Characterization of the Secretome of Aggregatibacter actinomycetemcomitans

The interaction between bacteria and the environment plays a significant role in growth and colonization.  The export of proteins across the bacterial membrane is a factor that modulates this interaction. In our laboratory, inactivation of the gene encoding morphogenesis protein C (MorC) of the oral and opportunistic pathogen Aggregatibacter actinomycetemcomitans impacts the secretion of leukotoxin, an immunomodulatory protein.  The effect of this mutation on other secreted proteins has not been determined. Objective:   To identify the secreted proteins of A. actinomycetemcomitans and to determine if the secretion of other proteins is affected by disruption of morC. Methods: A chemically defined medium supplemented with 0.3% Trypticase Soy Broth was employed for growth of bacterial strains. Cells were removed from the medium by centrifugation following 24 hrs of growth.  The spent media was filtered to remove any intact cells and concentrated by precipitation with 70% ammonium sulfate.  The precipitated proteins were resuspended, dialyzed, and separated using two-dimensional gel electrophoresis.  The individual protein spots, detected by silver staining, were excised and analyzed using liquid chromatography coupled with mass spectrometry (LCMS). Results: Greater than 80 individual proteins were identified in the medium of the wild type strain.  A subset of these proteins has been analyzed by LCMS.  In addition, qualitative differences have been observed in the medium proteins of the morC mutant as compared with the wild type secretome.  Conclusions: The secretion defect associated with the morC mutant strain is not restricted to leukotoxin.  The inactivation of this gene may result in a general protein secretion defect.