Evolve and Resequencing (E & R) of Toxoplasma Gondii During Lab-Adaptation to Identify Virulence Factors

Abstract

The two type I genotype T. gondii strains, RH, a lab-adapted strain, and GT1, a non-lab-adapted strain, have a genetic difference of only 0.002%, but show remarkable phenotypic differences in vitro. For example, it has long been known that RH’s in vitro virulence (i.e. plaquing capacity) and extracellular survival is far superior to that of GT1, likely due to several decades of adaptation to the in vitro environment (i.e. lab-adaptation). The genetic basis of these phenotypes, however, remains largely unknown despite previous allele-swapping experiments, thus inspiring two hypotheses: 1) epistatic interactions between two or more alleles and/or 2) gene regulatory mechanisms are responsible for lab-adaptive phenotypes. Uncovering the molecular basis underlying lab-adaptive phenotypes will support our growing understanding of T. gondii virulence and suggest therapeutic targets that affect the parasites lytic cycle in a host-independent manner. To answer this question, we applied Evolve and Resequencing (E&R) of GT1 during the first 1500 generations of its lab-adaptation in order to chronologically identify emerging genotype-phenotype correlations. Indeed, lab-adaptation augmented GT1’s in vitro virulence by improving its extracellular survival and reinvasion capabilities- both extracellular phenotypes of the lytic cycle. DNA-sequencing of parallel GT1 populations at multiple evolutionary timepoints (i.e. passages) identified a polymorphic phospholipid flippase gene whose gene expression is critical for in vitro virulence but, unfortunately, the evolved mutations could not be functionally characterized due to technical limitations. RNA-seq of both intracellular and extracellular parasites across several passages identified hundreds of “pro-tachyzoite” differentially expressed genes (DEGs), but only in extracellular parasites, paralleling our phenotypic observations. Interestingly, several upregulated DEGs are connected to fatty acid biosynthesis. Lastly, genetic KO of five seemingly non-related DEGs indicates that GT1’s lab-adaptive in vitro virulence is a complex and polygenic phenotype that is largely controlled by mechanisms independent of genomic mutations.