The cagE gene sequence as a diagnostic marker to identify JP2 and non-JP2 highly leukotoxic Aggregatibacter actinomycetemcomitans serotype b strains.

BACKGROUND AND OBJECTIVE: Aggregatibacter actinomycetemcomitans is involved in oral and systemic infections, and is associated with, eg aggressive forms of periodontitis and with endocarditis. The cagE gene encodes a ≈39 kDa putative exotoxin expressed by A. actinomycetemcomitans. The level of conservation of cagE, and its possible significance in periodontal disease, has not yet been thoroughly investigated. In the present study, the role of the cagE gene as a diagnostic marker has been investigated. MATERIAL AND METHODS: We have used conventional polymerase chain reaction (PCR), quantitative PCR and whole genome sequencing data to determine the prevalence of cagE in A. actinomycetemcomitans based on analysis of: (i) 249 isolates, collected and cultivated in a Ghanaian longitudinal cohort study; (ii) a serotype b collection of 19 strains; and (iii) the 36 A. actinomycetemcomitans genomes available in the NCBI database. RESULTS: Whereas cagE was absent in the other serotypes, our data support that this gene sequence is linked to a virulent and highly leukotoxic group of serotype b strains, including both JP2 and non-JP2 genotypes of A. actinomycetemcomitans. CONCLUSION: We propose that cagE has the potential to be used as a PCR-based gene marker for the identification of a virulent and highly leukotoxic group of serotype b strains, including both JP2 and non-JP2 genotypes. This finding might be of importance in the risk assessment of the development of periodontal attachment loss in young individuals and hence suggested to be a relevant discovery in future development of new diagnostic tools and/or treatment strategies.

Cellular and molecular response of human macrophages exposed to Aggregatibacter actinomycetemcomitans leukotoxin.

Aggregatibacter (Actinobacillus) actinomycetemcomitans is a facultative anaerobic gram-negative bacterium associated with severe forms of periodontitis. A leukotoxin, which belongs to the repeats-in-toxin family, is believed to be one of its virulence factors and to have an important role in the bacterium's pathogenicity. This toxin selectively kills human leukocytes by inducing apoptosis and lysis. Here, we report that leukotoxin-induced cell death of macrophages proceeded through a process that differs from the classical characteristics of apoptosis and necrosis. A. actinomycetemcomitans leukotoxin-induced several cellular and molecular mechanisms in human macrophages that led to a specific and excessive pro-inflammatory response with particular secretion of both interleukin (IL)-1ß and IL-18. In addition, this pro-inflammatory cell death was inhibited by oxidized ATP, which indicates involvement of the purinergic receptor P2X(7) in this process. This novel virulence mechanism of the leukotoxin may have an important role in the pathogenic potential of this bacterium and can be a target for future therapeutic agents.

Antibacterial effect of ozone on cariogenic bacterial species.

OBJECTIVE: The aim was to evaluate the antibacterial effect of ozone on cariogenic bacterial species with and without the presence of saliva and a possible effect on the salivary proteins. METHODS: Suspensions of Actinomyces naeslundii (ACTCC 12104(T)), Lactobacilli casei (N CTC 151) and Streptococcus mutans (NCTC 10449), in salt buffer or in saliva, were exposed to ozone gas delivered by the ozone generator Healozone 2130C. Aliquots of the suspensions were taken after 10, 30 and 60s ozone exposures and cultivated on agar plates. Initial number of bacteria per ml was 8.0 x 10(7) (SD 2.2 x 10(7)) (A. naeslundii), 1.0 x 10(8) (SD 3.1 x 10(6)) (L. casei) and 1.0 x 10(8) (SD 7.0 x 10(5)) (S. mutans), respectively. The proteins were separated by SDS electrophoresis and visualized by silver staining. RESULTS: In salt buffer 92%, 73% and 64% of the initial numbers of A. naeslundii, S. mutans and L. casei, respectively, were killed already after 10s ozone exposure, while approximately 99.9% of the bacteria were dead after a 60s exposure. After 10 and 30s, but not after 60s exposure to ozone, S. mutans and L. casei were less efficiently killed in saliva compared to the salt buffer. Various saliva proteins were degraded by ozone after a 60s exposure. CONCLUSIONS: The cariogenic species S. mutans, L. casei and A. naeslundii were almost eliminated following 60s of ozone treatment. This killing was reduced in the presence of saliva although increasing the ozone application time to 60s overcame these reductants in saliva. Detection of altered salivary proteins indicates that saliva components constitute additional targets for ozone.

Mutans streptococci and lactobacilli in plaque on a leucite-reinforced dental ceramic and on a calcium aluminate cement.

In this in vivo study, the proportions of mutans streptococci and lactobacilli in plaque were examined (1) on proximal surfaces of bonded, leucite-reinforced ceramic crowns and (2) on class V restorations of calcium aluminate cement (CAC). The examined proportions were intraindividually compared with those of resin composite and enamel. Mutans streptococci and lactobacilli in samples from plaque that was accumulated for 10 days on the following surfaces were determined by cultivation on blood agar plates and species-selective plates: (1) proximal leucite-reinforced ceramic crown, class II composite and enamel (n=11); and (2) class V restoration of CAC and composite, and enamel (n=17). Mutans streptococci and lactobacilli in the samples were distributed in three groups: 0, >0-1, and >1% of total bacteria. The surfaces with detected mutans streptococci were similarly distributed between the materials and enamel. The highest proportion of mutans streptococci and lactobacilli were observed on ceramic followed by composite and enamel. A higher proportion of lactobacilli, but not of mutans streptococci, was detected on enamel compared to CAC and composite. However, no significant differences were found between the surfaces. Conclusively, the materials investigated did not show different relative proportions of mutans streptococci and lactobacilli in plaque, compared to enamel.

Abundant secretion of bioactive interleukin-1beta by human macrophages induced by Actinobacillus actinomycetemcomitans leukotoxin.

Actinobacillus actinomycetemcomitans produces a leukotoxin that selectively kills human leukocytes. Recently, we reported that macrophages are highly sensitive to leukotoxin and that their lysis involves activation of caspase 1. In this study, we show that leukotoxin also induces the production and release of proinflammatory cytokines from human macrophages. The macrophages were challenged with leukotoxin or lipopolysaccharide (LPS) from A. actinomycetemcomitans or LPS from Escherichia coli, and the production and secretion of interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) were determined at the mRNA and protein levels by reverse transcription-PCR and enzyme-linked immunosorbent assay, respectively. Leukotoxin (1 to 30 ng/ml) induced abundant production and secretion of IL-1beta, while the effects on IL-6 and TNF-alpha production were limited. Leukotoxin (1 ng/ml) caused a 10-times-higher release of IL-1beta than did LPS (100 ng/ml). The secreted IL-1beta was mainly the bioactive 17-kDa protein. At higher concentrations (>30 ng/ml), leukotoxin caused secretion of mainly inactive cytokine, the 31-kDa pro-IL-1beta. The presence of specific antibodies to IL-1beta or of a caspase 1 inhibitor blocked the secretion and production of the cytokine. Supernatants of leukotoxin-challenged macrophages stimulated bone resorption when tested in a mouse calvarial model. The activity could be blocked by an IL-1 receptor antagonist or specific antibodies to IL-1beta. We concluded that A. actinomycetemcomitans leukotoxin can trigger abundant production and secretion of bioactive IL-1beta by human macrophages, which is mediated by activation of caspase 1.

Subgingival plaque microbiota in Saudi Arabians after use of miswak chewing stick and toothbrush.

BACKGROUND: The chewing stick, the miswak, is used in many developing countries as the traditional means for oral hygiene. It is prepared from the roots, twigs and stem of Salvadora persica or other alternative local plants. OBJECTIVES: To compare the effects of the chewing stick miswak (from S. persica) and toothbrush on subgingival plaque microflora among Saudi Arabian individuals. Further, to investigate whether components extracted from S. persica may interfere with the subgingival plaque micro-organisms. MATERIAL AND METHODS: Fifteen healthy Saudi Arabian male volunteers aged 21-36 years were included in a single-blind, randomized cross-over study. The participants were taught how to use each device properly. Plaque sampling for DNA test was performed at the baseline, 1 week after professional tooth cleaning, and after 3 weeks of either miswak or toothbrush use. Identification and quantification of microbial species were performed by the checkerboard method, using whole genomic, digoxigenin-labelled DNA probes. Inhibition zones around miswak were examined on agar plates with Actinobacillus actinomycetemcomitans and the leukotoxicity of this bacterium was analyzed in a bioassay with macrophages+/-extracts of miswak. RESULTS: Miswak and toothbrushing had a similar influence on the levels of the subgingival microbiota. However, A. actinomycetemcomitans was significantly more reduced by miswak (p<0.05) than by toothbrushing. These results were supported by our in vitro results which, indicated that extracts from S. persica might interfere with the growth and leukotoxicity of A. actinomycetemcomitans. CONCLUSIONS: In contrast to toothbrush use, miswak use significantly reduced the amount of A. actinomycetemcomitans in the subgingival plaque.

Caspase 1 involvement in human monocyte lysis induced by Actinobacillus actinomycetemcomitans leukotoxin.

Actinobacillus actinomycetemcomitans, an oral bacterium implicated in the etiology of periodontal diseases, produces a leukotoxin that selectively lyses primate neutrophils and monocytes, the major populations of defense cells in the periodontium. Though lysis requires expression of the receptor lymphocyte function-associated molecule 1 (LFA-1) on the cell surface, not all LFA-1-expressing leukocyte populations are equally susceptible to the toxin. In this study, the susceptibility of human leukocytes to leukotoxin-induced lysis is compared to their expression of LFA-1 and the activity of caspase 1. Cytolysis was determined by the activity of lactate dehydrogenase released from peripheral human leukocytes after 1-h exposure to leukotoxin. Monocytes were lysed at leukotoxin concentrations of > or = 5 ng/ml, while the corresponding values for neutrophils and lymphocytes were approximately 10 times greater. Similar LFA-1 expression was found in all susceptible cell populations irrespective of their degree of sensitivity to the toxin. Exposure of monocytes to leukotoxin increased their caspase 1 activity about fivefold within 10 to 20 min. Presence of the caspase 1 inhibitor Ac-YVAD-CMK significantly blocked the leukotoxin-induced lysis of monocytes only. At sublytic concentrations, leukotoxin induced no apoptotic activity in monocytes, as revealed by the lack of caspase 3 activation and DNA fragmentation. Monocytes are the most lysis-sensitive leukocytes for A. actinomycetemcomitans leukotoxin. Their lysis by this toxin depends on caspase 1 activation and proceeds through a process that differs from classical apoptosis.

Serum-mediated release of leukotoxin from the cell surface of the periodontal pathogen Actinobacillus actinomycetemcomitans.

The leukotoxin of the periodontopathogen Actinobacillus actinomycetemcomitans is an important virulence factor that lyses human neutrophils and monocytes and thus, it may enable the bacterium to evade the local host defense. The toxin also induces degranulation of neutrophils and cytokine release in monocytes. To trigger these biological activities, leukotoxin has to be released from the bacterium and diffuse into the periodontal tissues. To date, the conditions found to cause toxin release have been artificial and have included high ion concentration and alkaline conditions. To study the release of the toxin under conditions mimicking the natural environment of the periodontium the ability of human serum to enable leukotoxin release from the bacterial surface was examined. Suspensions of leukotoxic A. actinomycetemcomitans strains were incubated with various concentrations of human serum or serum albumin. The suspensions were centrifuged and the leukotoxin in the supernatants or the cell pellets was detected by gel electrophoresis and immunoblotting. Serum was found to cause the rapid release of leukotoxin from the bacteria in a concentration-dependent manner. Pure albumin exhibited a similar effect. The leukotoxin released was active against human neutrophils. Only a minor proportion of it was associated with membranous vesicles produced by the bacteria. The results indicate that serum, a fluid closely related to the exudate in inflamed periodontal pockets, releases leukotoxin from the cell surface of A. actinomycetemcomitans. The process may enable the diffusion of the toxin from the bacterial biofilm into the surrounding tissues, where it can exert its biological effect.

Release and activation of matrix metalloproteinase 8 from human neutrophils triggered by the leukotoxin of Actinobacillus actinomycetemcomitans.

Matrix metalloproteinase 8 (MMP 8) degrades type I collagen and may be involved in the pathogenesis of periodontitis. Latent MMP 8 is stored in neutrophil granules and can be activated when released extracellularly. The periodontitis-associated bacterium Actinobacillus actinomycetemcomitans produces an RTX-toxin, leukotoxin, that degranulates and lyses human neutrophils. This study deals with the ability of leukotoxic A. actinomycetemcomitans to trigger the release and activation of MMP 8. Whole bacteria of three A. actinomycetemcomitans strains or leukotoxin purified from the highly toxic strain HK 1519 were incubated with human neutrophils. The extracellularly released latent and active forms of MMP 8 were detected by an immunoblot technique using specific antibodies against the protease. The activity of MMP 8 was determined by a collagen degradation assay. All strains induced release and activation of MMP 8. The effect was more pronounced under aerobic than anaerobic conditions and correlated with the leukotoxicity of the strains. Pure leukotoxin also induced MMP 8 release and activation in a concentration-dependent manner. Under aerobic conditions, oxidising substances formed by the neutrophils contributed to the rapid activation of the latent enzyme. Upon anaerobic incubation, the activation was slow and mainly caused by other proteases released during neutrophil degranulation. The activation was totally abolished in the presence of serum, probably due to the serum-protease inhibitors. Compared to the calcium ionophore A 23187, a well-known stimulus of neutrophil degranulation, leukotoxin was a more powerful inducer of MMP 8 release, since it triggered the process at a 1000-fold lower concentration. The present findings reveal a specific mechanism that can be induced by A. actinomycetemcomitans leukotoxin and which may contribute to the degradation of periodontal tissues under certain conditions.

Lack of lipoprotein-dependent effects on the cytotoxic interactions of Actinobacillus actinomycetemcomitans leukotoxin with human neutrophils.

A high odds ratio has been reported for hyperlipidemia and periodontal diseases in humans, and the severity of periodontitis seems to correlate with the hyperlipidemic status of the patients. Early studies indicated that the lipoprotein-containing fraction of the serum enhances the leukotoxic activity of the periodontopathogen Actinobacillus actinomycetemcomitans against human polymorphonuclear leukocytes (PMNL). The protease inhibitors of normal serum account for this enhancement, while delipidated serum has no effect on the leukotoxin-dependent PMNL cytolysis. No information exists for the effect of serum lipoproteins or hyperlipidemic serum. The aim of this study was to evaluate the role of serum lipoproteins in the interaction of the leukotoxin of A. actinomycetemcomitans with human PMNL. Purified leukotoxin was mixed with human PMNL prepared from venous blood of healthy subjects and various varying amounts of hyperlipidemic or delipidated serum, or purified serum lipoproteins. The cytolytic activity of leukotoxin was determined by activity of the cytosol enzyme lactate dehydrogenase released from injured PMNL. The degranulating activity of the toxin was measured through the release of the granule components elastase and lactoferrin. Normal human serum without leukotoxin-neutralizing antibodies caused a 4-fold enhancement of the leukotoxic activity when present at concentrations of 5-10% in the reaction mixture. Serum lipoproteins had no effect when added at concentrations that occur normally in serum. At high concentrations, purified low density and very low-density lipoproteins increased the leukotoxicity of the mixture. Nevertheless, hyperlipidemic serum prepared from a normal serum by the addition of autologous lipoproteins had no influence on the leukotoxin-caused cytolysis compared to the normal serum. Pre-incubation of PMNL for 1 h in hyperlipidemic or delipidated serum had no effect on the leukotoxin-induced degranulation of PMNL. The results indicate that the cytotoxic interactions of A. actinomycetemcomitans leukotoxin against human PMNL are not influenced by the presence of serum lipoproteins.

Protease inhibitors, the responsible components for the serum-dependent enhancement of Actinobacillus actinomycetemcomitans leukotoxicity.

Serum enhances the leukotoxic activity of Actinobacillus actinomycetemcomitans against human polymorphonuclear leukocytes (PMNL) by a mechanism that still is unknown. Early attempts to identify the serum components responsible for this enhancement gave no conclusive results, but indicated that the lipoprotein-containing fraction of the serum was involved in the interaction. This study aimed to clarify the role of serum lipoproteins in the leukotoxin interaction, and to identify other serum components involved. The main hypothesis examined was that the leukotoxicity enhancement might depend on serum protease inhibitors that block proteolytic cleavage of leukotoxin by enzymes released from the leukocytes. PMNL were isolated from human peripheral blood and incubated with purified leukotoxin in the presence of serum or purified serum components or lipoprotein-deficient serum. Leukotoxin was also incubated with purified elastase and cathepsin G or with enzyme mixtures from degranulated PMNL. The leukotoxic activity in these mixtures was determined as the extracellular release of lactate dehydrogenase from PMNL. Cleavage of the toxin was showed by gel electrophoresis and Western blot. Morphological changes in PMNL from the above mixtures were examined by electron microscopy. Enzymes from degranulated PMNL cleaved leukotoxin to non-cytotoxic fragments. Elastase and cathepsin G were mainly responsible for the cleavage. Inhibition of leukotoxin degradation was found in the presence of whole serum or of the serum protease inhibitors alpha2-macroglobulin and alpha1-proteinase inhibitor. Under these conditions enhanced PMNL lysis was also observed. A similar enhancement of PMNL lysis was found when PMNL degranulation was blocked by EDTA. On the other hand, lipoprotein-deficient serum had no influence on the leukotoxic activity. The results indicate that the increased leukotoxicity of A. actinomycetemcomitans observed in the presence of human serum is caused by the serum protease inhibitors that counteract proteolytic degradation of leukotoxin. The degradation is caused by enzymes from degranulated PMNL triggered by leukotoxin.

Polymorphonuclear leukocyte degranulation induced by leukotoxin from Actinobacillus actinomycetemcomitans.

The ability of leukotoxin from Actinobacillus actinomycetemcomitans to induce release of lysosomal constituents was studied with human polymorphonuclear leukocytes (PMNL). Leukotoxin purified from A. actinomycetemcomitans or bacterial cells of a leukotoxic strain were mixed with human PMNL and the suspension was incubated under anaerobic conditions. Samples were taken at certain time intervals to examine the cell morphology of PMNL by electron microscopy and the extracellular concentrations of the granule components lactoferrin and elastase by enzyme-linked immunosorbent assay (ELISA). Electron microscopy revealed that within 10 min of exposure to leukotoxin, the number of intracellular granules was markedly reduced and the remaining granules were translocated to the periphery in PMNL. At the same time, the extracellular concentrations of lactoferrin and elastase were elevated, while that of the cytosolic enzyme lactate dehydrogenase, an indicator of cell lysis, remained low. The lysosome molecules CD63 and CD66b were also exposed on the PMNL surface, indicating fusion of lysosomes with the plasma membrane. These effects were completely abolished by the addition of anti-leukotoxin serum. Pre-incubation of PMNL with monoclonal antibodies to CD11a and CD18 that recognize alpha- and beta-chains of the LFA-1 integrin, a leukotoxin receptor on PMNL, inhibited the cytolysis, but not the release of granule components. The present results demonstrate the ability of A. actinomycetemcomitans leukotoxin to trigger a rapid release of lysosomal compounds in human PMNL. The release is due to an active process stimulated by the interaction of PMNL with the toxin or toxin-carrying bacteria.

Anaerobic neutrophil-dependent killing of Actinobacillus actinomycetemcomitans in relation to the bacterial leukotoxicity.

The effect of leukotoxin on the interaction of Actinobacillus actinomycetemcomitans with human polymorphonuclear leukocytes (PMNL) was studied under anaerobic conditions with strains able to produce high or low amounts of leukotoxin. PMNL morphology, phagocytosis and killing were examined by transmission electron microscopy and bioassays, respectively. At ratios of > or =25 bacteria/PMNL, a highly toxic A. actinomycetemcomitans strain completely destroyed the PMNL within 7 min, resulting in bacterial survival. Lowering the bacteria/PMNL-ratio enabled phagocytosis and killing of this highly toxic strain. A. actinomycetemcomitans strains with low leukotoxicity were effectively killed by PMNL under any condition. Presence of specific antibodies against A. actinomycetemcomitans or of anti-leukotoxin serum protected PMNL from being injured and allowed phagocytosis to occur. Pre-incubation of the leukotoxic strain with Porphyromonas gingivalis, a bacterium that destroys leukotoxin, abolished lysis of PMNL and inhibited phagocytosis of A. actinomycetemcomitans. The results show that leukotoxin may protect A. actinomycetemcomitans against phagocytosis by human PMNL. The protection occurs at a population level and in relation to the bacterial load. The size of the bacterial population required to counteract phagocytosis is dependent on the leukotoxin production of the strain.

Gingival crevicular fluid from patients with periodontitis contains bone resorbing activity.

Inflammatory processes occurring in the vicinity of bone tissue often result in stimulation of osteoclast activity and loss of skeletal mass. The aim of the current study was to determine if inflammatory exudates collected from gingival pockets in patients with periodontitis contain factors capable of stimulating resorptive activity. The degree of bone mineral mobilization and bone matrix degradation was assessed by analysis of the release of 45Ca and 3H from bones prelabelled with 45CaCl2 and [3H]proline, respectively. Gingival crevicular washings from six patients with signs of periodontitis stimulated 45Ca or 3H release from the calvarial bones. The stimulatory effect of the gingival crevicular washings on 45Ca release was concentration- and time-dependent, and reduced by calcitonin, a specific osteoclast inhibitor. These data demonstrate that crevicular fluid contains factor(s) which can stimulate osteoclastic degradation of bone in vitro. The bone resorbing activity was partially retained after extensive dialysis. Analysis of the concentrations of prostaglandin E2, interleukin-1alpha and interleukin-1beta in the crevicular fluids, and comparisons of these agents as stimulators of 45Ca release in the mouse calvarial assay, suggest that prostaglandin E2 is not the sole factor responsible for the bone resorbing activity of the exudates. The data indicate that other factors, such as IL-1, may play key roles in the stimulation of osteoclastic activity by gingival crevicular washings.

Relative role of chloramines, hypochlorous acid, and proteases in the activation of human polymorphonuclear leukocyte collagenase.

The activation of collagenase released by polymorphonuclear leukocytes (PMNs) has been extensively studied in vitro, but the activation of the enzyme in vivo is not fully understood. For further evaluation of the relative role of oxidative and proteolytic mechanisms in the activation of collagenase, PMNs were stimulated by serum-opsonized zymosan under both aerobic and anaerobic conditions. The results showed that similar amounts of collagenase were released by the PMNs under aerobic and anaerobic conditions, but the activity of the released collagenase was twice as high under aerobic conditions as under anaerobic conditions. Under aerobic conditions the enzyme was rapidly activated by hypochlorous acid and chloramines, which are products of the myeloperoxidase-H2O2-chloride system of the PMNs. There was also a slow proteolytic activation of the enzyme, which could be ascribed to cathepsin G and possibly to some other serine proteases of PMNs. When extrapolating these findings to in vivo conditions, it seems probable that the oxidative activation of collagenase will proceed mainly by chloramines, which are more long-lived in the tissue than hypochlorous acid. In poorly oxygenated tissues, collagenase may be mainly activated by proteolytic mechanisms.

The economic cost of a streptococcal tonsillitis episode.

OBJECTIVE: To estimate the cost of a streptococcal tonsillitis episode from the data of a questionnaire. SETTING: Five primary health centres in the west of Sweden. PARTICIPANTS: 101 consecutive patients treated for streptococcal tonsillitis. MAIN OUTCOME MEASURE: The cost estimation included costs for physician visit and drug, travel costs to and from the primary health centre, cost of lost production resulting from the patient's or the guardian's absence from work for physician visit or sick-leave, and cost of telephone consultation with a physician or nurse. RESULTS: The period of illness was on average seven days, time to recovery after treatment five days, and the mean period of sick-leave 2.5 days. The total cost of a tonsillitis episode was about SEK 3,300 (385 USD). Of this sum, the cost for the antibiotic accounted for only 3% and loss of production for 75%. CONCLUSION: Differences in the cost of drugs only have a minor influence on the total cost, while factors causing loss of production, such as efficacy and side effects of the drug, have a greater influence. Economic evaluation of pharmaceuticals will be more relevant in the future, and in the search for the most effective treatment, cost effective studies will be integrated with clinical trials.

Competition for peptides and amino acids among periodontal bacteria.

We recently studied the utilization of glutathione (L-gamma-glutamyl-L-cysteinylglycine), L-cysteinylglycine and L-cysteine by anaerobic bacteria. The rate of hydrogen sulfide formation from these compounds was determined and it was concluded that Peptostreptococcus micros and Fusobacterium nucleatum subsp. nucleatum had an active transport of small peptides. In the present study it is shown that methyl mercaptan formation from L-methionine and L-methionyl-containing peptides can also be used to study peptide utilization. There were differences among the periodontal bacteria P. micros, F. nucleatum subsp. nucleatum, and Porphyromonas gingivalis in their capacity to use L-cysteine and L-methionine and peptides containing these amino acids. The peptides were used more efficiently by P. micros and F. nucleatum subsp. nucleatum than by P. gingivalis. All three species used the peptides more efficiently than the free amino acids. The efficiency in utilizing various amino acids and peptides may be among the key determinants of the periodontal microbial ecology.

Oxygen-dependent modulation of release and activity of polymorphonuclear leukocyte granule products.

Polymorphonuclear leukocytes are important in the defense against the anaerobic microflora of infected gingival pockets. One part of this defense is release of antibacterial granule products by polymorphonuclear leukocytes into the pockets. The aim of the present study was to compare the efficiency of polymorphonuclear leukocytes in releasing granule products under aerobic and anaerobic conditions. Polymorphonuclear leukocytes were exposed to serum-opsonized zymosan under aerobic and anaerobic conditions. The levels of released granule products were determined by combining measurements of activity with enzyme-linked immunosorbent assays. The level of released elastase was twice as high in anaerobic as in aerobic reaction mixtures. A similar difference was not detected for myeloperoxidase. However, myeloperoxidase was inactivated after its release under aerobic conditions. The release of lactoferrin was an efficient under aerobic as under anaerobic conditions. The effect of aerobic conditions on the release of elastase and the inactivation of myeloperoxidase could be ascribed to oxidants formed in the myeloperoxidase-H2O2-chloride system. Also, the activity of the released cytoplasmic enzyme lactate dehydrogenase was inactivated by oxidants formed in the myeloperoxidase-H2O2-chloride system. These findings suggest that, in the anaerobic environment of the gingival pocket, elastase and possibly also other azurophilic granule products are released in higher amounts than under fully oxygenated conditions. In this environment, the released products may also escape inactivation by the myeloperoxidase-H2O2-chloride system.

Effect of anaerobiosis and sulfide on killing of bacteria by polymorphonuclear leukocytes.

Anaerobic microorganisms in periodontal pockets produce toxic amounts of hydrogen sulfide. The capacity of polymorphonuclear leukocytes to kill a capsulated and a non-capsulated variant of a group B streptococcal strain was studied in presence and absence of sulfide. The killing was equally efficient under aerobic and anaerobic conditions. However, in presence of sulfide the killing of the capsulated variant of the strain was significantly inhibited. Since this strain required higher serum concentrations to be killed by the polymorphonuclear leukocytes, it suggested that sulfide interfered with the opsonization of the bacteria. The capacity of sulfide to split the disulfide bonds of complement factor 3 and immunoglobulin G, deposited on the bacterial surface, was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was no detectable effect of 2 mM sulfide on immunoglobulin G. However, sulfide released from opsonized bacteria the beta-chain of C3b C3bi, and the C-terminal part of the alpha-chain of C3bi. This region of the alpha-chain of C3bi has been suggested to bind to the complement receptor 3 of polymorphonuclear leukocytes. The beta-chain of C3b/C3bi may augment the binding of opsonized bacteria to the complement receptors of polymorphonuclear leukocytes. The formation of sulfide by the microflora of the periodontal pockets may provide conditions for the bacteria to escape important parts of the host immune system.