Development of qPCR Based Method to Quantify Colonization of an Engineered Bacterium in Gut

Parkinson’s Disease is the leading neurodegenerative disease in the western world, caused by degeneration of neurons in the substantia nigra. This causes a decrease of dopamine in nigrostriatal neural circuitry, leading to motor and nonmotor symptoms which can be incredibly debilitating for the patient. Current dopamine supplementation therapy does not provide a steady state neurotransmitter level and can lead to dyskinesias and dystonia’s. However, Escherichia coli Nissle (EcN) bacteria have been shown to prove effective therapeutic treatments that can be engineered to specifically treat certain diseases, such as Phenylketonuria. EcN can be engineered to upregulate production of L-DOPA by adding an extra HpaBC gene. Administration of L-DOPA through ECN can maintain steady state dopamine level and thereby normalizing dopaminergic neural pathways for patients suffering from Parkinson’s Disease. The goal of this study was to monitor EcN-DOPA gut colonization in mouse models of Parkinson’s disease using a qPCR method. We generated a unique primer set for ECN-L-DOPA and determined its utility in measuring the colonization. Our results showed that ECN-L-DOPA primer effectively amplifies the gene and detected the engineered bacteria in the fecal sample. Further evaluation in Mitopark transgenic mouse model of Parkinson’s disease, qPCR method successfully detected colonization profile of ECN-DOPA and correlated with the plasma level with plasma L-DOPA level. Collectively, our results show qPCR method is very amenable for monitoring gut colonization in microbiome-based therapy