IADR Abstract Archives
Human-Serum Specific Activation of Alternative Sigma Factors in Aggregatibacter actinomycetemcomitans
Objectives: Periodontal disease is an inflammatory response of the periodontium to bacterial plaque. During disease progression, gingival crevices transform into periodontal pockets with inflammatory exudates from serum. A. actinomycetemcomitans (A.a.) is a known pathogen of periodontitis and endocarditis; elucidating the adaptation mechanisms of A.a. to changing in vivo environments may be critical to understand the pathogenesis of A.a.-associated infections. The objectives of this study are to examine the growth phenotypes and the adaptation mechanisms of A.a. to human serum.
Methods: Twenty-six strains of six serotypes (a-f) were chosen. Heat-inactivated, male, type AB plasma plus 50% trypticase soy broth and yeast extract (TSBYE) was used as the basic growth medium. TSBYE with/without serums from horse, bovine, porcine and sheep were used as controls. Three strains representing distinct growth phenotypes (high-, medium- and low-responders) in human serum were selected for whole transcriptomic analysis. Promoters of operons that specifically responded to human serum were analyzed using a lacZ reporter construct. SDS-PAGE and Mass-spectrometry were employed to study protein profiles of bacteria and bacterium-bound human serum proteins.
Results: Nine high-responder strains (serotypes c and f) responded to human serum by showing a fast climbing of the optical density after 10-13 hours of culture, and reached a final optical density 4-5 times higher than TSBYE with/without serums from other animal source. The top 20 up-regulated genes in the high-responder strain included three operons (rpoE, rpoH and degQ) specializing in stress responses. The promoter of alternative sigma factor rpoE in the reporter construct showed positive response to human serum, accompanying by a growth inhibition of the Escherichia coli host. SDS-PAGE revealed several human-serum proteins prominently bound to the high-responder strain, including apolipoprotein A1 (ApoA1) as determined by Mass-spectrometry.
Conclusions: Varied responses of A.a. strains to human serum suggest distinct mechanisms of in vivo adaptation of this microorganism in humans.
Methods: Twenty-six strains of six serotypes (a-f) were chosen. Heat-inactivated, male, type AB plasma plus 50% trypticase soy broth and yeast extract (TSBYE) was used as the basic growth medium. TSBYE with/without serums from horse, bovine, porcine and sheep were used as controls. Three strains representing distinct growth phenotypes (high-, medium- and low-responders) in human serum were selected for whole transcriptomic analysis. Promoters of operons that specifically responded to human serum were analyzed using a lacZ reporter construct. SDS-PAGE and Mass-spectrometry were employed to study protein profiles of bacteria and bacterium-bound human serum proteins.
Results: Nine high-responder strains (serotypes c and f) responded to human serum by showing a fast climbing of the optical density after 10-13 hours of culture, and reached a final optical density 4-5 times higher than TSBYE with/without serums from other animal source. The top 20 up-regulated genes in the high-responder strain included three operons (rpoE, rpoH and degQ) specializing in stress responses. The promoter of alternative sigma factor rpoE in the reporter construct showed positive response to human serum, accompanying by a growth inhibition of the Escherichia coli host. SDS-PAGE revealed several human-serum proteins prominently bound to the high-responder strain, including apolipoprotein A1 (ApoA1) as determined by Mass-spectrometry.
Conclusions: Varied responses of A.a. strains to human serum suggest distinct mechanisms of in vivo adaptation of this microorganism in humans.