Cellular barcoding of protozoan pathogens reveals the within-host population dynamics of Toxoplasma gondii host colonization.

Cellular barcoding techniques are powerful tools to understand microbial pathogenesis. However, barcoding strategies have not been broadly applied to protozoan parasites, which have unique genomic structures and virulence strategies compared with viral and bacterial pathogens. Here, we present a CRISPR-based method to barcode protozoa, which we successfully apply to Toxoplasma gondii and Trypanosoma brucei. 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 representing a minor fraction of the inoculum could become a dominant population in the brain by three months post-infection. These data establish a cellular barcoding approach for protozoa and evidence that the blood-brain barrier is not a major bottleneck to colonization by T. gondii.

Nutrient supplements boost yeast transformation efficiency.

Efficiency of yeast transformation is determined by the rate of yeast endocytosis. The aim of this study was to investigate the effect of introducing amino acids and other nutrients (inositol, adenine, or p-aminobenzoic acid) in the transformation medium to develop a highly efficient yeast transformation protocol. The target of rapamycin complex 1 (TORC1) kinase signalling complex influences the rate of yeast endocytosis. TORC signaling is induced by amino acids in the media. Here, we found that increasing the concentration of amino acids and other nutrients in the growth media lead to an increase yeast transformation efficiency up to 107 CFU per µg plasmid DNA and per 108 cells with a 13.8 kb plasmid DNA. This is over 130 times that of current published methods. This improvement may facilitate more efficient experimentation in which transformation efficiency is critical, such as yeast two-hybrid screening.