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Streptococcus mutans NCTC 10449 and Escherichia coli K-12 strain 37 were grown under aerobic and anaerobic conditions. In cell extracts of both strains, pyruvate dehydrogenase activity dependent on thiamine pyrophosphate, coenzyme A, and NAD was shown. The enzyme was induced by pyruvate in the growth medium, and there was higher activity in aerobically grown cells than in anaerobically grown cells. Acetyl phosphate was a potent inhibitor of the activity. This inhibition was partly overcome by inorganic phosphate.

Lactoperoxidase catalyzes the oxidation of thiocyanate by hydrogen peroxide into hypothiocyanite, a reaction which can protect bacterial and mammalian cells from killing by hydrogen peroxide. The present study demonstrates, however, that lactoperoxidase in the presence of thiocyanate can actually potentiate the bactericidal and cytotoxic effects of hydrogen peroxide under specific conditions, such as when hydrogen peroxide is present in the reaction mixtures in excess of thiocyanate. The toxic agent was also formed in the absence of lactoperoxidase in a reaction between hypothiocyanite and hydrogen peroxide. Sulfate, sulfite, cyanate, carbonate, and ammonia, which have been postulated to be formed in the chemical oxidation of hypothiocyanite by hydrogen peroxide, were not bactericidal and did not potentiate the bactericidal effect of hydrogen peroxide. Cyanosulfurous acid, the only other postulated product of the chemical oxidation of hypothiocyanite by hydrogen peroxide, may be the killing agent.

Lactoperoxidase and thiocyanate were shown to protect Escherichia coli and three oral streptococcal species from the bactericidal effect of hydrogen peroxide under aerobic conditions. Lactoperoxidase in the absence of thiocyanate was also protective for two of the bacterial species in a dilution medium but potentiated hydrogen peroxide toxicity for the other two under the same conditions. The products of the reaction between hydrogen peroxide and thiocyanate in the presence of lactoperoxidase were not bactericidal except in the case of E. coli, and then only under special conditions. The results demonstrate the effectiveness of lactoperoxidase and thiocyanate in protecting living cells from hydrogen peroxide toxicity. Although the effect on human cells was not examined in this study, extrapolation of these results to the cells of the oral mucosa would suggest an important protective role of lactoperoxidase and thiocyanate against the toxic effects of hydrogen peroxide in the oral cavity.

L-Cysteine potentiates 100-fold the hydrogen peroxide-induced killing of a growing culture of Escherichia coli K-12 (Berglin et al., J. Bacteriol. 152:81-88). In the present study it is shown that hydrogen sulfide is formed from L-cysteine and that sodium sulfide could substitute for L-cysteine in the potentiation of hydrogen peroxide-induced killing of E. coli K-12. Addition of an amino acid, L-leucine, L-valine, or L-alanine, to an L-cysteine-containing medium with a growing culture of E. coli K-12 inhibited hydrogen sulfide formation and the potentiation of hydrogen peroxide-induced killing. These amino acids did not inhibit hydrogen sulfide formation from L-cysteine by a cell extract, and they did not inhibit the potentiation by sulfide of hydrogen peroxide-induced killing. This indicated that the amino acids protected the culture from L-cysteine-potentiated, hydrogen peroxide-induced killing by inhibiting the transport of L-cysteine into the cell. The potentiation by sodium sulfide of hydrogen peroxide-induced killing was abolished by the metal ion chelator 2,2'-bipyridyl. This indicated that metal ions, in addition to sulfide, were involved in the killing. Toxic effects of hydrogen peroxide are often presumed to be mediated by hydroxyl radicals formed in iron-catalyzed reactions. It was demonstrated that iron sulfide was more efficient than ferrous iron in catalyzing the formation of hydroxyl radicals from hydrogen peroxide. It was suggested that hydrogen sulfide formed in polymicrobial infections may play an important role in the host defense by potentiating the antimicrobial effect of hydrogen peroxide produced by phagocytic cells.

Polymorphonuclear leukocytes (PMN) isolated from human blood were exposed to various levels of hydrogen sulfide. The effect on respiratory burst, myeloperoxidase activity, and capacity to phagocytose and kill bacteria were studied. A 1-h exposure of the PMN to 1 mM sulfide did not decrease their myeloperoxidase activity or their capacity to initiate a respiratory burst. Actually the products of the respiratory burst rapidly oxidized sulfide. The phagocytosis and killing of bacteria in the presence of 1 mM sulfide was only decreased to a minor extent. Myeloperoxidase in cell extract was, however, almost completely inhibited by 1 microM sulfide. These results indicate that hydrogen sulfide does not easily permeate PMN. PMN may be able to function in infected sites with high sulfide levels such as in the gingival pockets of periodontal disease. In the oxygenated areas of these sites the PMN may actually help in the detoxification of sulfide.

Approved type strains of Streptococcus sanguis, S. mitis, S. mutans, and S. salivarius were grown under aerobic and anaerobic conditions. The rate of hydrogen peroxide excretion, oxygen uptake, and acid production from glucose by washed-cell suspensions of these strains were studied, and the levels of enzymes in cell-free extracts which reduced oxygen, hydrogen peroxide, or hypothiocyanite (OSCN-) in the presence of NADH or NADPH were assayed. The effects of lactoperoxidase-thiocyanate-hydrogen peroxide on the rate of acid production and oxygen uptake by intact cells, the activity of glycolytic enzymes in cell-free extracts, and the levels of intracellular glycolytic intermediates were also studied. All strains consumed oxygen in the presence of glucose. S. sanguis, S. mitis, and anaerobically grown S. mutans excreted hydrogen peroxide. There was higher NADH oxidase and NADH peroxidase activity in aerobically grown cells than in anaerobically grown cells. NADPH oxidase activity was low in all species. Acid production, oxygen uptake, and, consequently, hydrogen peroxide excretion were inhibited in all the strains by lactoperoxidase-thiocyanate-hydrogen peroxide. S. sanguis and S. mitis had a higher capacity than S. mutans and S. salivarius to recover from this inhibition. Higher activity in the former strains of an NADH-OSCN oxidoreductase, which converted OSCN- into thiocyanate, explained this difference. The change in levels of intracellular glycolytic intermediates after inhibition of glycolysis by OSCN- and the actual activity of glycolytic enzymes in cell-free extracts in the presence of OSCN- indicated that the primary target of OSCN- in the glycolytic pathway was glyceraldehyde 3-phosphate dehydrogenase.

The effect of Bacteroides gingivalis W83 on various key components of the human plasma proteinase cascade systems was studied. When purified C1-inhibitor was incubated with the bacterium, the inhibitor was rapidly inactivated by limited proteolytic cleavage. In citrated whole plasma, C1-inhibitor, antithrombin, plasminogen, prekallikrein, prothrombinase complex, the clotting factor X, and most of the alpha 2-antiplasmin were functionally eliminated after 30 min of incubation with the bacterium. Fibrinogen disappeared from the plasma almost immediately upon mixing with the bacterial suspension. In contrast, there was no appreciable decrease in the bulk of other plasma proteins, such as various transport proteins (albumin, prealbumin, transferrin) and immunoglobulins, during 4 h of incubation with the bacterium. Most of the observed effects can be assigned to the proteolytic activity of the bacterium itself, since there was little evidence for generation of intrinsic plasma proteinase activity, despite the loss of proteinase inhibitory activities. B. gingivalis W83 thus seems to be equipped with proteolytic enzyme systems which selectively recognize and rapidly inactivate the most important proteinase inhibitors and proenzymes present in human plasma. This bacterium therefore seems to be able to efficiently paralyze the host's various defenses against invading microorganisms.

We have previously reported that a wild strain of Francisella tularensis is much less efficiently killed by human polymorphonuclear leukocytes than is an attenuated strain. In the present study, the killing of the attenuated strain was found to be strictly oxygen dependent. The wild and the attenuated strains both induced a respiratory burst in the leukocytes. The difference between the strains in susceptibility to agents produced at the burst could be explained by a difference in susceptibility to hypochlorous acid.

Various strains of black-pigmented Bacteroides species were grown on horse blood agar and suspended in human serum. After various times of incubation the effect of the bacteria on the serum was evaluated by polyacrylamide gel electrophoresis and "rocket" immunoelectrophoresis. The formation of trichloroacetic acid-soluble material in the suspensions and the capacity of the treated sera to inhibit the activity of trypsin were also determined. The two tested strains of Bacteroides gingivalis (W83, H185) degraded most serum proteins, including the plasma proteinase inhibitors alpha-1-antitrypsin and alpha-2-macroglobulin. They did not, however, degrade alpha-1-antichymotrypsin. Bacteroides intermedius NCTC 9336, Bacteroides asaccharolyticus NCTC 9337, and an asaccharolytic oral strain different from B. gingivalis (BN11a-f) did not degrade the plasma proteinase inhibitors. These strains were, however, able to inactivate the capacity of serum to inhibit the activity of trypsin.

Twenty-three individuals were vaccinated with a viable attenuated strain of Francisella tularensis, and blood was collected at various time intervals during 4 weeks. To demonstrate opsonizing antibodies, a mixture of serum and vaccine bacteria was incubated, whereafter the chemiluminescence response of polymorphonuclear (PMN) leukocytes to this mixture was recorded. No opsonizing antibodies against F. tularensis were found in sera obtained before vaccination. Eleven days after vaccination, sera from nine individuals, and 21 days after vaccination, sera from all 23 individuals contained antibodies. Antibodies were demonstrated earlier with the chemiluminescent technique that with the agglutination reaction. Heat treatment (56 degrees C, 30 min) or removal of complement component C3 from immune serum reduced the chemiluminescent response of the leukocytes. A high chemiluminescent response of the leukocytes was induced by immunoglobulin G (IgG)- and IgM-enriched fractions of immune serum in the presence of complement. In the absence of complement, the IgG fraction induced a low chemiluminescent response; the IgM fraction induced no response at all.

Vaccine bacteria of Francisella tularensis were mixed with human serum containing specific antibodies against the same organism, and the mixture was incubated at 37 degrees C for 30 min. The mixture induced a two-peak chemiluminescent response in polymorphonuclear leukocytes. The initial peak was induced by a soluble agent, formed during incubation. The formation of this agent involved the activation of complement component C5. The second peak of the chemiluminescent response was induced by the opsonized bacteria; this peak was augmented by complement component C3, whereas C5 had no influence.