Development of a novel screen to dissect Toxoplasma gondii egress

• Main Author: Eidell, Keith
• Format: Others
• Language: English
• Published: Boston College 2010
• Subjects: Toxoplasma gondii
• Online Access: http://hdl.handle.net/2345/1732

Thesis advisor: 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 with 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 destruction 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 followed by egress of motile parasites in order to infect neighboring host cells. Egress is an increasingly more appreciated aspect of the lytic cycle for which three physiological triggers have been identified. All three triggers converge on the release of Ca2+ stores within the parasite. Large sections of the signaling pathways and molecular players 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 response to pharmaceutically induced egress. The biggest caveat to such a screen is the ability to separate intracellular from extracellular parasites, which is hampered by the stickiness of parasites to host cells as well as their fast reinvasion capacity. This hurdle was overcome by saturating the parasite's surface receptors with the glycan heparin to prevent attachment to the host cell. Simultaneously, the oxidizing agent pyrrolidine dithiocarbamate (PDTC) was applied to specifically kill extracellular parasites. The enrichment power of the screen was assessed by diluting a previously identified 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 capacity 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 mutagenesis was applied and dithiotreitol (DTT) that artificially creates the reducing environment triggering egress was used to screen mutants. Three chemically induced constitutive egress mutants and one insertional mutant were isolated. As expected, all mutants displayed resistance to DTT induced egress. In addition, cross resistance to two other egress inducers upstream of Ca2+ 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 complementation will help to fill in important blanks in the egress mechanisms and will ultimately lead to a better understanding of intracellular parasitism. This gained understanding will potentially lead to therapies to combat the destructive effects of apicomplexan parasites. === Thesis (MS) — Boston College, 2010. === Submitted to: Boston College. Graduate School of Arts and Sciences. === Discipline: Biology.