Rolf Claesson

Caspase 1 Involvement in Human Monocyte Lysis Induced by Actinobacillus actinomycetemcomitans Leukotoxin

ABSTRACT 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 ≥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.

Oral microbial profile discriminates breast-fed from formula-fed infants

Objectives: Little is known about the effect of diet on the oral microbiota of infants, although diet is known to affect the gut microbiota. The aims of the present study were to compare the oral microbiota in breast-fed and formula-fed infants, and investigate growth inhibition of streptococci by infant-isolated lactobacilli. Methods: A total of 207 mothers consented to participation of their 3-month-old infants. A total of 146 (70.5%) infants were exclusively and 38 (18.4%) partially breast-fed, and 23 (11.1%) were exclusively formula-fed. Saliva from all of their infants was cultured for Lactobacillus species, with isolate identifications from 21 infants. Lactobacillus isolates were tested for their ability to suppress Streptococcus mutans and S sanguinis. Oral swabs from 73 infants were analysed by the Human Oral Microbe Identification Microarray (HOMIM) and by quantitative polymerase chain reaction for Lactobacillus gasseri. Results: Lactobacilli were cultured from 27.8% of exclusively and partially breast-fed infants, but not from formula-fed infants. The prevalence of 14 HOMIM-detected taxa, and total salivary lactobacilli counts differed by feeding method. Multivariate modelling of HOMIM-detected bacteria and possible confounders clustered samples from breast-fed infants separately from formula-fed infants. The microbiota of breast-fed infants differed based on vaginal or C-section delivery. Isolates of L plantarum, L gasseri, and L vaginalis inhibited growth of the cariogenic S mutans and the commensal S sanguinis: L plantarum >L gasseri >L vaginalis. Conclusions: The microbiota of the mouth differs between 3-month-old breast-fed and formula-fed infants. Possible mechanisms for microbial differences observed include species suppression by lactobacilli indigenous to breast milk.

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 bacteriums 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)-1β and IL-18. In addition, this pro-inflammatory cell death was inhibited by oxidized ATP, which indicates involvement of the purinergic receptor P2X7 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.

Abundant Secretion of Bioactive Interleukin-1beta by Human Macrophages Induced by Actinobacillus actinomycetemcomitans Leukotoxin

ABSTRACT 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-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α) 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-1β, while the effects on IL-6 and TNF-α production were limited. Leukotoxin (1 ng/ml) caused a 10-times-higher release of IL-1β than did LPS (100 ng/ml). The secreted IL-1β 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-1β. The presence of specific antibodies to IL-1β 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-1β. We concluded that A. actinomycetemcomitans leukotoxin can trigger abundant production and secretion of bioactive IL-1β by human macrophages, which is mediated by activation of caspase 1.

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.

Progression of attachment loss is strongly associated with presence of the JP2 genotype of Aggregatibacter actinomycetemcomitans: a prospective cohort study of a young adolescent population

AIM To assess the progression of attachment loss (AL) during a 2-year period according to the presence of JP2 and non-JP2 genotypes of Aggregatibacter actinomycetemcomitans in a Ghanaian adolescent population. METHODS A total of 500 adolescents (mean age 13.2 years, SD ± 1.5) were enrolled in the study. After 2 years, 397 (79.4%) returned for a periodontal re-examination, including the measurement of AL. The carrier status of the JP2 and non-JP2 genotypes of A. actinomycetemcomitans was evaluated in a baseline examination 2 years earlier. RESULTS Individuals who carried the JP2 genotype of A. actinomycetemcomitans had a significantly increased risk [relative risk (RR) = 7.3] of developing AL ≥ 3 mm. The mean AL at the follow-up and the mean 2-year progression of AL were significantly higher in the JP2 genotype-positive group (n = 38) compared with the group positive for the non-JP2 genotypes of A. actinomycetemcomitans (n = 169), and the group of A. actinomycetemcomitans-negative individuals (n = 190). The JP2 genotype was strongly associated with the progression of AL ≥ 3 mm (OR = 14.3). The non-JP2 genotypes of A. actinomycetemcomitans were also, however, less pronounced, associated with the progression of AL ≥ 3 mm (OR = 3.4). CONCLUSION The JP2 genotype of A. actinomycetemcomitans is strongly associated with the progression of AL.

Leukotoxic Activity of Aggregatibacter actinomycetemcomitans and Periodontal Attachment Loss

Aggregatibacter actinomycetemcomitans is a Gram-negative periodontitis-associated bacterium that expresses a toxin that selectively affects leukocytes. This leukotoxin is encoded by an operon belonging to the core genome of this bacterial species. Variations in the expression of the leukotoxin have been reported, and a well-characterized specific clonal type (JP2) of this bacterium with enhanced leukotoxin expression has been isolated. In particular, the presence of the JP2 genotype significantly increases the risk for the progression of periodontal attachment loss (AL). Based on these findings we hypothesized that variations in the leukotoxicity are linked to disease progression in infected individuals. In the present study, the leukotoxicity of 239 clinical isolates of A. actinomycetemcomitans was analysed with different bioassays, and the genetic peculiarities of the isolates were related to their leukotoxicity based on examination with molecular techniques. The periodontal status of the individuals sampled for the presence of A. actinomycetemcomitans was examined longitudinally, and the importance of the observed variations in leukotoxicity was evaluated in relation to disease progression. Our data show that high leukotoxicity correlates with an enhanced risk for the progression of AL. The JP2 genotype isolates were all highly leukotoxic, while the isolates with an intact leukotoxin promoter (non-JP2 genotypes) showed substantial variation in leukotoxicity. Genetic characterization of the non-JP2 genotype isolates indicated the presence of highly leukotoxic genotypes of serotype b with similarities to the JP2 genotype. Based on these results, we conclude that A. actinomycetemcomitans harbours other highly virulent genotypes besides the previously described JP2 genotype. In addition, the results from the present study further highlight the importance of the leukotoxin as a key virulence factor in aggressive forms of periodontitis.

Presence of JP2 and Non-JP2 Genotypes of Aggregatibacter actinomycetemcomitans and Attachment Loss in Adolescents in Ghana

BACKGROUND Limited data are reported concerning the presence of A. actinomycetemcomitans and attachment loss (AL) in sub-Saharan countries. The authors investigate the carrier frequency of JP2 and non-JP2 genotypes of A. actinomycetemcomitans and the presence of AL in Ghanaian adolescents and evaluate socioeconomic conditions and oral hygiene practices. METHODS Five hundred individuals (mean ± SD age: 13.2 ± 1.5 years) in public and private schools were interviewed about demographic characteristics and oral hygiene practices and were given a full-mouth periodontal examination. Subgingival plaque samples were obtained from periodontal sites around permanent first molars and incisors. The carrier status of A. actinomycetemcomitans at the individual level was determined based on results obtained by cultivation and polymerase chain reaction. RESULTS The findings of this study show a relatively high carrier rate of JP2 and non-JP2 genotypes of Aggregatibacter actinomycetemcomitans in the Ghanaian adolescent population and the presence of this bacterium is associated with the occurrence of AL. The overall carrier rate of A. actinomycetemcomitans was 54.4%, and the highly leukotoxic JP2 genotype was detected in 8.8% of the study population. A total of 107 (21.4%) individuals had ≥ 1 tooth with AL ≥ 3 mm. The majority of the individuals carrying A. actinomycetemcomitans (80.1%) (P <0.001) and of the periodontally diseased individuals (91.6%) (P <0.001) were found in public schools. CONCLUSIONS A. actinomycetemcomitans and AL were frequently found in Ghanaian adolescents. The school type was the strongest predictor of both presence of A. actinomycetemcomitans and AL.

Cytolethal Distending Toxin in Isolates of Aggregatibacter actinomycetemcomitans from Ghanaian Adolescents and Association with Serotype and Disease Progression

Background and Objectives The cytolethal distending toxin (Cdt) is a highly conserved exotoxin that are produced by a number of Gram negative bacteria, including Aggregatibacter actinomycetemcomitans, and affects mammalian cells by inhibiting cell division and causing apoptosis. A complete cdt-operon is present in the majority of A. actinomycetemcomitans, but the proportion of isolates that lack cdt-encoding genes (A, B and C) varies according to the population studied. The objectives of this study were to examine serotype, Cdt-genotype, and Cdt-activity in isolates of A. actinomycetemcomitans collected from an adolescent West African population and to examine the association between the carrier status of A. actinomycetemcomitans and the progression of attachment loss (AL). Materials and Methods A total of 249 A. actinomycetemcomitans isolates from 200 Ghanaian adolescents were examined for serotype and cdt-genotype by PCR. The activity of the Cdt-toxin was examined by DNA-staining of exposed cultured cells and documented with flow cytometry. The periodontal status of the participants was examined at baseline and at a two-year follow-up. Results Presence of all three cdt-encoding genes was detected in 79% of the examined A. actinomycetemcomitans isolates. All these isolates showed a substantial Cdt-activity. The two different cdt-genotypes (with and without presence of all three cdt-encoding genes) showed a serotype-dependent distribution pattern. Presence of A. actinomycetemcomitans was significantly associated with progression of AL (OR = 5.126; 95% CI = [2.994–8.779], p<0.001). Conclusion A. actinomycetemcomitans isolated from the Ghanaian adolescents showed a distribution of serotype and cdt-genotype in line with results based on other previously studied populations. Presence of A. actinomycetemcomitans was significantly associated with disease progression, in particular the b serotype, whereas the association with disease progression was not particularly related to cdt-genotype, and Cdt-activity.

Characterization and in vitro properties of oral lactobacilli in breastfed infants

BackgroundLactobacillus species can contribute positively to general and oral health and are frequently acquired by breastfeeding in infancy. The present study aimed to identify oral lactobacilli in breast and formula-fed 4 month-old infants and to evaluate potential probiotic properties of the dominant Lactobacillus species detected. Saliva and oral swab samples were collected from 133 infants who were enrolled in a longitudinal study (n=240) examining the effect of a new infant formula on child growth and development. Saliva was cultured and Lactobacillus isolates were identified from 16S rRNA gene sequences. Five L. gasseri isolates that differed in 16S rRNA sequence were tested for their ability to inhibit growth of selected oral bacteria and for adhesion to oral tissues. Oral swab samples were analyzed by qPCR for Lactobacillus gasseri.Results43 (32.3%) infants were breastfed and 90 (67.7%) were formula-fed with either a standard formula (43 out of 90) or formula supplemented with a milk fat globule membrane (MFGM) fraction (47 out of 90). Lactobacilli were cultured from saliva of 34.1% breastfed infants, but only in 4.7% of the standard and 9.3% of the MFGM supplemented formula-fed infants. L. gasseri was the most prevalent (88% of Lactobacillus positive infants) of six Lactobacillus species detected. L. gasseri isolates inhibited Streptococcus mutans binding to saliva-coated hydroxyapatite, and inhibited growth of S. mutans, Streptococcus sobrinus, Actinomyces naeslundii, Actinomyces oris, Candida albicans and Fusobacterium nucleatum in a concentration dependent fashion. L. gasseri isolates bound to parotid and submandibular saliva, salivary gp340 and MUC7, and purified MFGM, and adhered to epithelial cells. L. gasseri was detected by qPCR in 29.7% of the oral swabs. Breastfed infants had significantly higher mean DNA levels of L. gasseri (2.14 pg/uL) than infants fed the standard (0.363 pg/uL) or MFGM (0.697 pg/uL) formula.ConclusionsLactobacilli colonized the oral cavity of breastfed infants significantly more frequently than formula-fed infants. The dominant Lactobacillus was L. gasseri, which was detected at higher levels in breastfed than formula-fed infants and displayed probiotic traits in vitro.