Metabolic adaptation during infection in the intestinal parasite \u3ci\u3eEntamoeba histolytica\u3c/i\u3e: roles of PFK enzymes

Entamoeba histolytica is a water- and food-borne intestinal parasite that causes diarrheal disease in an estimated 90 million people each year worldwide. This pathogen encounters many different environments during infection and must adjust its metabolic activities specifically to that environment. Entamoeba lacks many common metabolic pathways and must ingest other cells to obtain many of the cellular building blocks such as amino acids that it cannot synthesize itself. E. histolytica relies on breakdown of the sugar glucose as the main means for obtaining energy. This is done through glycolysis, a metabolic pathway that is present in essentially all organisms. A key enzyme in glycolysis is phosphofructokinase (PFK). E. histolytica has four PFKs: PFK4 is thought to be the primary enzyme in glycolysis but the roles of PFK1, PFK2, and PFK3 are as yet unknown. PFK4 utilizes pyrophosphate, which increases the energy output of glucose breakdown by glycolysis; however, PFKs1-3 all use ATP, the main energy currency in the cell. In order to understand the role of the ATP-PFKs, we are silencing the genes encoding them and determining the effect on cell growth on glucose and other substrates. We are also characterizing the activity and regulation of purified PFK1, PFK2, and PFK3. Currently, the model systems for infection of E.histolytica are not quite advanced and an animal model that mimics human infection is lacking. Thus, no one has been achieved a full understanding of metabolism during the different stages of infection. Our studies will contribute to a better understanding of energy metabolism in E. histolytica and will fill a gap in our knowledge of how this parasite survives and thrives in the different environments encountered during infection in humans leading to symptomatic disease

Heat Stress Response and Excystation in Entamoeba histolytica

Entamoeba histolytica is a water- and food-borne intestinal protozoan parasite that causes amoebiasis and liver abscess and is responsible for symptomatic disease in approximately 100 million people each year leading to ~ 100,000 deaths. The most common disease transmission follows the oral-fecal route, but it can also be transmitted by mechanical vectors such as animals carrying the amoeba from contaminated sources to water systems. In rare cases, disease transmission has been recorded in some patients in which men-to-men sexual practices were preferred. The life cycle of E. histolytica starts through ingestion of infectious cysts, which are non-dividing, quadri-nucleated structures surrounded by a chitinous cell wall. The chitin cell wall protects the cyst from damage, including the harsh acidic environment of the stomach. Once the cyst enters the nutrient-rich duodenum of the small intestine, excystation takes place to produce proliferative trophozoites. Trophozoites, the motile disease-causing form of E. histolytica, move along the digestive tract and then colonize the large intestine. Trophozoites must encyst back to cysts to survive outside of the host body, so their life cycle requires both successful encystation and excystation. Over the decades, Entamoeba invadens, a related species that is a pathogen of reptiles, has been used as a model to study encystation and excystation because the E. histolytica life cycle could not be replicated in the laboratory. Even though the disease ranked as the third most common cause of death from parasitic infections with a significant global burden, it is consequently less well studied. These two species do not share a high genome sequence identity, and humans and reptiles provide very different host environments, limiting the applicability of E. invadens findings to E. histolytica. Our research group (Wesel et al., (2021)) recently established a means for in vitro encystation of E. histolytica in axenic culture, allowing us now to study encystation and excystation directly in the human parasite. E. histolytica encystation is induced by nutrient starvation with high cell density, and the resulting cysts displayed the four defining cyst characteristics: detergent resistance, a chitinous cell wall, small round cell morphology, and tetranucleation. In this dissertation, I further examined the processes of encystation and excystation in E. histolytica. The heat stress response and encystation overlap in Entamoeba, and here I have investigated the effect of heat stress on encystation to better understand the intersection of the general stress response and encystation. I also examined the signals that induce excystation of non-motile cysts back to motile and actively proliferating trophozoites. These studies will lead to a better understanding of the life cycle of E. histolytica, its infection mechanisms, and potential drug targets

Reproducibility of fluorescent expression from engineered biological constructs in E. coli

We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices.Peer reviewe