IADR Abstract Archives
Phage Induction by Human Serum in Aggregatibacter Actinomycetemcomitans
Objectives: A. actinomycetemcomitans causes periodontitis and infective endocarditis and has evolved mechanisms to survive in the blood environment during the disease process. Our work indicates that selective clinical strains demonstrate increased sensitivity to killing by human serum. This study was developed to explore the hypothesis that the bacteriophage contributes to the sensitivity of this microorganism to serum.
Methods: One serum-sensitive and two serum-resistant strains were identified for this investigation. Each fimbriated strain was inoculated from agar to liquid culture medium using polystyrene culture tubes without serum and grown overnight. The adherent cells were subsequently exposed to fresh media with or without the addition of 50% human serum. The bacteria were collected using cell scrapers at selected times over a 24-hour period. The RNA was isolated for RNAseq and proteins were analyzed using blue native gel electrophoresis followed by mass spectrometry. The corresponding culture supernatants were also collected for analysis.
Results: A 44-kbp Aggregatibacter phage S1249 was identified in the serum-sensitive strain and 20% of the phage genes were up-regulated over 10-fold in the presence of serum. PCR analysis of the supernatants utilizing two primer sets targeting the phage tmp (tail length tape measure protein) and bpp (baseplate protein) genes indicated that the release of viral particles peaked after six-hour exposure to serum. Consistently, total bacterial DNA synthesis peaked at approximately the same time and produced two-fold more DNA in serum versus without. Blue native gel electrophoresis followed by mass spectrometric analysis identified that a 1.5-megadalton protein complex, corresponding to the pyruvate dehydrogenase (PDH) complex, was only up-regulated in the serum-sensitive strain six hours post serum exposure.
Conclusions: Our data suggest that human serum induces the replication and release of phage S1249 resulting in cell lysis. We propose that the up-regulated PDH complex leads to an increase of ATP production to support phage replication.
Methods: One serum-sensitive and two serum-resistant strains were identified for this investigation. Each fimbriated strain was inoculated from agar to liquid culture medium using polystyrene culture tubes without serum and grown overnight. The adherent cells were subsequently exposed to fresh media with or without the addition of 50% human serum. The bacteria were collected using cell scrapers at selected times over a 24-hour period. The RNA was isolated for RNAseq and proteins were analyzed using blue native gel electrophoresis followed by mass spectrometry. The corresponding culture supernatants were also collected for analysis.
Results: A 44-kbp Aggregatibacter phage S1249 was identified in the serum-sensitive strain and 20% of the phage genes were up-regulated over 10-fold in the presence of serum. PCR analysis of the supernatants utilizing two primer sets targeting the phage tmp (tail length tape measure protein) and bpp (baseplate protein) genes indicated that the release of viral particles peaked after six-hour exposure to serum. Consistently, total bacterial DNA synthesis peaked at approximately the same time and produced two-fold more DNA in serum versus without. Blue native gel electrophoresis followed by mass spectrometric analysis identified that a 1.5-megadalton protein complex, corresponding to the pyruvate dehydrogenase (PDH) complex, was only up-regulated in the serum-sensitive strain six hours post serum exposure.
Conclusions: Our data suggest that human serum induces the replication and release of phage S1249 resulting in cell lysis. We propose that the up-regulated PDH complex leads to an increase of ATP production to support phage replication.