Evolutionary lineages of Actinobacillus actinomycetemcomitans bear diverse traits to support roles as a member of normal flora as a pathogen

8 REVIEW OF THE LITERATURE 10 1. Periodontitis 10 1.1 General characteristics and classification 10 1.2 Oral biofilms 10 1.3 Actinobacillus actinomycetemcomitans in periodontitis 12 2. Evolutionary lineages of A. actinomycetemcomitans 14 2.1 Population structure 14 2.2 Natural transformation 16 2.3 Intraspecies diversity 17 2.4 Serotypes and serotype-specific antigens 19 2.5 Serotypes vs. genotypes 22 3. Actinobacillus actinomycetemcomitans and systemic host responses 23 3.1 Periodontitis and cardiovascular diseases 24 3.2 Actinobacillus actinomycetemcomitans lipopolysaccharide as a stimulant of host responses related to cardiovascular diseases 27 AIMS OF THE STUDY 30 MATERIALS AND METHODS 31 1. Subjects and bacterial strains 31 2. Eukaryotic cells and cell cultures 32 3. Methods 32 3.1 Serotyping 32 3.2 Genotyping 33 3.3 Isolation of lipopolysaccharide 35 3.4 Natural transformation 35 3.5 Isolation of serotype-specific antigen 36 3.6 Characterization of serotype-specific antigen 36 3.7 Isolation and labeling of low density lipoprotein 37 3.8 Low density lipoprotein uptake by macrophages 37 3.9 Cytokine expression 37 3.10 RNA isolation 37 3.11 Real-time PCR 38 3.12 Statistics 38 RESULTS AND DISCUSSION 39 1. Actinobacillus actinomycetemcomitans serotypes in relation to their proportion in subgingival flora 39 2. Natural transformation among Actinobacillus actinomycetemcomitans serotypes 40 3. Serotype-specific antigen of serotype d 42 3.1 Western blot of Actinobacillus actinomycetemcomitans lipopolysaccharide 42 3.2 Characterization of the affinity-purified antigen 42 4. Effects of Actinobacillus actinomycetemcomitans lipopolysaccharide on murine macrophages 44 4.1 Foam cell formation 44 4.2 Expression of SR-BI and ABCA-1 mRNA 45