The bacterium Aggregatibacter actinomycetemcomitans (Aa) grows in the human oral cavity and is a causitive agent of periodontitis. In the oral cavity, Aa must grow in close proximity to oral microbial flora, the most numerous of which are often oral streptococci, such as Streptococcus gordonii (Sg), that are able to ferment sugars into organic acids and release hydrogen peroxide (H2O2). Our preliminary results show that Aa responds to H2O2 from Sg by upregulating a gene (aplA) involved in defense against innate immunity. Thus, Aa appears to be using H2O2 as an anticipatory signal to increase resistance to host innate immunity. The goal of our proposal is to understand how co-culture with oral streptococci impacts Aa survival in the presence of host innate immune factors, and we propose two aims to accomplish this goal.
My first aim tests if co-culture with Sg diminishes opsonization and phagocytosis of Aa. I hypothesize that co-culture will reduce opsonization and phagocytosis and plan to test this by quantifying opsonizing proteins Aa cell surface by immunofluoresence as well as measuring levels of uptake of Aa and Sg by phagocytic cells.
Our second aim tests if co-culture biofilm increases protection from innate immunity, whether by biofilm architecture or spatial proximity of cells to induce genes involved in immune defense. We hypothesize that co-culture biofilms will be more resistant to innate immune factors. To test this hypothesis we will measure Aa and Sg cell survival in co-culture vs pure culture biofilms by plate count assay and microscopy after exposure to cxjmplement active serum and phagocytic cells. We will also utilize fluorescent reporter fusions to the Aa aplA promoter to determine if its expression is dependent on proximity to Sg in the co-culture biofilm and if this impacts survival of either species while in close proximity. Understanding these events provides us with a better knowledge of interactions Aa must have with its neighbors that allow it to resist the human immune system. Public Health Relevance: The mission of the National Institute of Dental and Craniofacial Research (NIDCR) is to improve oral, dental and craniofacial health through research. We fulfill that mission by determining key relationships that an oral pathogen must have in its natural environment and how they impact survival from host immunity. Bacterial infections are often studied in pure culture despite the fact that multi-species infections occur often in the human body. We wish to further characterize the unique observation that different bacterial species impact another's resistance to the human immune system and my lead to new strategies for antimicrobial therapy.
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