Blank, Matthew
(2020)
Exploiting Toxoplasma gondii MAF1 locus diversity to identify essential host proteins required for mitochondrial sequestration and manipulation.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
Recent genomic comparisons identified multiple expanded loci in Toxoplasma gondii that are unique compared to close Apicomplexan relatives. One of these loci, mitochondrial association factor 1 (MAF1), encodes distinct paralogs of secreted dense granule effector proteins, some of which mediate the host mitochondrial association (HMA) phenotype. MAF1b drives HMA, MAF1a does not. Through sequence and functional analysis of multiple MAF1 paralogs, we have identified regions of the protein that have undergone paralog-specific selection-driven diversification. Using structure and alignment-guided site-directed mutagenesis of MAF1b and MAF1a, we identified three critical residues in the C-terminal helix that are required for HMA. Using this MAF1b mutant as a new tool to probe the function of MAF1b and HMA in T. gondii interactions, we performed an unbiased quantitative mass spectrometry screen comparing co-IP samples of MAF1b, MAF1a, and MAF1b mutant parasite infections. Of the 1,360 proteins identified in all samples, 13 candidate proteins were uniquely enriched in the MAF1b samples compared to both MAF1a and MAF1b mutant samples. Of these 13 candidate host binding proteins, nine play roles in mitochondrial biology. RNAi knockdown of each of the nine candidates followed by either Type II:MAF1b infection or GFP-MAF1b expression identified TOM70 and its mitochondrial chaperone, HSPA9, as partners required for MAF1b-mediated HMA. TOM70 enrichment at the T. gondii vacuolar membrane and parasite exclusion of the ER at this interface illustrate an intricate manipulative strategy on the part of parasite. Both TOM70 and HSPA9 are implicated in specific ER-mitochondrial contact site functions like immune modulation, mitochondrial dynamics, autophagy, and calcium homeostasis. The requirement of both TOM70 and HSPA9 could explain key phenotypes previously described in an HMA+ T. gondii infection such as an in vivo growth advantage, modulated immune response, and mitochondrial fusion around the vacuole. Taken together, the neofunctionalization of the MAF1 locus drove the evolution of an intermolecular network capable of mediating intimate interactions between host mitochondria and the T. gondii-containing vacuole.
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Details
| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
|
| Date: |
8 June 2020 |
| Date Type: |
Publication |
| Defense Date: |
3 March 2020 |
| Approval Date: |
8 June 2020 |
| Submission Date: |
4 February 2020 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
139 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Biological Sciences |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
toxoplasma gondii, tom70, mitochondria, host mitochondria association, HMA |
| Date Deposited: |
08 Jun 2020 15:46 |
| Last Modified: |
08 Jun 2020 15:46 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/38223 |
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