IADR Abstract Archives
Type VI-Secretion-Dependent Pathobiont Killing by Oral Commensal Aggregatibacter Aphrophilus
Objectives: The Type VI secretion system (T6SS) is a bacterial mechanism that delivers effector proteins that can eliminate competitors and modulate host responses. We recently discovered that the oral symbiont Aggregatibacter aphrophilus uses its T6SS to kill the pathobiont Aggregatibacter actinomycetemcomitans. This study aimed to identify specific effectors and characterize host responses to the A. aphrophilus T6SS.
Methods: Two potential T6SS effectors, a glycosyl hydrolase (Glh), and a Tle5 phospholipase, were identified through in silico analysis. Mutant strains of A. aphrophilus HK83 were generated with allelic replacements of glh and tle5, and a double mutant was constructed in addition. These strains were introduced into a six-species biofilm and co-cultured with an immortal oral epithelial cell line. Species-specific counts of the biofilm were determined by colony-forming units, and proteomic analyses of epithelial cells were conducted using liquid chromatography-tandem mass spectrometry. Proteins were quantified using Progenesis QI, and those with a fold change >2 and a p-value <0.05 considered significantly regulated.
Results: A. aphrophilus significantly reduced A. actinomycetemcomitans levels in the multi-species biofilms, with this effect depending on the presence of both effectors, Glh and Tle5, indicating a potential effector synergism. In co-cultures with epithelial cells, we identified 3,189 proteins in total, with regulation trends showing that both A. aphrophilus and A. actinomycetemcomitans induced an inflammatory response, characterized by elevated pro-inflammatory proteins including S100A8, IL1A, and ICAM1. Interestingly, the co-presence of both bacterial species appeared to reduce inflammation, which could be due to the A. aphrophilus T6SS-dependent killing of A. actinomycetemcomitans. This anti-inflammatory effect was lost in strains lacking the T6SS core protein Hcp or both effectors, with mutant strains showing no significant effect on inflammation compared to the wild-type strain.
Conclusions: Our findings highlight the pivotal role of the T6SS of A. aphrophilus and its effectors in targeted bacterial killing and modulation of host inflammation.
Methods: Two potential T6SS effectors, a glycosyl hydrolase (Glh), and a Tle5 phospholipase, were identified through in silico analysis. Mutant strains of A. aphrophilus HK83 were generated with allelic replacements of glh and tle5, and a double mutant was constructed in addition. These strains were introduced into a six-species biofilm and co-cultured with an immortal oral epithelial cell line. Species-specific counts of the biofilm were determined by colony-forming units, and proteomic analyses of epithelial cells were conducted using liquid chromatography-tandem mass spectrometry. Proteins were quantified using Progenesis QI, and those with a fold change >2 and a p-value <0.05 considered significantly regulated.
Results: A. aphrophilus significantly reduced A. actinomycetemcomitans levels in the multi-species biofilms, with this effect depending on the presence of both effectors, Glh and Tle5, indicating a potential effector synergism. In co-cultures with epithelial cells, we identified 3,189 proteins in total, with regulation trends showing that both A. aphrophilus and A. actinomycetemcomitans induced an inflammatory response, characterized by elevated pro-inflammatory proteins including S100A8, IL1A, and ICAM1. Interestingly, the co-presence of both bacterial species appeared to reduce inflammation, which could be due to the A. aphrophilus T6SS-dependent killing of A. actinomycetemcomitans. This anti-inflammatory effect was lost in strains lacking the T6SS core protein Hcp or both effectors, with mutant strains showing no significant effect on inflammation compared to the wild-type strain.
Conclusions: Our findings highlight the pivotal role of the T6SS of A. aphrophilus and its effectors in targeted bacterial killing and modulation of host inflammation.