited bactericidal activity against Vibrio cholerae O1. The tea extract inhibited the haemolysin activity of V. cholerae O1, El Tor and the morphological changes of Chinese hamster ovary cells induced by cholera toxin. Tea extract also reduced fluid accumulation induced by cholera toxin in sealed adult mice and by V. cholerae O1 in ligated intestinal loops of rabbits. These findings suggest that tea has protective activity against V. cholerae O1.

Mechanism of membrane damage by El Tor hemolysin of Vibrio cholerae O1.

El Tor hemolysin (ETH; molecular mass, 65 kDa) derived from Vibrio cholerae O1 spontaneously assembled oligomeric aggregates on the membranes of rabbit erythrocyte ghosts and liposomes. Membrane-associated oligomers were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting into two to nine bands with apparent molecular masses of 170 to 350 kDa. ETH assembled oligomers on a liposomal membrane consisting of phosphatidylcholine and cholesterol, but not on a membrane of phosphatidylcholine alone. Cholesterol could be replaced with diosgenin or ergosterol but not with 5alpha-cholestane-3-one, suggesting that sterol is essential for the oligomerization. The treatment of carboxyfluorescein-encapsulated liposomes with ETH caused a rapid release of carboxyfluorescein into the medium. Because dextrin 20 (molecular mass, 900 Da) osmotically protected ETH-mediated hemolysis, this hemolysis is likely to be caused by pore formation on the membrane. The pore size(s) estimated from osmotic protection assays was in the range of 1.2 to 1.6 nm. The pore formed on a rabbit erythrocyte membrane was confirmed morphologically by electron microscopy. Thus, we provide evidence that ETH damages the target by the assembly of hemolysin oligomers and pore formation on the membrane.

The protective activity of tea catechins against experimental infection by Vibrio cholerae O1.

Tea catechins inhibited the fluid accumulation induced by cholera toxin in sealed adult mice. The catechins also reduced fluid accumulation by Vibrio cholerae O1 in ligated intestinal loops of rabbits. These findings suggest that tea catechins may possess protective activity against V. cholerae O1.

Effect of temperature on antibacterial activity of lidocaine to Staphylococcus aureus and Pseudomonas aeruginosa.

The effect of temperature on the antibacterial activity of lidocaine to Staphylococcus aureus and Pseudomonas aeruginosa was investigated in vitro. At 10 C at which S. aureus organisms do not grow and might be metabolically inactive, the antibacterial activity of lidocaine to S. aureus was not observed in a concentration of 1%, which was quite antibacterial to S. aureus at 37 C. On the other hand, at 40 C a conspicuously increased antibacterial activity to S. aureus of lidocaine was observed in a concentration of 0.25% which was not antibacterial to S. aureus organisms at 37 C. Similar results were obtained when P. aeruginosa organisms were examined in place of S. aureus, although P. aeruginosa was found to be less susceptible to lidocaine than S. aureus. The clinical significance of the thermal effect on the antibacterial activity of lidocaine was discussed in brief.