Hydrogen peroxide-mediated killing of Caenorhabditis elegans by Streptococcus pyogenes

Caenorhabditis elegans is currently introduced as a new, facile, and cheap model organism to study the pathogenesis of gram-negative bacteria such as Pseudomonas aeruginosa and Salmonella enterica serovar Typhimurium. The mechanisms of killing involve either diffusible exotoxins or infection-like processes. Recently, it was shown that also some gram-positive bacteria kill C. elegans, although the precise mechanisms of killing remained open. We examined C. elegans as a pathogenesis model for the gram-positive bacterium Streptococcus pyogenes, a major human pathogen capable of causing a wide spectrum of diseases. We demonstrate that S. pyogenes kills C. elegans, both on solid and in liquid medium. Unlike P. aeruginosa and S. enterica serovar Typhimurium, the killing by S. pyogenes is solely mediated by hydrogen peroxide. Killing required live streptococci; the killing capacity depends on the amount of hydrogen peroxide produced, and killing can be inhibited by catalase. Major exotoxins of S. pyogenes are not involved in the killing process as confirmed by using specific toxin inhibitors and knockout mutants. Moreover, no accumulation of S. pyogenes in C. elegans is observed, which excludes the involvement of infection-like processes. Preliminary results show that S. pneumoniae can also kill C. elegans by hydrogen peroxide production. Hydrogen peroxide-mediated killing might represent a common mechanism by which gram-positive, catalase-negative pathogens kill C. elegans.     

Role of catalase in Campylobacter jejuni intracellular survival

The ability of Campylobacter jejuni to penetrate normally nonphagocytic host cells is believed to be a key virulence determinant. Recently, kinetics of C. jejuni intracellular survival have been described and indicate that the bacterium can persist and multiply within epithelial cells and macrophages in vitro. Studies conducted by Pesci et al. indicate that superoxide dismutase contributes to intraepithelial cell survival, as isogenic sod mutants are 12-fold more sensitive to intracellular killing than wild-type strains. These findings suggest that bacterial factors that combat reactive oxygen species enable the organism to persist inside host cells. Experiments were conducted to determine the contribution of catalase to C. jejuni intracellular survival. Zymographic analysis indicated that C. jejuni expresses a single catalase enzyme. The gene encoding catalase (katA) was cloned via functional complementation, and an isogenic katA mutant strain was constructed. Kinetic studies indicate that catalase provides resistance to hydrogen peroxide in vitro but does not play a role in intraepithelial cell survival. Catalase does however contribute to intramacrophage survival. Kinetic studies of C. jejuni growth in murine and porcine peritoneal macrophages demonstrated extensive killing of both wild-type and katA mutant strains shortly following internalization. Long-term cultures (72 h postinfection) of infected phagocytes permitted recovery of viable wild-type C. jejuni; in contrast, no viable katA mutant bacteria were recovered. Accordingly, inhibition of macrophage nitric oxide synthase or NADPH oxidase permitted recovery of katA mutant C. jejuni. These observations indicate that catalase is essential for C. jejuni intramacrophage persistence and growth and suggest a novel mechanism of intracellular survival.     

Virulence effect of Enterococcus faecalis protease genes and the quorum-sensing locus fsr in Caenorhabditis elegans and mice

The expression of two Enterococcus faecalis extracellular virulence-related proteins, gelatinase (GelE) and serine protease (SprE), has been shown to be positively regulated by the fsr quorum-sensing system. We recently developed a novel system for studying E. faecalis pathogenicity that involves killing of the nematode worm Caenorhabditis elegans and showed that an E. faecalis fsrB mutant (strain TX5266) exhibited attenuated killing. We explore here the role of the fsr/gelE-sprE locus in pathogenicity by comparing results obtained in the nematode system with a mouse peritonitis model of E. faecalis infection. Insertion mutants of fsrA (TX5240) and fsrC (TX5242), like fsrB (TX5266), were attenuated in their ability to kill C. elegans. A deletion mutant of gelE (TX5264) and an insertion mutant of sprE (TX5243) were also attenuated in C. elegans killing, although to a lesser extent than the fsr mutants. Complementation of fsrB (TX5266) with a 6-kb fragment containing the entire fsr locus restored virulence in both the nematode and the mouse peritonitis models. The fsr mutants were not impaired in their ability to colonize the nematode intestine. These data show that extracellular proteases and the quorum-sensing fsr system are important for E. faecalis virulence in two highly divergent hosts: nematodes and mice.     

Catalases of Aspergillus fumigatus

Upon infection of a host, the pathogenic fungus Aspergillus fumigatus is attacked by the reactive oxygen species produced by phagocytic cells. Detoxification of hydrogen peroxide by catalases was proposed as a way to overcome this host response. A. fumigatus produces three active catalases; one is produced by conidia, and two are produced by mycelia. The mycelial catalase Cat1p was studied previously. Here we characterized the two other catalases, their genes, and the phenotypes of gene-disrupted mutants. CatAp, a spore-specific monofunctional catalase, is resistant to heat, metal ions, and detergent. This enzyme is a dimeric protein with 84.5-kDa subunits. The 749-amino-acid polypeptide exhibits high levels of similarity to the Aspergillus nidulans CatA catalase and to bacterial catalase HPII of Escherichia coli. In spite of increased sensitivity to H(2)O(2), killing of DeltacatA conidia by alveolar macrophages and virulence in animals were similar to the killing of conidia by alveolar macrophages and virulence in animals observed for the wild type. In contrast to the Cat1p and CatAp catalases, the mycelial Cat2p enzyme is a bifunctional catalase-peroxidase and is sensitive to heat, metal ions, and detergent. This enzyme, an 82-kDa monomer, is homologous to catalase-peroxidases of several fungi and bacteria. Surprisingly, mycelium of the double Deltacat1Deltacat2 mutant with no catalase activity exhibited only slightly increased sensitivity to H(2)O(2) and was as sensitive to killing by polymorphonuclear neutrophils as mycelium of the wild-type strain. However, this mutant exhibited delayed infection in the rat model of aspergillosis compared to infection by the wild-type strain. These results indicate that conidial catalase is not a virulence factor and that mycelial catalases transiently protect the fungus from the host.     

Competition and resilience between founder and introduced bacteria in the Caenorhabditis elegans gut

The microbial communities that reside within the intestinal tract in vertebrates are complex and dynamic. In this report, we establish the utility of Caenorhabditis elegans as a model system for identifying the factors that contribute to bacterial persistence and for host control of gut luminal populations. We found that for N2 worms grown on mixed lawns of bacteria, Salmonella enterica serovar Typhimurium substantially outcompeted Escherichia coli, even when E. coli was initially present at 100-fold-higher concentrations. To address whether innate immunity affects the competition, the daf-2 and daf-16 mutants were studied; their total gut bacterial levels reflect overall capacity for colonization, but Salmonella outcompeted E. coli to an extent similar to wild-type worms. To address the role of virulence properties, Salmonella Δspi-1 Δspi-2 was used to compete with E. coli. The net differential was significantly less than that for wild-type Salmonella; thus, spi-1 spi-2 encodes C. elegans colonization factors. An E. coli strain with repeated in vivo passage had an enhanced ability to compete against an in vitro-passed E. coli strain and against Salmonella. Our data provide evidence of active competition for colonization niches in the C. elegans gut, as determined by bacterial factors and subject to in vivo selection.     

Human vascular smooth muscle cells and endothelial cells lack catalase activity and are susceptible to hydrogen peroxide

⁵Cr release as lytic and cell detachment as nonlytic injury were employed to estimate neutrophil-mediated injury of cultured human vascular smooth muscle cells and endothelial cells. The reagents hydrogen peroxide or hypoxanthine-xanthine oxidase produced dose-dependent killing and nonlytic cell detachment, which were specifically inhibited by catalase but not by superoxide dismutase. The concentration of hydrogen peroxide or xanthine oxidase to induce cell detachment was less than lytic dose, suggesting that cell detachment was a much more sensitive assay of injury. Neutrophil-mediated cell lysis averaged 15% at most and was mostly dependent on hydrogen peroxide, while neutrophil-mediated cell detachment was nearly 100% and its dependency on hydrogen peroxide varied from 46% to 60%. These results suggest that vascular smooth muscle cells and endothelial cells in neutrophil-mediated events are destroyed by a hydrogen peroxide-dependent process, mainly via a nonlytic cell detachment mechanism. There was no striking difference of sensitivity to hydrogen peroxide between vascular smooth muscle cells and endothelial cells. Vascular smooth muscle ceils and endothelial cells contained fairly high concentrations of superoxide dismutase, but not catalase, activity. The sensitivity of these cells to hydrogen peroxide but not to superoxide may arise from the fact that these cells lack intracellular catalase activity. The injury of vascular cells, which constitute important components of blood vessels, may lead to vascular injury and subsequent tissue damage.     

Cloning and Sequencing of a Candida albicans Catalase Gene and Effects of Disruption of This Gene

Catalase plays a key role as an antioxidant, protecting aerobic organisms from the toxic effects of hydrogen peroxide, and in some cases has been postulated to be a virulence factor. To help elucidate the function of catalase in Candida albicans, a single C. albicans-derived catalase gene, designated CAT1, was isolated and cloned. Degenerate PCR primers based on highly conserved areas of other fungal catalase genes were used to amplify a 411-bp product from genomic DNA of C. albicans ATCC 10261. By using this product as a probe, catalase clones were isolated from genomic libraries of C. albicans. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 487 amino acid residues. Construction of a CAT1-deficient mutant was achieved by using the Ura-blaster technique for sequential disruption of multiple alleles by integrative transformation using URA3 as a selectable marker. Resulting mutants exhibited normal morphology and comparable growth rates of both yeast and mycelial forms. Enzymatic analysis revealed an abundance of catalase in the wild-type strain but decreasing catalase activity in heterozygous mutants and no detectable catalase in a homozygous null mutant. In vitro assays showed the mutant strains to be more sensitive to damage by both neutrophils and concentrations of exogenous peroxide that were sublethal for the parental strain. Compared to the parental strain, the homozygous null mutant strain was far less virulent for mice in an intravenous infection model of disseminated candidiasis. Definitive linkage of CAT1 with virulence would require restoration of activity by reintroduction of the gene into mutants. However, initial results in mice, taken together with the enhanced susceptibility of catalase-deficient hyphae to damage by human neutrophils, suggest that catalase may enhance the pathogenicity of C. albicans.     

The enigmatic physiological roles of AhpCF,Gpx, Npr and Kat in peroxide stress response of Enterococcus faecium

In this work, the physiological roles of the primary peroxide scavenging activities of Enterococcus faecium AUS0004 strain were analysed. This healthcare-associated pathogen harbours genes encoding putative NADH peroxidase (Npr), alkyl hydroperoxide reductase (AhpCF), glutathione peroxidase (Gpx) and manganese-dependent catalase (Mn-Kat). Gene expression analyses showed that npr and kat genes are especially and significantly induced in cells treated with hydrogen peroxide (H₂O₂) and cumene hydroperoxide (CuOOH), which suggested an important function of these enzymes to protect E. faecium against peroxide stress. Mutants affected in one or several predicted anti-oxidative activities mentioned above showed that neither the peroxidases nor the catalase are implicated in the defence against peroxide challenges. However, our investigations allowed us to show that Npr is responsible for the degradation of approximately 45% of metabolically derived H₂O₂ which avoids accumulation of the peroxide to lethal concentrations.     

Reduction of ferricytochrome C may underestimate superoxide production by monocytes

Superoxide production by stimulated phagocytes is commonly measured by reduction of ferricytochrome C, with specificity of the assay assumed if the reaction is inhibited by superoxide dismutase (SOD). Most preparations of ferricytochrome C contain a small proportion in the reduced (ferro) form, and this is also formed by the reaction of ferricytochrome C with superoxide. The generation of other reactive oxygen intermediates, such as hydrogen peroxide or hydroxyl radical, could cause oxidation of ferrocytochrome C and consequent underestimation of superoxide production. In support of this, it has been demonstrated that exogenous catalase enhanced the reduction of ferricytochrome C by phorbol myristate acetate (PMA)-stimulated human monocytes. Control experiments confirmed that this was due to enhanced detection rather than increased production of superoxide. In addition, SOD was found to promote oxidation of ferrocytochrome C by PMA-stimulated human monocytes, but this was also inhibited by catalase. These effects of catalase and SOD on ferricytochrome C reduction/ferrocytochrome C oxidation were also demonstrated when superoxide was produced independently of monocytes by a xanthine and xanthine oxidase generating system. It is concluded that the assay of superoxide, using 'SOD inhibitable' reduction of ferricytochrome C, underestimates superoxide production.     

An operon in Streptococcus pneumoniae containing a putative alkylhydroperoxidase D homologue contributes to virulence and the response to oxidative stress

Analysis of the pneumococcal genome sequences from strains R6 and TIGR4 identified a putative alkylhydroperoxidase homologue RT-PCR showed this gene to be expressed in an operon with the downstream open reading frame. No probable function for this second gene is suggested although it appears to be an integral membrane protein. An allelic replacement mutant lacking this two-gene operon in strain D39 was attenuated in competitive infections with the wild type parent. This operon is, therefore, a novel pneumococcal virulence determinant. In line with a role in the response to oxidative stress, this mutant showed enhanced resistance to killing by hydrogen peroxide, a phenotype shared by alkylhydroperoxidase mutants in other bacterial species. The analysis of non-polar single mutants shows that both genes contribute to these phenotypes. Finally, an important role in pneumococcal biology is suggested by the presence of this operon in all 20 clinical isolates examined and the highly conserved sequence of the two genes.     

Inhibitory and bactericidal effects of hydrogen peroxide production by Streptococcus pneumoniae on other inhabitants of the upper respiratory tract

An inverse correlation between colonization of the human nasopharynx by Streptococcus pneumoniae and Haemophilus influenzae, both common upper respiratory pathogens, has been reported. Studies were undertaken to determine if either of these organisms produces substances which inhibit growth of the other. Culture supernatants from S. pneumoniae inhibited growth of H. influenzae, whereas culture supernatants from H. influenzae had no effect on the growth of S. pneumoniae. Moreover, coculture of S. pneumoniae and H. influenzae led to a rapid decrease in viable counts of H. influenzae. The addition of purified catalase prevented killing of H. influenzae in coculture experiments, suggesting that hydrogen peroxide may be responsible for this bactericidal activity. H. influenzae was killed by concentrations of hydrogen peroxide similar to that produced by S. pneumoniae. Hydrogen peroxide is produced by the pneumococcus through the action of pyruvate oxidase (SpxB) under conditions of aerobic growth. Both an spxB mutant and a naturally occurring variant of S. pneumoniae, which is downregulated in SpxB expression, were unable to kill H. influenzae. A catalase-reversible inhibitory effect of S. pneumoniae on the growth of the respiratory tract pathogens Moraxella catarrhalis and Neisseria meningitidis was also observed. Elevated hydrogen peroxide production, therefore, may be a means by which S. pneumoniae is able to inhibit a variety of competing organisms in the aerobic environment of the upper respiratory tract.     

Nitrate sensing and metabolism modulate motility, biofilm formation, and virulence in Pseudomonas aeruginosa

Infection by the bacterial opportunist Pseudomonas aeruginosa frequently assumes the form of a biofilm, requiring motility for biofilm formation and dispersal and an ability to grow in nutrient- and oxygen-limited environments. Anaerobic growth by P. aeruginosa is accomplished through the denitrification enzyme pathway that catalyzes the sequential reduction of nitrate to nitrogen gas. Mutants mutated in the two-component nitrate sensor-response regulator and in membrane nitrate reductase displayed altered motility and biofilm formation compared to wild-type P. aeruginosa PAO1. Analysis of additional nitrate dissimilation mutants demonstrated a second level of regulation in P. aeruginosa motility that is independent of nitrate sensor-response regulator function and is associated with nitric oxide production. Because motility and biofilm formation are important for P. aeruginosa pathogenicity, we examined the virulence of selected regulatory and structural gene mutants in the surrogate model host Caenorhabditis elegans. Interestingly, the membrane nitrate reductase mutant was avirulent in C. elegans, while nitrate sensor-response regulator mutants were fully virulent. The data demonstrate that nitrate sensing, response regulation, and metabolism are linked directly to factors important in P. aeruginosa pathogenesis.     

Characterization and virulence analysis of catalase mutants of Haemophilus influenzae.

In addition to detoxifying peroxides generated by aerobic metabolism, the catalases of pathogenic bacteria have also been hypothesized to serve as virulence factors by enabling microorganisms to resist the oxidative bursts of host inflammatory cells. Using transposon mutagenesis of the hktE gene, encoding the Haemophilus influenzae structural gene for catalase, we constructed defined catalase mutants of H. influenzae strains Rd- and Eagan b+. These mutants show no detectable catalase production during exponential or stationary phases or following induction with hydrogen peroxide or ascorbic acid, indicating that hktE is the only functional hydroperoxidase gene present in these two strains of H. influenzae. Exponential-phase cultures of hktE mutants are 8- to 25-fold more sensitive to hydrogen peroxide than the wild type. Using the infant rat model, hktE mutants of strain Eagan b+ were 2.3-fold less virulent than the wild type following intraperitoneal inoculation (P = 0.07). When administered intranasally, the Eagan b+ hktE mutant produced wild-type levels of bacteremia and nasal colonization. The results of this study show that while the H. influenzae hktE gene is important for survival in the presence of peroxides, deletion of the gene produces only a modest reduction in ability to cause lethal sepsis following parenteral challenge and no change in ability to colonize following intranasal inoculation in the infant rat model of infection.     

Virulence of Staphylococcus aureus small colony variants in the Caenorhabditis elegans infection model

Small colony variants (SCVs) of Staphylococcus aureus are slow-growing morphological variants that have been implicated in persistent, relapsing, and antibiotic-resistant infections. The altered phenotype of SCVs in most strains has been attributed to defects in electron transport due to mutations in hemin or menadione biosynthesis. The pathogenic capacity of SCVs compared to phenotypically normal strains is variable depending on the attribute examined, with some studies showing reduced virulence of SCVs and others demonstrating normal or heightened virulence. Recently, the nematode Caenorhabditis elegans has been successfully employed as an alternative host to investigate virulence mechanisms of a variety of bacterial pathogens, including S. aureus. In this study, we show that clinical SCVs as well as hemB- and menD-deficient mutants of S. aureus are greatly reduced in virulence in the C. elegans infection model.     

Virulence of Streptococcus pneumoniae: PsaA mutants are hypersensitive to oxidative stress

psaA encodes a 37-kDa pneumococcal lipoprotein which is part of an ABC Mn(II) transport complex. Streptococcus pneumoniae D39 psaA mutants have previously been shown to be significantly less virulent than wild-type D39, but the mechanism underlying the attenuation has not been resolved. In this study, we have shown that psaA and psaD mutants are highly sensitive to oxidative stress, i.e., to superoxide and hydrogen peroxide, which might explain why they are less virulent than the wild-type strain. Our investigations revealed altered expression of the key oxidative-stress response enzymes superoxide dismutase and NADH oxidase in psaA and psaD mutants, suggesting that PsaA and PsaD may play important roles in the regulation of expression of oxidative-stress response enzymes and intracellular redox homeostasis.     

Bactericidal mechanisms in rabbit alveolar macrophages: evidence against peroxidase and hydrogen peroxide bactericidal mechanisms.

The role of peroxidase-mediated bacterial killing by rabbit alveolar macrophages was examined. During 3 h of incubation in vitro, alveolar macrophages ingested and killed greater than 88% of the Streptococcus faecalis, Proteus mirabilis, or Streptococcus pneumoniae present in the incubation mixture. Preincubation of alveolar macrophages with inhibitors of catalase, 3-amino-1,2,4-triazole or sodium nitrite, did not alter their bactericidal potential. Iodination of ingested zymosan particles, a peroxidase-dependent and hydrogen peroxide-dependent reaction, was not observed, in spite of vigorous phagocytosis by alveolar macrophages. Furthermore, iodination by alveolar macrophages was not significantly increased when peroxidase-coated zymosan particles were ingested. The results suggest that hydrogen peroxide may not be available to the phagocytic vacuole for microbial killing. Since tetrazolium dye reduction reflects the activity of an oxidase responsible for stimulated oxygen consumption by polymorphonuclear leukocytes, this reaction was also measured. Rabbit alveolar macrophages incubated with latex particles did not exhibit an increased dye reduction compared with resting cells. The absence of significant stimulation of tetrazolium dye reduction indicates that the oxidase reaction does not occur in the proximity of the phagocytic vacuole of alveolar macrophages.     

Susceptibility of Entamoeba histolytica to oxidants.

The effect of hydrogen peroxide and hypochlorite on culture forms of Entamoeba histolytica trophozoites was examined by using two strains of E. histolytica, virulent (IP:0682:1) and nonvirulent (DKB). The amoebae were incubated with various concentrations of hydrogen peroxide and hypochlorite, and their viability was determined at different times after incubation. When the viability of the virulent and nonvirulent strains was compared to different oxidant strengths, it became apparent that the virulent strain was less susceptible than the nonvirulent one to the cytotoxic effect of hydrogen peroxide and hypochlorite. Our studies further showed that the toxic effect was both time and dose dependent. To confirm that the killing of amoebae in this system was associated with the presence of hydrogen peroxide, amoebae were incubated with hydrogen peroxide and catalase. Catalase reduced the killing effect of hydrogen peroxide to the control level. These data confirmed previous observations of the susceptibility of amoebic trophozoites to hydrogen peroxide and also demonstrated susceptibility to hypochlorite.     

Caenorhabditis elegans as a model host for Staphylococcus aureus pathogenesis

Staphylococcus aureus, an important pathogen of humans and other warm-blooded animals, is also capable of killing the nematode Caenorhabditis elegans. Here, we show that C. elegans organisms that are fed S. aureus die over the course of several days in a process that is correlated with the accumulation of bacteria within the nematode digestive tract. Several S. aureus virulence determinants known or speculated to be important in mammalian pathogenesis, including the quorum-sensing global virulence regulatory system agr and the global virulence regulator sarA, the alternative sigma factor sigma(B), alpha-hemolysin, and V8 serine protease, are required for full pathogenicity in nematodes. In addition, several defined C. elegans mutants were examined for susceptibility to S. aureus infection. Enhanced susceptibility to S. aureus killing was observed with loss-of-function mutations in the C. elegans genes esp-2/sek-1 and esp-8/nsy-1, which encode components of a conserved p38 MAP kinase signaling pathway involved in nematode defense against multiple pathogens. These results suggest that key aspects of S. aureus pathogenesis have been conserved, irrespective of the host, and that specific C. elegans host factors can alter susceptibility to this gram-positive human pathogen.     

The contribution of hydrogen peroxide resistance to virulence of Mycobacterium tuberculosis during the first six days after intravenous infection of normal and BCG-vaccinated guinea-pigs.

The course of infection with Mycobacterium tuberculosis strains H37Rv, H37Ra and their isoniazid-resistant, hydrogen peroxide-susceptible mutants in guinea-pig spleen and lung were assessed by measuring changes in number of viable bacteria during the first and second 3-day intervals after i.v. infection of normal and BCG-vaccinated animals. Vaccination had no effect on bacterial survival in the first 3 days of infection. The peroxide-susceptible mutants were killed or inhibited more than their parent strains; in normal animals this enhanced susceptibility was expressed equally during the first and second 3-day intervals while in vaccinated animals the effect was greater in the second 3-day interval. The results suggest that hydrogen peroxide is generated in significant amounts in the environment of tubercle bacilli lodged in normal tissues and in enhanced amounts when acquired immunity becomes expressed after a few days'' lodgement in the tissues of vaccinated animals. Thus hydrogen peroxide resistance may contribute to virulence by protecting against both normal resident and immunologically activated macrophages.     

Hydrogen peroxide excretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogen peroxide

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.