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A Toxoplasma gondii locus required for the direct manipulation of host mitochondria has maintained multiple ancestral functions.

ABSTRACT: The Toxoplasma gondii locus mitochondrial association factor 1 (MAF1) encodes multiple paralogs, some of which mediate host mitochondrial association (HMA). Previous work showed that HMA was a trait that arose in T. gondii through neofunctionalization of an ancestral MAF1 ortholog. Structural analysis of HMA-competent and incompetent MAF1 paralogs (MAF1b and MAF1a, respectively) revealed that both paralogs harbor an ADP ribose binding macro-domain, with comparatively low (micromolar) affinity for ADP ribose. Replacing the 16 C-terminal residues of MAF1b with those of MAF1a abrogated HMA, and we also show that only three residues in the C-terminal helix are required for MAF1-mediated HMA. Importantly these same three residues are also required for the in vivo growth advantage conferred by MAF1b, providing a definitive link between in vivo proliferation and manipulation of host mitochondria. Co-immunoprecipitation assays reveal that the ability to interact with the mitochondrial MICOS complex is shared by HMA-competent and incompetent MAF1 paralogs and mutants. The weak ADPr coordination and ability to interact with the MICOS complex shared between divergent paralogs may represent modular ancestral functions for this tandemly expanded and diversified T. gondii locus.

SUBMITTER: Blank ML

PROVIDER: S-EPMC6245547 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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A Toxoplasma gondii locus required for the direct manipulation of host mitochondria has maintained multiple ancestral functions.

Blank Matthew L ML Parker Michelle L ML Ramaswamy Raghavendran R Powell Cameron J CJ English Elizabeth D ED Adomako-Ankomah Yaw Y Pernas Lena F LF Workman Sean D SD Boothroyd John C JC Boulanger Martin J MJ Boyle Jon P JP

Molecular microbiology 20180411 5

The Toxoplasma gondii locus mitochondrial association factor 1 (MAF1) encodes multiple paralogs, some of which mediate host mitochondrial association (HMA). Previous work showed that HMA was a trait that arose in T. gondii through neofunctionalization of an ancestral MAF1 ortholog. Structural analysis of HMA-competent and incompetent MAF1 paralogs (MAF1b and MAF1a, respectively) revealed that both paralogs harbor an ADP ribose binding macro-domain, with comparatively low (micromolar) affinity fo ...[more]

PMID: 29505111

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Project description:Apicomplexa are obligate intracellular parasites that cause important diseases in humans and animals. Manipulating the pathogen genome is the most direct way to understand the functions of specific genes in parasite development and pathogenesis. In Toxoplasma gondii, nonhomologous recombination is typically highly favored over homologous recombination, a process required for precise gene targeting. Several approaches, including the use of targeting vectors that feature large flanks to drive site-specific recombination, have been developed to overcome this problem. We have generated a new large-insert repository of T. gondii genomic DNA that is arrayed and sequenced and covers 95% of all of the parasite's genes. Clones from this fosmid library are maintained at single copy, which provides a high level of stability and enhances our ability to modify the organism dramatically. We establish a robust recombineering pipeline and show that our fosmid clones can be easily converted into gene knockout constructs in a 4-day protocol that does not require plate-based cloning but can be performed in multiwell plates. We validated this approach to understand gene function in T. gondii and produced a conditional null mutant for a nucleolar protein belonging to the NOL1/NOP2/SUN family, and we show that this gene is essential for parasite growth. We also demonstrate a powerful complementation strategy in the context of chemical mutagenesis and whole-genome sequencing. This repository is an important new resource that will accelerate both forward and reverse genetic analysis of this important pathogen.Toxoplasma gondii is an important genetic model to understand intracellular parasitism. We show here that large-insert genomic clones are effective tools that enhance homologous recombination and allow us to engineer conditional mutants to understand gene function. We have generated, arrayed, and sequenced a fosmid library of T. gondii genomic DNA in a copy control vector that provides excellent coverage of the genome. The fosmids are maintained in a single-copy state that dramatically improves their stability and allows modification by means of a simple and highly scalable protocol. We show here that modified and unmodified fosmid clones are powerful tools for forward and reverse genetics.

Project description:UnlabelledToxoplasma gondii is a human obligate intracellular parasite that has infected over 20% of the world population and has a vast intermediate host range compared to those of its nearest relatives Neospora caninum and Hammondia hammondi. While these 3 species have highly syntenic genomes (80 to 99%), in this study we examined and compared species-specific structural variations, specifically at loci that have undergone local (i.e., tandem) duplication and expansion. To do so, we used genomic sequence coverage analysis to identify and curate T. gondii and N. caninum loci that have undergone duplication and expansion (expanded loci [ELs]). The 53 T. gondii ELs are significantly enriched for genes with predicted signal sequences and single-exon genes and genes that are developmentally regulated at the transcriptional level. We validated 24 T. gondii ELs using comparative genomic hybridization; these data suggested significant copy number variation at these loci. High-molecular-weight Southern blotting for 3 T. gondii ELs revealed that copy number varies across T. gondii lineages and also between members of the same clonal lineage. Using similar methods, we identified 64 N. caninum ELs which were significantly enriched genes belonging to the SAG-related surface (SRS) antigen family. Moreover, there is significantly less overlap (30%) between the expanded gene sets in T. gondii and N. caninum than would be predicted by overall genomic synteny (81%). Consistent with this finding, only 59% of queried T. gondii ELs are similarly duplicated/expanded in H. hammondi despite over 99% genomic synteny between these species.ImportanceGene duplication, expansion, and diversification are a basis for phenotypic differences both within and between species. This study represents the first characterization of both the extent and degree of overlap in gene duplication and locus expansion across multiple apicomplexan parasite species. The most important finding of this study is that the locus duplications/expansions are quantitatively and qualitatively distinct, despite the high degree of genetic relatedness between the species. Given that these differential expansions are prominent species-specific genetic differences, they may also contribute to some of the more striking phenotypic differences between these species. More broadly, this work is important in providing further support for the idea that postspeciation selection events may have a dramatic impact on locus structure and copy number that overshadows selection on single-copy genes.

Dataset Information

A Toxoplasma gondii locus required for the direct manipulation of host mitochondria has maintained multiple ancestral functions.

Publications

A Toxoplasma gondii locus required for the direct manipulation of host mitochondria has maintained multiple ancestral functions.

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