Integration of nutrient availability, TCA cycle activity, and colonization factor expression in Campylobacter jejuni - Project Summary Campylobacter jejuni is the leading cause of bacterial-derived gastroenteritis in the world. Human infection often occurs through the ingestion of contaminated food, especially poultry, which leads to the bacterium colonizing the colon and causing severe inflammation and diarrhea. While infection and disease is often self-limiting, persistent colonization and chronic diseases do occur. Despite the significant impacts of C. jejuni on human health, very little is known about the interactions that occur at the host-pathogen interface during infection, including how C. jejuni senses and adapts to the host intestinal environment to promote infection. This is primarily due to the evolutionary divergence of this organism from other gastrointestinal pathogens, which limits the relevance of findings from those organisms and necessitates specific study of the Campylobacter genus. To that end, our group previously identified a unique regulator in C. jejuni, which we call HeuR, that promotes maximum colonization of a natural avian host and was subsequently found to positively or negatively regulate several genetic determinants, including those involved in the acquisition of iron from host heme and the biosynthesis of methionine. In addition, HeuR and its downstream targets are required for efficient invasion or persistence in human colonocytes, which suggests these mechanisms need to be better understood as they are clearly involved in infection of animals. We preliminarily determined that this novel regulator binds several TCA intermediates and may sense TCA cycle activity to control expression of colonization determinants. First, we will define all direct targets of HeuR and examine whether TCA intermediates impact the ability of HeuR to bind those DNA targets and impact gene expression. Additionally, we will identify the ligand binding motif of this novel regulator and how it facilitates HeuR activity. Second, because we have determined that C. jejuni TCA intermediate abundance is affected by iron-restriction, we will use mass isotopomer analysis to identify the points along the C. jejuni TCA cycle that are affected by iron-limitation and determine whether altering TCA cycle activity affects HeuR-dependent colonization determinant expression. In addition, we will directly determine the levels at which each TCA enzyme indicated by mass isotopomer analysis is affected by iron-limitation. Lastly, one of the direct targets of HeuR we identified that may be impacted by TCA intermediate-dependent binding is the heme utilization system of C. jejuni. This system is poorly characterized in C. jejuni and we will examine whether this system facilitates iron acquisition during animal infection and will work to fully characterize the process of heme utilization so that it can be leveraged in future studies to inhibit the infection potential of C. jejuni.
