Engineering bioluminescent bacteria for in vivo immune response tracking in D. melanogaster 2333028

Abstract Introduction Immune activation is a tightly controlled process, as overactivation leads to autoimmunity, while underactivation allows bacteria to grow unchecked. Genetically identical D. melanogaster, which rely solely on an innate immune response, infected with the same number of bacteria will die at varying times due to random variation in pathogen growth and immune response. Through indirect observation, previous research suggests that variation in survival arises from variation in the onset of the immune response in the fly. However, how exactly bacterial growth and immune response determine the outcome of an infection has yet to be observed due to the difficulty of obtaining individual and real-time data. Methods Dilution plating can estimate bacterial growth but loses the nuance of individual immunity over time. To continuously track individual flies, we generated bioluminescent bacteria using the iLux plasmid, which has a broad host range and confers auto-bioluminescence. As a model, we used P. rettgeri, a gram-negative bacteria that is moderately lethal to flies, leading to a variety of infection outcomes.We tracked P. rettgeri growth in infected flies in a high-throughput manner as a proxy for fly immune function. Results Flies individually housed in a 96-well plate and injected with bioluminescent P. rettgeri were imaged every 10-30 minutes. Among these flies, three outcomes were observed: clearance of the infection to undetectable levels, stabilization of infection at a chronic load, or death from an uncontrolled infection. Conclusion Using D. melanogaster and bioluminescent P. rettgeri, we can observe host-pathogen interactions in a model system ideal for studying the conserved features of innate immunity in the absence of an adaptive immune response. In future work we will image immune reporter flies injected with bioluminescent bacteria, tracking how the bacterial signal changes in response to directly observable immune activation and generating models for the determinants of infection outcome. Funding Source This research was supported by a Kilachand Fellowship funded by the Boston University Multicellular Design Program Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)