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Differential proteomic analysis in a host–parasite interaction of host cells infected with Toxoplasma gondii

ABSTRACT: Toxoplasma gondii, a representative model organism of the phylum Apicomplexa, can infect almost all warm-blooded organisms including humans. Invasion of host cells in a host–parasite interaction is the key step necessary for T. gondii to complete its life cycle. Here, we investigate global proteomic changes based on TMT analysis in both the host cells (human foreskin fibroblasts, HFFs) and T. gondii during intracellular infection. A total of 3477 and 1434 proteins were quantified, of which 378 and 1100 proteins were differentially expressed (p-value<0.05 and ≥1.5-fold change)in Homo sapiens and T. gondii proteins, respectively. In HFF cells, Gene Ontology (GO) and KEGG pathway analyses revealed a large number of differentially expressed proteins (DEPs) enriched in metabolic processes and immune-associated signaling pathways, such as NF-κB, cAMP, and Rap1 signaling pathways. T. gondii DEPs involved in pathway analysis and GO annotations included cellular protein metabolic process, peptide metabolic process, and peptide biosynthetic processes and indicated that cell biosynthesis and metabolism were increased during T. gondii infection. These findings reveal new proteomic differences in the parasite–host interaction during T. gondii infection.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: hui sun

LAB HEAD: hui sun

PROVIDER: PXD021736 | Pride | 2021-05-10

REPOSITORIES: Pride

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Project description:The population structure of Toxoplasma gondii includes three highly prevalent clonal lineages, types I, II, and III, which differ greatly in virulence in the mouse model. Previous studies have implicated a family of serine threonine protein kinases found in rhoptries (ROPs) as important in mediating virulence differences between types I vs. III and II vs. III. Here, we explored the genetic basis of differences in virulence between the highly virulent type I lineage and moderately virulent type II based on a new genetic cross and linkage mapping. Genome-wide association revealed a single quantitative trait locus controls the > 4 log difference in lethality between these strains. Neither ROP16 nor ROP18, previously implicated in virulence differences in T. gondii, were found to contribute to differences between types I and II. Instead, the major virulence locus contained a cluster of pseudokinases denoted as rhoptry protein 5 (ROP5); this locus contains a tandem cluster of polymorphic alleles that differed in expression levels between strains. ROP5 alleles contained only part of the catalytic triad of canonical S/T kinases, and consistent with this they lack demonstrable kinase activity in vitro. Genetic disruption of the rop5 locus in the type I lineage lead to a > 5 log increase in the lethal dose, and surviving mice developed lasting immunity and were protected from an otherwise lethal challenge. These findings reveal that amplification of a polymorphic cluster of pseudokinases plays an important role in pathogenesis of toxoplasmosis in the mouse model. We used a new genotyping protocol based on hybridization of parental and recombinant progeny genomic DNA (gDNA) to Affymetrix gene arrays constructed for T. gondii (http://ancillary.toxodb.org/docs/Array-Tutorial.html). We identified 1,603 single feature polymorphisms (SFP) based on probes that successfully discriminated perfect match (type II) from mismatch (type I) hybridization across all clones and provided reliable SFP markers for this cross.

Project description:Toxoplasma gondii transmitted via various route and rapidly duplicated during the acute infection, which caused the important zoonosis regarding to medicine and veterinary, toxoplasmosis. T. gondii possessed innate capability of producing nitric oxide (NO) and nitric oxide derivatives and S-nitrosylation not only participated in their signaling transduction, but also was a regulatory mechanism of protein post-translation. To date, S-nitrosylation proteome of T .gondii remains a mystery. In present study, we reported the first S-nitrosylated proteomic profile in T. gondii using mass spectrometry in combination with resin-assisted enrichment. 637 proteins were found to be S-nitrosylated and more than half of these S-nitrosylated proteins were localized in nucleus or cy-toplasm. Motif analysis identified seven motifs including five motifs containing lysine and two motif harboring isoleucine. GO enrichment revealed that S-nitrosylated proteins were mainly lo-cated in the mitochondrial inner membrane and organelle inner membrane in the cell. Those S-nitrosylated proteins were related with diverse biological and metabolic processes including or-ganic acid binding, carboxylic acid binding ribose and phosphate biosynthetic process. Glycoly-sis/gluconeogenesis and aminoacyl-tRNA biosynthesis were two remarkable pathways in which T. gondii S-nitrosylated proteins engaged. Twenty seven ribosomal proteins and eleven microneme proteins out of 22 secretory proteins were identified as S-nitrosylated proteins, which suggested that proteins in ribosome and microneme were S-nitrosylated with high proportion. PPI analysis identified three subnetworks with high relevancy ribosome, RNA transport and chaperonin com-plex component. These results implied that S-nitrosylated proteins in T. gondii were associated with protein translation in ribosome, gene transcription, invasion and proliferation of T. gondii. Our research firstly identified the S-nitrosylated proteomic profile of T. gondii and will provide a valuable resource for deep-going investigation of the functions of S-nitrosylation in T. gondii.

Project description:The phylum Apicomplexa comprises an ancient group of early divergent eukaryotes, including some of the most deadly pathogens of medical and veterinary importance. Plasmodium species are responsible for malaria, which causes as many as 700,000 deaths per year, while Toxoplasma gondii chronically infects up to 30% of the human population, with immunocompromised patients and pregnant women at risk for adverse outcomes, such as toxoplasmic encephalitis and spontaneous abortion, respectively. T. gondii is considered a model system not only for its pathogenic relatives but also for intracellular parasitism and infection biology in general. T. gondii has common eukaryotic organelles, including the nucleus, endoplasmic reticulum (ER), and a single Golgi stack, but also specific secretory organelles named dense granules, micronemes, and rhoptries that contain parasite-derived factors required for host infection. Rhoptries and micronemes are formed de novo during parasite replication, and this process requires significant protein and lipid trafficking through the secretory pathway. The trafficking mechanisms employed by T. gondii retain several typical eukaryote components as well as evolving divergent features. Protein trafficking of this parasite is mediated by entry into a canonical ER followed by vesicle packaging through a single Golgi complex. Post-Golgi protein sorting to specific organelles requires the function of dynamin-related protein B, which is involved in fission events. Downstream Rab-GTPases function throughout the parasite secretory pathway. T. gondii soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins in docking and fusion at target membranes have also been described. However, unlike in mammalian cells, T. gondii ER is reduced so that the nuclear envelope itself contributes to a substantial proportion of its total volume. Whereas in mammalian cells hundreds of Golgi stacks occupy the perinuclear area, the Golgi apparatus is limited to a single discrete structure in T.gondii. The post-Golgi system also named the endosome-like compartment (ELC) is involved in the trafficking of microneme proteins. The ELC is decorated by the small GTPases, Rab5 and Rab7, which are typically associated with the endosomal system. Nevertheless, classical endocytosis has not yet been validated in T. gondii. This parasite has no lysosomes; rather the parasite harbors acidic vesicles that were thought to be precursors of the rhoptry organelles. The parasite lacks most components of endosomal sorting complexes (ESCRT), which are known for their roles in forming multivesicular bodies that deliver ubiquitinated membrane proteins and lipids to lysosomes for degradation. The machinery required for caveogenesis and caveola-dependent invaginations have not yet been identified in the parasite. Furthermore, while evidence of conventional clathrin-dependent endocytosis by T.gondii is lacking, clathrin is present exclusively in post-Golgi compartments where its function is restricted to post-Golgi trafficking, and the uptake of cytosol proteins by the tachyzoites of T. gondii has recently been described using an endocytosis assay. However, the mechanisms underlying the events of this unconventional endocytosis in the parasite remain to be determined. Clearly, the secretory pathway of T. gondii can be considered a stripped-down version of the more complex trafficking machinery that characterizes higher eukaryotes. Despite this minimal trafficking machinery, the parasites actively rely on a membrane vesicle formation and transport during its intracellular lifecycle; however, to date, comparatively little is known about the mechanisms involved in trafficking pathways in T. gondii. We previously reported a T. gondii sortilin-like receptor (TgSORTLR) that regulates protein transport and is essential for apical secretory organelle biogenesis and host infection17. Moreover, the C-terminal tail of TgSORTLR was shown to be involved in recruiting many cytosolic cargo proteins including two homologues of the core retromer components, Vps26 and Vps35, which are known to regulate retrograde transport from endosomes to the trans-Golgi Network (TGN) in yeast and mammals. Here, we report that a singular architecture with a trimer Vps35-Vps26-Vps29 core complex acts as the major cargo endosomal recycling machinery and is required for parasite integrity and more specifically for secretory organelle biogenesis and maintenance of a multiple ligand-binding transporter at the T. gondii membrane. Our findings provide strong evidence that the unconventional TgSORTLR-containing endosome-like compartment is involved in distinct mechanisms for the delivery of major retromer-dependent cargo. In this current work, we demonstrate a definitive role for the endocytic recycling pathway in T. gondii pathogenesis.

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Differential proteomic analysis in a host–parasite interaction of host cells infected with Toxoplasma gondii

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