RT Journal Article SR Electronic T1 Cellular barcoding of protozoan pathogens reveals the within-host population dynamics of Toxoplasma gondii host colonization JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.08.06.239822 DO 10.1101/2020.08.06.239822 A1 Ceire J. Wincott A1 Gayathri Sritharan A1 Henry J. Benns A1 Farzana B. Liakath A1 Carla Gilabert-Carbajo A1 Monique Bunyan A1 Aisling R. Fairweather A1 Eduardo Alves A1 Ivan Andrew A1 Laurence Game A1 Eva M. Frickel A1 Calvin Tiengwe A1 Sarah E. Ewald A1 Matthew A. Child YR 2022 UL http://biorxiv.org/content/early/2022/02/21/2020.08.06.239822.abstract AB Molecular barcoding techniques have emerged as powerful tools to understand microbial pathogenesis. However, barcoding strategies have not been extended to protozoan parasites, which have unique genomic structures and virulence strategies compared to viral and bacterial pathogens. Here, we present a versatile CRISPR-based method to barcode protozoa, which we successfully apply to Toxoplasma gondii and Trypanosoma brucei. The murine brain is an important transmission niche for T. gondii, and brain persistence is a clinically untreatable feature of infection. The blood-brain barrier is expected to physically restrict parasite colonization of this niche, resulting in a selection bottleneck. Using libraries of barcoded T. gondii we evaluate shifts in the population structure from acute to chronic infection of mice. Contrary to expectation, most barcodes were present in the brain one-month post-intraperitoneal infection in both inbred CBA/J and outbred Swiss mice. Although parasite cyst number and barcode diversity declined over time, barcodes that represented a minor fraction of the inoculum could become a dominant population in the brain by three months post-infection. Together, these data establish the first, robust molecular barcoding approach for protozoa and evidence that the blood-brain barrier does not represent a major bottleneck to colonization by T. gondii.Competing Interest StatementThe authors have declared no competing interest.