Aggregatibacter actinomycetemcomitans: virulence of its leukotoxin and association with aggressive periodontitis.
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ABSTRACT:
SUBMITTER: Aberg CH
PROVIDER: S-EPMC4601274 | biostudies-other | 2015
REPOSITORIES: biostudies-other
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Project description:ObjectivesWe and others have previously shown that epigallocatechin gallate (EGCg) inhibits the activity of an important virulence factor, leukotoxin (LtxA), produced by the oral bacterium Aggregatibacter actinomycetemcomitans, suggesting the potential use of this molecule as an anti-virulence strategy to treat periodontal infections. Here, we sought to better understand the effects of EGCg on toxin secretion and A. actinomycetemcomitans pathogenicity in a co-culture model.MethodsWe used a quantitative immunoblot assay to determine the concentrations of LtxA in the bacterial supernatant and on the bacterial cell surface. Using a co-culture model, consisting of A. actinomycetemcomitans and THP-1 cells, we studied the impact of EGCg-mediated changes in LtxA secretion on the toxicity of A. actinomycetemcomitans.Key findingsEGCg increased production of LtxA and changed the localization of secreted LtxA from the supernatant to the surface of the bacterial cells. In the co-culture model, a single low dose of EGCg did not protect host THP-1 cells from A. actinomycetemcomitans-mediated cytotoxicity, but a multiple dosing strategy had improved effects.ConclusionsTogether, these results demonstrate that EGCg has important, but complicated, effects on toxin secretion and activity; new dosing strategies and comprehensive model systems may be required to properly develop these anti-virulence activities.
| S-EPMC7947220 | biostudies-literature
Project description:This study aims to investigate the prevalence of the highly leukotoxic JP2 sequence versus the minimally leukotoxic non-JP2 sequence of Aggregatibacter actinomycetemcomitans within a cohort of 180 young African Americans, with and without localized aggressive periodontitis (LAP), in north Florida. The study included patients aged 5 to 25 y: 60 LAP patients, 60 healthy siblings (HS), and 60 unrelated healthy controls (HC). Subgingival plaque was collected from LAP sites-diseased (PD ≥5 mm with bleeding on probing) and healthy (PD ≤3 mm with no bleeding on probing)-and from healthy sites of HS and HC. Plaque DNA was extracted and analyzed by polymerase chain reaction for the detection of the JP2 and non-JP2 sequences of A. actinomycetemcomitans. Overall, 90 (50%) subjects tested positive for the JP2 sequence. Fifty (83.33%) LAP subjects were carriers of the highly leukotoxic JP2 sequence, detected in 45 (75%) diseased sites and 34 (56.67%) healthy sites. Additionally, JP2 carriage was found in 16 HS (26.67%) and 24 HC (40%; P < 0.0001, among groups). The non-JP2 sequence was detected in 26 (14.44%) total subjects: 17 (28.33%) LAP patients detected in equal amounts of diseased and healthy sites (n = 11, 18.33%), 6 (10%) HS sites, and 3 (5%) HC sites (P < 0.05, among groups). The JP2 sequence was strongly associated with LAP-diseased sites in young African Americans, significantly more so than the non-JP2 (ClinicalTrials.gov NCT01330719). Knowledge Transfer Statement: Clinicians may use the results of this study to identify susceptible individuals to aggressive periodontitis, potentially leading to more appropriate selection of therapeutic choices.
| S-EPMC5576056 | biostudies-literature
Project description:Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its ?2 integrin receptor (lymphocyte function-associated antigen-1 (LFA-1)) results in the clustering of the toxin·receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by 4 orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid consensus (CRAC) sites; CRAC(336) ((333)LEEYSKR(339)) is highly conserved among RTX toxins, whereas CRAC(503) ((501)VDYLK(505)) is unique to LtxA. A peptide corresponding to CRAC(336) inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC(336) and CRAC(503) bind cholesterol, only CRAC(336) competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC(336) site was essential for LtxA cytotoxicity. The conservation of CRAC(336) among RTX toxins suggests that this mechanism may be conserved among RTX toxins.
| S-EPMC3949334 | biostudies-literature
Project description:ObjectiveThe aim of this work was to explore the association between Aggregatibacter actinomycetemcomitans (A actinomycetemcomitans) infection and disease activity amongst those with rheumatoid arthritis (RA) with or without periodontitis (PD) in a Chinese population.MethodsA case-control study was conducted from November 2017 to March 2019. The correlation coefficients between A actinomycetemcomitans positivity and RA-related examination indicators as well as periodontal examination parameters were calculated by using the Spearman correlation analysis.ResultsA total of 115 patients with RA were recruited: 67 patients with RA only and 48 with RA + PD. The percentage of A actinomycetemcomitans positivity was significantly higher in the RA + PD group compared with the RA-only group (P = .007 for positive percentage; P = .020 for percentage of A actinomycetemcomitans positivity in the total oral microbiome). Furthermore, RA-related measures such as Disease Activity Score 28, rheumatoid factor, anticyclic citrullinated peptide, and anticitrullinated protein antibodies were all positively correlated with the percentage of A actinomycetemcomitans positivity (P range: .002∼.041). In addition, significant correlations were observed amongst A actinomycetemcomitans positivity and probing pocket depth (P = .027) and gingival index (P = .043), whereas null correlations were found amongst the percentage of A actinomycetemcomitans positivity and plaque index (P = .344), clinical attachment loss (P = .217), and bleeding on probing (P = .710).ConclusionsA actinomycetemcomitans infection may be related to the development of PD amongst patients with RA.
| S-EPMC10829364 | biostudies-literature
Project description:The oral pathogen, Aggregatibacter actinomycetemcomitans, produces a number of virulence factors, including a leukotoxin (LtxA), which specifically kills human white blood cells, to provide a colonization advantage to the bacterium. Strains of A. actinomycetemcomitans that produce more LtxA have been more closely linked to disease, indicating that this toxin plays a key role in pathogenesis of the bacterium. Disruption of the activity of LtxA thus represents a promising approach to reducing the pathogenicity of the bacterium. Catechins are polyphenolic molecules derived from plants, which have shown potent antibacterial and antitoxin activities. We have previously shown that galloylated catechins are able to prevent LtxA delivery to host cells by altering the toxin's secondary structure and preventing binding to cholesterol on the host cell membrane. Here, we have investigated how one particular galloylated catechin, epigallocatechin gallate (EGCg), affects A. actinomycetemcomitans growth and toxin secretion. Our results demonstrate that EGCg, at micromolar concentrations, inhibits A. actinomycetemcomitans growth, as has been reported for other bacterial species. At subinhibitory concentrations, EGCg promotes LtxA production, but the toxicity of the bacterial supernatant against human immune cells is reduced. The results of our biophysical studies indicate that this seemingly contradictory result is caused by an EGCg-mediated enhancement of LtxA affinity for the bacterial cell surface. Together, these results demonstrate the potential of EGCg in the treatment of virulent A. actinomycetemcomitans infections.
| S-EPMC7015128 | biostudies-literature
Project description:Repeats-in-toxin leukotoxin (LtxA) produced by the oral bacterium Aggregatibacter actinomycetemcomitans kills human leukocytes in a lymphocyte function-associated antigen 1 (LFA-1, integrin αL /β2 )-dependent manner, although the mechanism for this interaction has not been identified. The LtxA internalisation by LFA-1-expressing cells was explored with florescence resonance energy transfer (FRET) microscopy using a cell line that expresses LFA-1 with a cyan fluorescent protein-tagged cytosolic αL domain and a yellow fluorescent protein-tagged β2 domain. Phorbol 12-myristate 13-acetate activation of LFA-1 caused transient cytosolic domain separation. However, addition of LtxA resulted in an increase in FRET, indicating that LtxA brings the cytosolic domains closer together, compared with the inactive state. Unlike activation, this effect was not transient, lasting more than 30 min. Equilibrium constants of LtxA binding to the cytoplasmic domains of both αL and β2 were determined using surface plasmon resonance. LtxA has a strong affinity for the cytosolic domains of both the αL and β2 subunits (Kd = 15 and 4.2 nM, respectively) and a significantly lower affinity for the cytoplasmic domains of other integrin αM , αX , and β3 subunits (Kd = 400, 180, and 230 nM, respectively), used as controls. Peptide fragments of αL and β2 show that LtxA binds membrane-proximal domain of αL and intermediate domain of β2 .
| S-EPMC6657692 | biostudies-literature
Project description:The cyclic-AMP receptor protein (CRP) acts as a global regulatory protein among bacteria. Here, the CRP regulon has been defined in Aggregatibacter actinomycetemcomitans using microarray analysis of A. actinomycetemcomitans strain JP2 wild type cells compared to an isogenic crp deletion mutant. Genes whose expression levels changed at least 2-fold with p≤0.05 were considered significant. Of the 300 genes identified as being CRP-regulated, 139 were CRP-activated, including leukotoxin, with the remaining being CRP-repressed. The 300 genes represent 14.2% of ORFs probed which is significantly higher than what has been reported for CRP regulons in other bacteria. If the CRP-regulated genes are put into 17 functional classes, all 17 categories had at least 1 CRP-regulated gene. Several functional categories, mainly transport and binding proteins and energy metabolism proteins, were disproportionately represented in the CRP-regulated subset of genes relative to their overall representation in the genome. This is similar to the patterns seen in other bacteria. Finally, quantitative RT-PCR was used to show that the leukotoxin RNA levels were repressed 16-fold in the CRP mutant indicating that CRP activates leukotoxin transcription. However, this regulation appears to be acting through another regulatory protein since the leukotoxin promoter, unlike ∼129 other promoters of CRP-regulated genes, does not have a match to the consensus CRP-binding site. Several candidate genes for this intermediary transcription factor have been identified in the CRP regulon.
| S-EPMC3120918 | biostudies-literature
Project description:Background:Aggregatibacter actinomycetemcomitans has been reported in higher proportions in subgingival microbiota of individuals with aggressive periodontitis (AgP) compared with those with chronic periodontitis (ChP) and healthy controls. The major virulence factors are the ones that help in colonization and evasion of host's defenses. Hence, this study was aimed to assess the prevalence of A. actinomycetemcomitans 16S rRNA and its virulent genotypes (leukotoxin [lktA] and fimbria-associated protein [fap]). Materials and Methods:In this case- control study We performed periodontal examination and measured probing depth and clinical attachment level (CAL). Subgingival plaque samples from 200 (ChP: n = 128 and AgP: n = 72) periodontitis patients and 200 healthy controls were screened for the presence of A. actinomycetemcomitans 16S rRNA, lktA, and fap genotypes by polymerase chain reaction. The prevalence of genotypes between periodontitis patients and healthy controls was compared with Pearson's Chi-square test. P < 0.05 was considered statistically significant. Results:Mean pocket probing depth and CAL were high as compared to the healthy controls. The prevalence of A. actinomycetemcomitans in ChP (n = 128), AgP (n = 72), and healthy individuals (n = 200) was 32.0%, 61.1%, and 2.5%, respectively. A. actinomycetemcomitans lktA genotype prevalence was 71.8% among periodontitis patients, while A. actinomycetemcomitans fap genotype showed 31.8% prevalence. The prevalence of these genotypes was insignificant in healthy controls. Conclusion:The high odds ratio for A. actinomycetemcomitans prevalence suggests its strong link to periodontitis. Detection of A. actinomycetemcomitans lktA + genotype may be a useful marker for AgP as its prevalence was found to be high in AgP.
| S-EPMC5958535 | biostudies-literature
Project description:Aggregatibacter actinomycetemcomitans is frequently associated with localized aggressive periodontitis (LAP); however, longitudinal cohort studies relating A. actinomycetemcomitans to initiation of LAP have not been reported. A periodontal assessment was performed on 1,075 primarily African-American and Hispanic schoolchildren, ages 11 to 17 years. Samples were taken from each child for A. actinomycetemcomitans. A cohort of 96 students was established that included a test group of 38 A. actinomycetemcomitans-positive students (36 periodontally healthy and 2 with periodontal pockets) and 58 healthy A. actinomycetemcomitans-negative controls. All clinical and microbiological procedures were repeated at 6-month intervals. Bitewing radiographs were taken annually for definitive diagnosis of LAP. At the initial examination, clinical probing attachment measurements indicated that 1.2% of students had LAP, while 13.7% carried A. actinomycetemcomitans, including 16.7% of African-American and 11% of Hispanic students (P = 0.001, chi-square test). A. actinomycetemcomitans serotypes a, b, and c were equally distributed among African-Americans; Hispanic students harbored predominantly serotype c (P = 0.05, chi-square test). In the longitudinal phase, survival analysis was performed to determine whether A. actinomycetemcomitans-positive as compared to A. actinomycetemcomitans-negative students remained healthy ("survived") or progressed to disease with attachment loss of >2 mm or bone loss (failed to "survive"). Students without A. actinomycetemcomitans at baseline had a significantly greater chance to remain healthy (survive) compared to the A. actinomycetemcomitans-positive test group (P = 0.0001). Eight of 38 A. actinomycetemcomitans-positive and none of 58 A. actinomycetemcomitans-negative students showed bone loss (P = 0.01). A. actinomycetemcomitans serotype did not appear to influence survival. These findings suggest that detection of A. actinomycetemcomitans in periodontally healthy children can serve as a risk marker for initiation of LAP.
| S-EPMC2168549 | biostudies-literature
Project description:BackgroundAggregatibacter actinomycetemcomitans is the etiological agent of periodontitis, and there is a strong association between clone JP2 and aggressive periodontitis in adolescents of African descent. The JP2 clone has an approximately 530-bp deletion (∆530) in the promoter region of the lkt/ltx gene, which encodes leukotoxin, and this clone has high leukotoxic activity. Therefore, this clone is very important in aggressive periodontitis. To diagnose this disease, culture methods and conventional PCR techniques are used. However, quantitative detection based on qPCR for the JP2 clone has not been developed due to genetic difficulties. In this study, we developed a qPCR-based quantification method specific to the JP2 clone.MethodsBased on our analysis of the DNA sequence of the lkt/ltx gene and its flanking region, we designed a reverse primer specific for the ∆530 deletion border sequence and developed a JP2-specific PCR-based quantification method using this primer. We also analyzed the DNA sequence of the ∆530 locus and found it to be highly conserved (97-100%) among 17 non-JP2 strains. Using the ∆530 locus, we designed a qPCR primer-probe set specific to non-JP2 clones. Next, we determined the numbers of JP2 and non-JP2 clone cells in the periodontal pockets of patients with aggressive periodontitis.ResultsThe JP2-specific primers specifically amplified the genomic DNA of the A. actinomycetemcomitans JP2 clone and did not react with other bacterial DNA, whereas the non-JP2 specific primers reacted only with A. actinomycetemcomitans non-JP2 clones. Samples from the 88 periodontal sites in the 11 patients with aggressive periodontitis were analyzed. The bacterial cell numbers in 88 periodontal sites ranged from 0 to 4.8 × 10(8) (mean 1.28 × 10(7)) for JP2 clones and from 0 to 1.6 × 10(6) for non-JP2 clones (mean 1.84 × 10(5)). There were significant differences in the JP2 cell number between a clinical attachment level (CAL) ≤6 mm and a level ≥7 mm (p < 0.01). Our new qPCR-based JP2- and non-JP2-specific quantitative detection assay is applicable to the diagnosis of aggressive periodontitis with A. actinomycetemcomitans.ConclusionsWe successfully developed a quantitative and discriminative PCR-based method for the detection of A. actinomycetemcomitans JP2 and non-JP2 clones. This technique will contribute to future analyses of the quantitative relationship between this organism and aggressive periodontitis.
| S-EPMC3523965 | biostudies-literature