Development of a novel screen to dissect Toxoplasma gondii egress

DEVELOPMENT OF A NOVEL SCREEN TO DISSECT TOXOPLASMA GONDII EGRESS By Keith Eidell Thesis Advisor: Dr. Marc-Jan Gubbels The Apicomplexa comprise a group of obligate intracellular parasites some of which cause severe diseases in humans with malaria the most notorious representative. Toxoplasma gondii infection is the most widespread apicomplexan infection, which is mostly symptomless in healthy people but is associated wit h a variety of birth defects upon congenital infection and can become life threatening in immunocompromised patients. In addition, T. gondii has been established as a model for the study of intracellular parasitism by Apicomplexa. The lytic destruct ion of host cells underlies the pathogenesis of all apicomplexan diseases. The T. gondii lytic cycle involves host cell invasion, several rounds of intracellular replication, and is followe d by egress of motile parasites in order to infect neighboring host cells. Egress i s an increasingly more appreciated aspect of the lytic cycle for which thre e physiological triggers have been identified. All three triggers converge on the release of Ca stores within the parasite. Large sections of the signaling pathways and molecular playe s associated with egress and intracellular calcium release remain unknown. The objective of this thesis was to develop and employ a novel enrichment screening procedure that would efficiently isolate egress mutants in re ponse to pharmaceutically induced egress. The biggest caveat to such a screen is the abilit y to separate intracellular from extracellular parasites, which is hamper ed by the stickiness of parasites to host cells as well as their fast reinvasion capacity. This hurdl e was overcome by saturating the parasite’s surface receptors with the glycan hepari n to prevent attachment to the host cell. Simultaneously, the oxidizing agent pyrrolidine dithiocarbamate (PDTC) was applied to specifically kill extracellul ar parasites. The enrichment power of the screen was assessed by diluting a previously identifi e temperature-sensitive egress mutant called F-P2 in wild type parasites. The screen’s enrichment power was assessed by flow cytometry and a 1000-fold enrichment capac ity to a 100% F-P2 population could routinely be achieved. Subsequently the screen was applied to generate mutants with defects in the poorly understood NTPase mediated egress-trigger pathway. Chemical mutagenesis as well as insertional m utagenesis was applied and dithiotreitol (DTT) that artificially creates the reducin g e vironment triggering egress was used to screen mutants. Three chemically induced cons titutive egress mutants and one insertional mutant were isolated. As expected, all mut ants displayed resistance to DTT induced egress. In addition, cross resistance to two o her egress inducers upstream of Ca 2+ release was observed, however all mutants egressed upon calcium ionophore treatment. Taken together, the developed enrichment procedure will enable the isolation of constitutive as well as conditional egress mutants. Future cosmid complementat ion will help to fill in important blanks in the egress mechanisms and will ultimate ly lead to a better understanding of intracellular parasitism. This gained understandin g will potentially lead to therapies to combat the destructive effects of apicomplex an parasites.