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Comparative genomic hybridization and transcriptome analysis with a pan-genome microarray reveal distinctions between JP2 and non-JP2 genotypes of Aggregatibacter actinomycetemcomitans.

ABSTRACT: It was postulated that the highly virulent JP2 genotype of Aggregatibacter actinomycetemcomitans may possess a constellation of distinct virulence determinants not found in non-JP2 genotypes. This study compared the genome content and the transcriptome of the serotype b JP2 genotype and the closely related serotype b non-JP2 genotype of A. actinomycetemcomitans. A custom-designed pan-genomic microarray of A. actinomycetemcomitans was constructed and validated against a panel of 11 sequenced reference strains. The microarray was subsequently used for comparative genomic hybridization of serotype b strains of JP2 (six strains) and non-JP2 (six strains) genotypes, and for transcriptome analysis of strains of JP2 (three strains) and non-JP2 (two strains). Two JP2-specific and two non-JP2-specific genomic islands were identified. In one instance, distinct genomic islands were found to be inserted into the same locus among strains of different genotypes. Transcriptome analysis identified five operons, including the leukotoxin operon, to have at least two genes with an expression ratio of 2 or greater between genotypes. Two of the differentially expressed operons were members of the membrane-bound nitrate reductase system (nap operon) and the Tol-Pal system of gram-negative bacterial species. This study is the first to demonstrate the differences in the full genome content and gene expression between A. actinomycetemcomitans strains of JP2 and non-JP2 genotypes. The information is essential for designing hypothesis-driven experiments to examine the pathogenic mechanisms of A. actinomycetemcomitans.

SUBMITTER: Huang Y

PROVIDER: S-EPMC3543495 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Comparative genomic hybridization and transcriptome analysis with a pan-genome microarray reveal distinctions between JP2 and non-JP2 genotypes of Aggregatibacter actinomycetemcomitans.

Huang Y Y Kittichotirat W W Mayer M P A MP Hall R R Bumgarner R R Chen C C

Molecular oral microbiology 20121012 1

It was postulated that the highly virulent JP2 genotype of Aggregatibacter actinomycetemcomitans may possess a constellation of distinct virulence determinants not found in non-JP2 genotypes. This study compared the genome content and the transcriptome of the serotype b JP2 genotype and the closely related serotype b non-JP2 genotype of A. actinomycetemcomitans. A custom-designed pan-genomic microarray of A. actinomycetemcomitans was constructed and validated against a panel of 11 sequenced refe ...[more]

PMID: 23194436

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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.

Project description:IntroductionMolar-incisor pattern periodontitis (MIPP) in the absence of significant local risk factors or systemic disease, is a rare, early onset periodontal disease phenotype, with 0.5% to 2.5% global prevalence. The condition is characterized by impaired neutrophil function and persistent Aggregatibacter actinomycetemcomitans (JP2 clone) infection. The aim of this study was to characterize neutrophil functional responses to JP2 and to investigate the neutrophil receptors involved.Materials and methodsNeutrophils were obtained from whole blood samples of periodontally healthy and MIPP subjects and incubated with the JP2 clone or a non-JP2 clone of A. actinomycetemcomitans. Bacterial survival was tested by blood agar culture; neutrophil death was tested with propidium iodide and flow cytometry; Reactive oxygen production (ROS) was measured with 2',7'-dichlorofluorescein diacetate and a fluorescence plate reader; the cytokinome was analysed using an array profiler, ELISA and RT-PCR. Receptors binding to JP2 were isolated using a novel immunoprecipitation assay and validated functionally using specific blocking antibodies.ResultsJP2 and non-JP2 survival was comparable between all the neutrophil groups. Resistance to neutrophil necrosis following exposure to JP2 was significantly lower in the MIPP group, than in all the other groups (p<0.0001). Conversely, MIPP neutrophils showed lower levels of ROS production in response to JP2 infection compared with that of healthy neutrophils (p<0.001). Furthermore, significantly lower levels of cytokines, such as IL8, IL10 and TNFα, were observed during JP2 incubation with MIPP neutrophils than upon incubation with periodontally healthy neutrophils. Various proteins expressed on neutrophils bind to JP2. Of these, CD18 was found to mediate neutrophil necrosis. The CD18 receptor on MIPP neutrophils acts differently from that on periodontally healthy patients neutrophils, and appears to reflect differential neutrophil reactions to JP2.ConclusionThis study portrays a fundamental difference in neutrophil response to JP2 infection between periodontally healthy and MIPP patients. This was evident in the resistance to necrosis, and lower ROS and cytokine production, despite the persistent presence of viable JP2. Whilst in periodontally healthy neutrophils, JP2 binds to CD18 on cell surfaces, this is not the case in MIPP neutrophils, suggesting a potential role for CD18 in the periodontal susceptibility of MIPP patients.

Dataset Information

Comparative genomic hybridization and transcriptome analysis with a pan-genome microarray reveal distinctions between JP2 and non-JP2 genotypes of Aggregatibacter actinomycetemcomitans.

Publications

Comparative genomic hybridization and transcriptome analysis with a pan-genome microarray reveal distinctions between JP2 and non-JP2 genotypes of Aggregatibacter actinomycetemcomitans.

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