Cellular barcoding of protozoan pathogens for within-host population dynamics and in vivo drug discovery

Abstract

The obligate intracellular apicomplexan parasite Toxoplasma gondii has broad infectious ability causing disease in humans and animals, some of which can be fatal. Existing treatments for T. gondii infections have notable side effects, and the emergence of resistance to first-line therapies is a growing concern. Understanding the fundamental aspects of T. gondii biology necessitates studying in vivo host-pathogen interactions. However, tracking parasite populations without artificially influencing infection dynamics has posed significant challenges. To address this, we propose a cellular barcoding technique combined with Next Generation Sequencing (NGS) technology to genetically identify and assess the representation of parasite populations. This approach can be applied not only to T. gondii but also to T. brucei and holds potential for future application to other pathogens. Using our cellular barcoding methodology, we conducted population dynamics studies to investigate T. gondii colonisation of the brain parenchyma. Surprisingly, we discovered that the blood-brain barrier (BBB) allows relatively unrestricted traversal by T. gondii, imposing a less stringent bottleneck than anticipated. Moreover, we observed the dynamic nature of chronic T. gondii infection, as brain cyst numbers continued to decrease over several months. Furthermore, we employed the cellular barcoding methodology to facilitate multiplexed in vivo drug screening. Through this approach, we successfully identified small molecule fragments with anti-parasitic effects. Our proof-of-concept data supports the use of this screening platform for iterative drug molecule development. Additionally, in concurrent studies, one of the identified hit fragments exhibited selective inhibition of translation in T. gondii compared to HEK293 cells, prompting further characterisation efforts.