David Warschkau (Berlin / DE), David Arranz-Solís (Davis, CA / US), Benedikt T. Fabian (Berlin / DE), Jeroen PJ Saeij (Davis, CA / US), Frank Seeber (Berlin / DE)
Abstract text
Introduction
Toxoplasma gondii oocysts are highly infectious, shed in large quantities by infected felines, are extremely stable in the environment and resistant to most inactivation procedures. Despite being a critical life stage for T. gondii transmission, the genetic basis for the environmental resistance of the oocysts and the contained sporozoites is largely unknown.
Objectives
Our objective was to test whether a set of sporozoite-specific proteins could determine T. gondii oocyst resilience to environmental stressors. Among them, we decided to investigate Late Embryogenesis Abundant (LEA)-related proteins, since LEAs are known to provide resistance to environmental stresses such as drought, salinity, and freezing in a variety of organisms. All four T. gondii LEA genes are located in a cluster (LEAc) on chromosome XII.
Materials & methods
We studied the cryoprotective effect of the four LEAs on T. gondii lactate dehydrogenase in vitro using recombinant proteins and assessing aggregation after repeated freeze-thaw cycles. Using an inducible expression system in E. coli, we tested whether the proteins confer resistance to low temperatures. Through biochemical and bioinformatic approaches, we analyzed the LEAs for features of intrinsically disordered proteins. By applying the CRISPR/Cas9 technology, we generated a LEAc knockout in a cat-compatible ME49 strain, and assessed the oocyst sensitivity to environmental stresses compared to that from wild-type parasites.
Results
Recombinantly expressed LEA proteins showed cryoprotective effects on lactate dehydrogenase in vitro and induced expression in E. coli improved viability at low temperatures. T. gondii LEAs showed features of intrinsically disordered proteins and amphoteric repeats, explaining some of the protein"s properties. Remarkably, LEAc knockout oocysts were more susceptible to high salinity, freezing and desiccation compared to oocysts from wild-type parasites.
Conclusion
We show for the first time that LEA proteins are important for the environmental resistance of T. gondii oocysts. Future studies using knockout strains for each of the LEA genes alone or in combination are now warranted to elucidate which LEAs are needed for the resistance phenotype.