Characterization of Selenomonas sputigena invasion, intracellular life cycle, and exocytosis in gingival epithelial cells. - PROJECT SUMMARY Periodontal disease (PD) is an inflammatory condition in which immune dysregulation causes inflammation and eventually results in tissue destruction. The onset of PD is caused by the outgrowth of pathobiont organisms that activate chronic immune stimulation. A numerous and complex variety of bacteria participate in the etiology of PD. One such member of the pathobiont community in disease is Selenomonas sputigena, the subject of this proposal. Numerous studies have reported that S. sputigena is associated with disease. Despite this, no other published research has directly investigated how the bacterium causes PD. Recently, our group has published the first ever characterization of how S. sputigena promotes inflammation in gingival keratinocytes. This study reports that S. sputigena activates the secretion of a broad range of important pro-inflammatory mediators. Importantly, this prior study also displays that S. sputigena stimulates immune cell recruitment; of which is an essential hallmark of PD etiology. In preliminary data reported here, we show that S. sputigena invades gingival epithelial cells (GECs). Here, we propose to elucidate i) the mechanism by which S. sputigena is endocytosed by GECs, ii) the biochemical and endosomal characteristics of the S. sputigena-enclosing vesicle, and iii) the intracellular lifecycle of the organism, including the time frame of bacterial viability and subsequent exocytosis. We hypothesize that S. sputigena is endocytosed by GECs via a clathrin-dependent mechanism and is ultimately trafficked to the nuclear membrane in a late endosome-resembling multivesicular body. Afterwards, we hypothesize that the organism survives within the endosome via inhibition of lysosomal fusion and exits the cell via ESCRT-dependent exocytosis. We propose two integrated Aims to investigate this hypothesis. In Aim 1, to investigate initial invasion, we will directly modulate clathrin-dependent endocytosis via pharmacological inhibitors. Also, in Aim 1 we will utilize immunofluorescence microscopy to observe S. sputigena vesicular trafficking during invasion. Last, we will perform transmission electron microscopy (TEM) in order to identify defining features of the S. sputigena-enclosing vesicle. In Aim 2 we will identify how long S. sputigena remains alive inside the cell. Also, we will investigate exocytosis of the S. sputigena-enclosing vesicle via immunofluorescence microscopy and, in so doing, identify a mechanism by which the bacterium may escape to re-infect naïve cells. Finally, we will infect a three-dimensional epithelial tissue model with S. sputigena to investigate tissue invasion via cell to cell spread. The data resulting from these Aims will elucidate the intracellular life cycle of S. sputigena in GECs. Thorough characterization of this process will establish the foundation through which to identify specific interactions between the bacterium and intracellular constituents. In this manner, the precise mechanism by which S. sputigena stimulates inflammation in GECs can be investigated and novel therapeutic targets can be considered. Understanding how S. sputigena causes PD will further the collective knowledge of how periopathogens interact with the host and will progress the current understanding of how the members of the complex plaque biofilm cause disease. Last, researching host-pathogen interactions in S. sputigena will allow for experiments that investigate bacterial community interactions between S. sputigena and more robustly characterized periopathogens like Porphyromonas gingivalis.
