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Identification of functional modules of AKMT, a novel lysine methyltransferase regulating the motility of Toxoplasma gondii.

ABSTRACT: The intracellular parasite Toxoplasma gondii is a leading cause of congenital neurological defects. To cause disease, it must reiterate its lytic cycle through host cell invasion, replication, and parasite egress. This requires the parasite to sense changes in its environment and switch between the non-motile (for replication) and motile (for invasion and egress) states appropriately. Recently, we discovered a previously unknown mechanism of motility regulation in T. gondii, mediated by a lysine methyltransferase, AKMT (for Apical complex lysine (K) methyltransferase). When AKMT is absent, activation of motility is inhibited, which compromises parasite invasion and egress, and thus severely impairs the lytic cycle. Although the methyltransferase activity of AKMT has been established, the phylogenetic relationship of AKMT with other better studied lysine methyltransferases (KMTs) was not known. Also unknown was the functional relationships between different domains of AKMT. In this work we carried out phylogenetic analyses, which show that AKMT orthologs form a new subfamily of KMTs. We systematically generated truncation mutants of AKMT, and discovered that the predicted enzymatic domain alone is a very poor enzyme and cannot complement the function of AKMT in vivo. Interestingly, the N- and C-terminal domains of the AKMT have drastically different impacts on its enzyme activity, localization as well as in vivo function. Our results thus reveal that AKMT is an unusual, parasite-specific enzyme and identified regions and interactions within this novel lysine methyltransferase that can be used as drug targets.

SUBMITTER: Sivagurunathan S

PROVIDER: S-EPMC3740173 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Identification of functional modules of AKMT, a novel lysine methyltransferase regulating the motility of Toxoplasma gondii.

Sivagurunathan Senthilkumar S Heaslip Aoife A Liu Jun J Hu Ke K

Molecular and biochemical parasitology 20130517 1-2

The intracellular parasite Toxoplasma gondii is a leading cause of congenital neurological defects. To cause disease, it must reiterate its lytic cycle through host cell invasion, replication, and parasite egress. This requires the parasite to sense changes in its environment and switch between the non-motile (for replication) and motile (for invasion and egress) states appropriately. Recently, we discovered a previously unknown mechanism of motility regulation in T. gondii, mediated by a lysine ...[more]

PMID: 23685344

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Project description:BackgroundLysine lactylation (Kla) is a novelposttranslational modification (PTM) identified in histone and nonhistone proteins of several eukaryotic cells that directly activates gene expression and DNA replication. However, very little is known about the scope and cellular distribution of Kla in apicomplexan parasites despite its significance in public and animal health care.MethodsToxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular apicomplexan parasite that can infect different nucleated cell types of animals and humans. We used this parasite as a model organism and extracted the total protein of tachyzoites to produce the first global lysine lactylome profile of T. gondii through liquid chromatography-tandem mass spectrometry. We also investigated the level and localization of the Kla protein in T. gondii using western blotting and the indirect fluorescent antibody test (IFA), respectively.ResultsA total of 983 Kla sites occurring on 523 lactylated proteins were identified in the total protein extracted from Toxoplasma tachyzoites, the acute toxoplasmosis-causing stage. Bioinformatics analysis revealed that the lactylated proteins were evolutionarily conserved and involved in a wide variety of cellular functions, such as energy metabolism, gene regulation and protein biosynthesis. Subcellular localization analysis and IFA results further revealed that most of the lactylated T. gondii proteins were localized in the nucleus, indicating the potential impact of Kla on gene regulation in the T. gondii model. Notably, an extensive batch of parasite-specific proteins unique to phylum Apicomplexa is lactylated in T. gondii.ConclusionsThis study revealed that Kla is widespread in early dividing eukaryotic cells. Lactylated proteins, including a batch of unique parasite proteins, are involved in a remarkably diverse array of cellular functions. These valuable data will improve our understanding of the evolution of Kla and potentially provide the basis for developing novel therapeutic avenues.

Project description:Toxoplasma gondii is a highly successful parasite that infects approximately one-third of the human population and can cause fatal disease in immunocompromised individuals. Systemic parasite dissemination to organs such as the brain and eye is critical to pathogenesis. T. gondii can disseminate via the circulation, and both intracellular and extracellular modes of transport have been proposed. However, the processes by which extracellular tachyzoites adhere to and migrate across vascular endothelium under the conditions of rapidly flowing blood remain unknown. We used microfluidics and time-lapse fluorescence microscopy to examine the interactions between extracellular T. gondii and primary human endothelial cells under conditions of physiologic shear stress. Remarkably, tachyzoites adhered to and glided on human vascular endothelium under shear stress conditions. Compared to static conditions, shear stress enhanced T. gondii helical gliding, resulting in a significantly greater displacement, and increased the percentage of tachyzoites that invaded or migrated across the endothelium. The intensity of the shear forces (from 0.5 to 10 dynes/cm(2)) influenced both initial and sustained adhesion to endothelium. By examining tachyzoites deficient in the T. gondii adhesion protein MIC2, we found that MIC2 contributed to initial adhesion but was not required for adhesion strengthening. These data suggest that under fluidic conditions, T. gondii adhesion to endothelium may be mediated by a multistep cascade of interactions that is governed by unique combinations of adhesion molecules. This work provides novel information about tachyzoite interactions with vascular endothelium and contributes to our understanding of T. gondii dissemination in the infected host. IMPORTANCE Toxoplasma gondii is a global parasite pathogen that can cause fatal disease in immunocompromised individuals. An unresolved question is how the parasites circulate in the body to tissues to cause disease. T. gondii parasites are found in the bloodstream of infected animals and patients, and they have been shown to adhere to and cross the endothelial cells that line blood vessel walls. To investigate these interactions, we devised a microfluidic system to visualize parasites interacting with vascular endothelium under conditions similar to those found in the bloodstream. Interestingly, parasite migration was significantly influenced by the mechanical force of shear flow. Furthermore, we identified a role for the parasite surface protein MIC2 in the initial phase of adhesion. Our study is the first to document T. gondii interactions with endothelium under shear stress conditions and provides a foundation for future studies on the molecules that mediate parasite interaction with the vasculature.

Dataset Information

Identification of functional modules of AKMT, a novel lysine methyltransferase regulating the motility of Toxoplasma gondii.

Publications

Identification of functional modules of AKMT, a novel lysine methyltransferase regulating the motility of Toxoplasma gondii.

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