Toxoplasma gondii is an obligate intracellular protozoan parasite capable of infecting almost any nucleated cell. It is an opportunistic pathogen that asymptomatically infects 10-20% of the world population and can cause severe disease in congenitally infected neonates and immuno-compromised individuals, in particular HIV and transplant patients. Reactivation of latent infections typically leads to toxoplasmic encephalitis, the pathology of which is associated with the parasite-mediated lytic destruction of infected host cells. Current anti-parasitic drugs, while effective, are limited in scope due to severe toxic side effects and the inability to eliminate or prevent chronic infection by encysted bradyzoites. Recent efforts to overcome such obstacles have focused on targeting parasite proteases, several of which have been shown to be critical for parasite survival and host cell invasion. A complete understanding of parasite-derived proteases and their involvement in key biological processes is necessary for developing new therapies to combat the disease. However, the identities of the protease(s) involved in Toxoplasma pathogenesis remain unknown for several reasons. First, while Toxoplasma has emerged as a model organism for the study of apicomplexan parasites due to the ease with which it can be genetically manipulated, ablation of critical genes involved in parasite survival is often difficult since the infective stage of the parasite is haploid. Furthermore, Toxoplasma is an intracellular parasite that undergoes rapid cell division inside a host making it a highly adaptable pathogen that may quickly find alternate survival mechanisms to compensate for loss of critical genes.
To overcome these limitations, we employed both reverse and forward chemical genetic strategies to identify and define roles for parasite-derived proteases in the lytic cycle of Toxoplasma gondii. We employed a reverse chemical genetic screen to develop high selective inhibitors to distinguish between the closely related TgCPB and TgCPL proteases (Chapter 3). Both proteases have been implicated as major players in host cell invasion. TgCPB has been implicated as a rhoptry protein maturase and treatment of parasites with the cathepsin B inhibitor, PRT2253F prevents infection in chick embryo model of congenital toxoplasmosis. However, the lack of highly selective inhibitors prevents determination of potential off-target effects. TgCPL has been implicated as a microneme protein maturase, however, the lack of highly selective reagents has hampered abilities to determine its precise function. To overcome these limitations, we used a positional scanning library of epoxide-based covalent inhibitors to query the specificity elements required in the S2, S3 and S4 substrate binding pockets of TgCPB and TgCPL. Using this information, we synthesized a series of selective inhibitors that can be used to dissect the specific functions of each protease.
Forward chemical genetics strategies offer several advantages over classical genetic and biochemical techniques. First, forward chemical genetic strategies identify compounds capable of perturbing key biological processes without knowledge of protein targets. Additionally, the use of small molecule inhibitors bypasses the need to genetically disrupt (knock down or knock out) genes of interest. Recently, a high throughput screen was undertaken to identify novel inhibitors targeting parasite proteins that play important roles in host cell invasion. This type of pharmacological study relied on screening large libraries (12,160) of unbiased, structurally diverse small molecules in an automated microscopy-based invasion assay. Several novel molecules were identified that perturb the process of host cell invasion and can now be used to begin to dissect this highly complex process. However, the resulting hits from the screen provide no clues as to the identity of the target protein or proteins responsible for the invasion phenotype.
To overcome this limitation to unbiased screening, we assembled a significantly smaller set (1500-2000) of highly focused small molecules known to be covalent inhibitors of cysteine and serine proteases (Chapter Four). Interestingly, we identified a small molecule, WRR-086, that inhibits Toxoplasma attachment to host cells. By coupling tandem orthogonal proteolysis-activity based protein profiling (TOP-ABPP) with site directed mutagenesis, we identified TgDJ-1 as the functionally relevant target of WRR-086. TgDJ-1 shares considerable sequence homology with human DJ-1, a gene that has been linked to autosomal recessive early onset Parkinson Disease. Relatively little is know about the exact function of this protein and therefore it is unlikely that TgDJ-1 would have been identified as a potential regulator of Toxoplasma attachment to host cells by sequence homology or other classical genetic methods. (Abstract shortened by UMI.)
|School Location:||United States -- California|
|Source:||DAI-B 70/10, Dissertation Abstracts International|
|Keywords:||Cell invasion, HIV, Microneme secretion, Protease, Protozoan parasite, Toxoplasma gondii|
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