Toxoplasma gondii is a widespread apicomplexan parasite that causes severe disease in the congenitally-infected fetus and in immunocompromised persons. T. gondii uses a unique form of gliding motility to invade host cells and disseminate through the body during infection, and motility is essential to parasite virulence. The machinery that powers gliding motility is well conserved in apicomplexan parasites, but very little is known about how the unconventional myosin motor protein at the heart of the motility machinery is regulated. A recent high-throughput screen identified 24 novel small-molecule inhibitors and six enhancers of host cell invasion by T. gondii. Strikingly, 21 of the 24 small molecules that inhibit invasion were found to inhibit parasite motility, and all six of the invasion enhancers enhance parasite motility. This proposal is focused on the detailed mechanism of action of one of the most promising of the motility inhibitors, compound 115556. When compound 115556 is added to T. gondii, it causes the single myosin light chain (TgMLC1) associated with the myosin motor that drives motility (TgMyoA) to undergo a pronounced electrophoretic mobility shift, due to an as yet uncharacterized posttranslational modification. Isolated T. gondii motor complexes containing the modified TgMLC1 show decreased motor activity in an in vitro motility assay. In the course of this work, TgMLC1 was also shown to be phosphorylated and dimethylated. The central hypothesis of this project is that posttranslational modifications of TgMLC1, including the modification induced by 115556, regulate the function of the TgMyoA motor and parasite motility.
The Specific Aims of the project are to: (1) Determine the nature of the 115556-induced modification to TgMLC1 and its effect on TgMyoA function and directly test the hypothesis that the 115556-induced modification of TgMLC1 is responsible for the inhibition of parasite motility by the compound;(2) Identify the target of 115556. The preliminary data suggest that 115556 does not bind to and act directly on TgMLC1. The direct target of 115556 will be identified, and the pathway connecting this target to the modification of TgMLC1 elucidated;(3) Determine the functional consequences of TgMLC1 phosphorylation and methylation on TgMyoA activity and parasite motility, and establish whether any of the other motility inhibitors or enhancers identified in the high-throughput screen act through phosphorylation or methylation of TgMLC1. This work will provide new insights into the proteins and pathways involved in the motility and invasion of host cells by T. gondii. A greater understanding of how motility is regulated in T. gondii and other apicomplexan parasites is likely to lead to new chemotherapeutic approaches to treating the devastating diseases these parasites cause.

Public Health Relevance

Toxoplasma gondii is an important human pathogen and a valuable model organism for other closely related but less experimentally accessible parasites, such as those that cause malaria and cryptosporidiosis. The goal of this project is to understand how the motility of T. gondii is regulated, at the level of the unconventional myosin motor protein that the parasite uses to force its way into cells of its host and disseminate through the body during infection. Parasite motility is essential for virulence, so an increased understanding of how the myosin motor protein is regulated may lead to new chemotherapeutic strategies to treating the diseases caused by this important group of human pathogens.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI054961-09
Application #
8468093
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Rogers, Martin J
Project Start
2003-03-01
Project End
2015-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
9
Fiscal Year
2013
Total Cost
$335,613
Indirect Cost
$115,539
Name
University of Vermont & St Agric College
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
Odell, Anahi V; Tran, Fanny; Foderaro, Jenna E et al. (2015) Yeast three-hybrid screen identifies TgBRADIN/GRA24 as a negative regulator of Toxoplasma gondii bradyzoite differentiation. PLoS One 10:e0120331
Leung, Jacqueline M; Rould, Mark A; Konradt, Christoph et al. (2014) Disruption of TgPHIL1 alters specific parameters of Toxoplasma gondii motility measured in a quantitative, three-dimensional live motility assay. PLoS One 9:e85763
Tilley, Lucas D; Krishnamurthy, Shruthi; Westwood, Nicholas J et al. (2014) Identification of TgCBAP, a novel cytoskeletal protein that localizes to three distinct subcompartments of the Toxoplasma gondii pellicle. PLoS One 9:e98492
Tang, Qing; Andenmatten, Nicole; Hortua Triana, Miryam A et al. (2014) Calcium-dependent phosphorylation alters class XIVa myosin function in the protozoan parasite Toxoplasma gondii. Mol Biol Cell 25:2579-91
Casapao, Anthony M; Davis, Susan L; Barr, Viktorija O et al. (2014) Large retrospective evaluation of the effectiveness and safety of ceftaroline fosamil therapy. Antimicrob Agents Chemother 58:2541-6
Bessoff, Kovi; Spangenberg, Thomas; Foderaro, Jenna E et al. (2014) Identification of Cryptosporidium parvum active chemical series by Repurposing the open access malaria box. Antimicrob Agents Chemother 58:2731-9
Leung, Jacqueline M; Tran, Fanny; Pathak, Ravindra B et al. (2014) Identification of T. gondii myosin light chain-1 as a direct target of TachypleginA-2, a small-molecule inhibitor of parasite motility and invasion. PLoS One 9:e98056
Bookwalter, Carol S; Kelsen, Anne; Leung, Jacqueline M et al. (2014) A Toxoplasma gondii class XIV myosin, expressed in Sf9 cells with a parasite co-chaperone, requires two light chains for fast motility. J Biol Chem 289:30832-41
Tran, Fanny; Odell, Anahi V; Ward, Gary E et al. (2013) A modular approach to triazole-containing chemical inducers of dimerisation for yeast three-hybrid screening. Molecules 18:11639-57
Hall, Carolyn I; Reese, Michael L; Weerapana, Eranthie et al. (2011) Chemical genetic screen identifies Toxoplasma DJ-1 as a regulator of parasite secretion, attachment, and invasion. Proc Natl Acad Sci U S A 108:10568-73

Showing the most recent 10 out of 18 publications