Mechanisms of LtxA intracellular trafficking and cell injury in human macrophages

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A Gram negative bacterium Aggregatibacter actinomycetemcomitans is a common inhabitant of the human upper aerodigestive and a causative agent of localized aggressive periodontitis (LAP). The bacterium produces an RTX toxin (LtxA). LtxA can be delivered to host cells in soluble and in outer membrane vesicles (OMVs)- associated forms. Soluble LtxA requires the presence of the β2 integrin LFA-1 and cholesterol-rich membrane rafts to exert its lethal effect on human immune cells. Recent data indicate that soluble LtxA damages lysosomes, causes release of proinflammatory cytokines and induces cell death in human macrophages. How soluble LtxA is delivered to the macrophages’ lysosomes and induces inflammatory response, is not known. The mechanism of the cell injury by OMV-delivered LtxA is completely unexplored. The objective of this study is to determine the mechanism of LtxA trafficking and intoxication in human macrophages. Our central hypothesis is that the destructive effect of LtxA can be halted by preventing LtxA trafficking. Therefore, we propose a set of experiments to understand intracellular localization of soluble versus OMV-associated LtxA in human macrophages. Our hypotheses will be tested in two specific aims: 1) To determine trafficking of and cell injury by soluble LtxA; 2) To determine trafficking of OMV-associated LtxA and the mechanism of intoxication. In our experimental plan, we will utilize confocal imaging coupled with immunological and biochemical approaches to investigate the mode of LtxA endocytic trafficking in macrophages and to determine downstream effects of LtxA on inflammasome activation. The toxin-induced macrophage damage will be investigated ex vivo using bacteria/macrophages complex and in vitro employing purified toxins, OMVs, and purified organelles. The proposed research is relevant to public health and the rationale underlying this proposal is to identify key targets for preventing LtxA trafficking which induces cell death in macrophages and periodontal inflammation. The project completion is expected to give rise to new types of treatments and diagnostics for periodontitis. Importantly, the results of this study will provide a basis that can be extended to understand pathogenic mechanisms of other RTX-toxins and open new avenues for diseases therapies.