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.

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.

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.

Catalase inhibition by sulfide and hydrogen peroxide-induced mutagenicity in Salmonella typhimurium strain TA102.

The lethal and mutagenic effects of hydrogen peroxide were studied in exponentially growing cultures of Salmonella typhimurium strain TA102. Exposure of the cultures to non-lethal levels of sodium sulfide significantly increased the lethality and mutagenicity of hydrogen peroxide. The catalase activity was decreased in cells exposed to sodium sulfide, but there were no changes in the cellular levels of superoxide dismutase, glutathione reductase, or NADPH-dependent alkyl hydroperoxide reductase. Hydrogen peroxide-induced mutagenesis and killing of S. typhimurium strain TA102 in the presence of sulfide may in part be explained by an inactivation of catalase by sulfide.

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.

Presence of Lactobacillus casei in saliva from children and adults using a new medium.

The aim of this study was to devise a medium for cultivation of L. casei and to compare the presence of L. casei in saliva from children and adults. The new medium was produced by omitting the sugars from Rogosa SL agar medium and supplementing this medium with melezitose. Although all tested strains of lactobacilli grew on the new medium, the facultatively homofermentative lactobacilli, L. casei and L. plantarum, could be identified by a typical colonial morphology. L. casei was the most common species of oral lactobacilli in saliva from children and made up a significantly higher proportion of the salivary lactobacillus flora in children than in adults.

Chemotaxis and degranulation of polymorphonuclear leukocytes in the presence of sulfide.

In polymicrobial infections such as periodontal disease, the polymorphonuclear leukocytes (PMN) may have to work in the absence of oxygen and in the presence of significant levels of hydrogen sulfide. There are conflicting results reported on the chemotactic capacity of PMN under anaerobic conditions. It is not known whether PMN are able to migrate and release the contents of their granules in the presence of sulfide. PMN were exposed to various levels of sulfide and their chemotaxis and degranulation were studied when they were stimulated with N-formyl-methionyl-leucyl-phenylalanine or zymosan-activated serum. Chemotaxis was evaluated with the agarose method. The release of granule markers, lactoferrin and myeloperoxidase, was evaluated with enzyme-linked immunosorbent assay. PMN had similar capacity for chemotaxis under aerobic and anaerobic conditions. The migration of PMN was only to a minor extent inhibited by 1-2 mM sulfide. The release of lactoferrin and myeloperoxidase was the same under aerobic and anaerobic conditions and was not significantly influenced by sulfide. PMN seem to be very well suited to defend the tissue against bacteria under the harsh conditions prevailing in the periodontal pocket.

The capacity of subgingival microbiotas to produce volatile sulfur compounds in human serum.

Hydrogen sulfide is formed by the subgingival microbiotas of periodontal pockets. The capacity of these microbiotas to form various volatile sulfur compounds in human serum was studied. Bacterial samples from nine deep periodontal pockets were incubated for 7 days in human serum and the amounts of volatile sulfur compounds and the degradation of serum proteins were determined. Hydrogen sulfide was the predominant volatile sulfur compound, but also methyl mercaptan was formed in significant amounts. Only traces of dimethyl sulfide and dimethyl disulfide were detected. There was an extensive degradation of the serum proteins. In most of the reaction mixtures hydrogen sulfide reached highly toxic levels.

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.

Presence of mutans streptococci and various types of lactobacilli in interdental spaces related to development of proximal carious lesions.

During a 2-yr study period samples from saliva, the tongue, and 276 interdental spaces were obtained from 23 7-yr-old children in order to (a) relate the presence of lactobacilli in various oral sites to the occurrence of lactobacilli in saliva, and (b) relate the presence of mutans streptococci and various types of lactobacilli interdentally to the development of proximal carious lesions. The results showed an increased number of interdental samples containing lactobacilli with an increasing number of salivary lactobacilli. Furthermore, lactobacilli were never found interdentally without the presence of mutans streptocci, lactobacilli proved to be the more suitable microorganism for prediction of proximal carious lesions. Neither the number nor the differentiation into different species of interdental lactobacilli seemed to be of importance, but simply whether they were present. The presence of lactobacilli probably reflects a caries-inducing environment (etiologic microflora + fermentable carbohydrates), thus explaining their high predictive ability compared to their rather limited etiologic importance in the initiation of decay.

Activity of polymorphonuclear leukocytes in the presence of sulfide.

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.

The formation of hydrogen sulfide and methyl mercaptan by oral bacteria.

The capacity to form volatile sulfur compounds was tested in bacteria isolated from subgingival microbiotas and in a representative number of reference strains. A majority of the 75 tested oral bacterial species and 7 unnamed bacterial taxa formed significant amounts of hydrogen sulfide from L-cysteine. The most active bacteria were found in the genera Peptostreptococcus, Eubacterium, Selenomonas, Centipeda, Bacteroides and Fusobacterium. Methyl mercaptan from L-methionine was formed by some members of the genera Fusobacterium, Bacteroides, Porphyromonas and Eubacterium. When incubated in serum for 7 d, the most potent producers of hydrogen sulfide were Treponema denticola and the black-pigmented species, Bacteroides intermedius, Bacteroides loescheii, Porphyromonas endodontalis and Porphyromonas gingivalis. P. endodontalis and P. gingivalis also produced significant amounts of methyl mercaptan in serum. No other volatile sulfur compound was detected in serum or in the presence of L-cysteine and L-methionine. These findings significantly increase the list of oral bacteria known to produce volatile sulfur compounds.

Production of volatile sulfur compounds by various Fusobacterium species.

In 12 species of Fusobacterium the following characteristics were studied; the desulfhydration of L-cysteine and L-methionine by resting cell suspensions, the formation of alpha-keto-acids from L-cysteine, D-cysteine and L-methionine by cell extracts, and the formation of hydrogen sulfide from L-cysteine, D-cysteine and L-cysteine by cell extracts separated by polyacrylamide gel electrophoresis. Multiple forms of L-cysteine desulfhydrase activity were found in most of the species. In some of them also D-cysteine desulfhydrase activity was demonstrated. Seven of the species had high L-methionine gamma-lyase activity. L-cysteine activity was present in 5 of the species.

Effect of sulfide ions on complement factor C3.

In infected sites such as the gingival pockets of patients with periodontal disease, sulfide levels up to 1 mmol/liter may be reached. There is little information, however, on how sulfide may interact with the host defense. In a previous study (R. Claesson, M. Granlund-Edstedt, S. Persson, and J. Carlsson, Infect. Immun. 57:2776-2781, 1989), it was shown that polymorphonuclear leukocytes were able to kill bacteria in the presence of 1 mM sulfide. However, sulfide seemed to interfere with the opsonization of the bacteria. It has been claimed that sulfide may be toxic by splitting disulfide bonds of proteins. In the present study, serum was exposed to 2 mM sulfide under anaerobic conditions, and the capacity of sulfide to split disulfide bonds of 10 serum proteins involved in opsonization was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection of the proteins after blotting. Sulfide had a low capacity to split the disulfide bonds of most proteins. Sulfide had, however, a pronounced effect on the complement component C3 in the form of C3bi. Sulfide released the C-terminal region of the alpha chain from C3bi. When C3 opsonizes bacteria, it is this region of C3bi which binds to complement receptor 3 (CR3) of the polymorphonuclear leukocytes. If sulfide has the same effect on C3bi deposited on the bacterial surface as it has on C3bi in solution, it will annihilate the very important contribution of C3bi to opsonization.

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.