The inner membrane complex through development of Toxoplasma gondii and Plasmodium

Plasmodium spp. and Toxoplasma gondii are important human and veterinary pathogens. These parasites possess an unusual double membrane structure located directly below the plasma membrane named the inner membrane complex (IMC). First identified in early electron micrograph studies, huge advances in genetic manipulation of the Apicomplexa have allowed the visualization of a dynamic, highly structured cellular compartment with important roles in maintaining the structure and motility of these parasites. This review summarizes recent advances in the field and highlights the changes the IMC undergoes during the complex life cycles of the Apicomplexa

Regulation of Plasmodium falciparum Glideosome Associated Protein 45 (PfGAP45) Phosphorylation

The actomyosin motor complex of the glideosome provides the force needed by apicomplexan parasites such as Toxoplasma gondii (Tg) and Plasmodium falciparum (Pf) to invade their host cells and for gliding motility of their motile forms. Glideosome Associated Protein 45 (PfGAP45) is an essential component of the glideosome complex as it facilitates anchoring and effective functioning of the motor. Dissection of events that regulate PfGAP45 may provide insights into how the motor and the glideosome operate. We found that PfGAP45 is phosphorylated in response to Phospholipase C (PLC) and calcium signaling. It is phosphorylated by P. falciparum kinases Protein Kinase B (PfPKB) and Calcium Dependent Protein Kinase 1 (PfCDPK1), which are calcium dependent enzymes, at S89, S103 and S149. The Phospholipase C pathway influenced the phosphorylation of S103 and S149. The phosphorylation of PfGAP45 at these sites is differentially regulated during parasite development. The localization of PfGAP45 and its association may be independent of the phosphorylation of these sites. PfGAP45 regulation in response to calcium fits in well with the previously described role of calcium in host cell invasion by malaria parasite

Computational Analysis and Experimental Validation of Gene Predictions in Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan that infects 20 to 90% of the population. It can cause both acute and chronic infections, many of which are asymptomatic, and, in immunocompromised hosts, can cause fatal infection due to reactivation from an asymptomatic chronic infection. An essential step towards understanding molecular mechanisms controlling transitions between the various life stages and identifying candidate drug targets is to accurately characterize the T. gondii proteome.We have explored the proteome of T. gondii tachyzoites with high throughput proteomics experiments and by comparison to publicly available cDNA sequence data. Mass spectrometry analysis validated 2,477 gene coding regions with 6,438 possible alternative gene predictions; approximately one third of the T. gondii proteome. The proteomics survey identified 609 proteins that are unique to Toxoplasma as compared to any known species including other Apicomplexan. Computational analysis identified 787 cases of possible gene duplication events and located at least 6,089 gene coding regions. Commonly used gene prediction algorithms produce very disparate sets of protein sequences, with pairwise overlaps ranging from 1.4% to 12%. Through this experimental and computational exercise we benchmarked gene prediction methods and observed false negative rates of 31 to 43%.This study not only provides the largest proteomics exploration of the T. gondii proteome, but illustrates how high throughput proteomics experiments can elucidate correct gene structures in genomes

Repression of Human T-lymphotropic virus type 1 Long Terminal Repeat sense transcription by Sp1 recruitment to novel Sp1 binding sites

Human T-lymphotropic Virus type 1 (HTLV-1) infection is characterized by viral latency in the majority of infected cells and by the absence of viremia. These features are thought to be due to the repression of viral sense transcription in vivo. Here, our in silico analysis of the HTLV-1 Long Terminal Repeat (LTR) promoter nucleotide sequence revealed, in addition to the four Sp1 binding sites previously identified, the presence of two additional potential Sp1 sites within the R region. We demonstrated that the Sp1 and Sp3 transcription factors bound in vitro to these two sites and compared the binding affinity for Sp1 of all six different HTLV-1 Sp1 sites. By chromatin immunoprecipitation experiments, we showed Sp1 recruitment in vivo to the newly identified Sp1 sites. We demonstrated in the nucleosomal context of an episomal reporter vector that the Sp1 sites interfered with both the sense and antisense LTR promoter activities. Interestingly, the Sp1 sites exhibited together a repressor effect on the LTR sense transcriptional activity but had no effect on the LTR antisense activity. Thus, our results demonstrate the presence of two new functional Sp1 binding sites in the HTLV-1 LTR, which act as negative cis-regulatory elements of sense viral transcription.info:eu-repo/semantics/publishe

Deciphering N-glycosylation Structures and Functions in Toxoplasma gondii

Toxoplasma gondii est un parasite protozoaire unicellulaire qui se développe à l’intérieur d’une cellule hôte. Chez les parasites Apicomplexa, peu de chose sont connues sur la N-glycosylation. Nous avons mis en évidence la présence de N-glycannes parasitaires totaux et démontré que ces N-glycannes sont de type riche en mannose. En utilisant une lectine, nous avons purifié de nombreuses N-glycoprotéines parasitaires intervenant majoritairement dans les mécanismes de motilité, d’invasion et de trafic intracellulaire. Nous avons démontré qu’un traitement par une drogue inhibant la synthèse des N-glycannes perturbe plusieurs processus biologiques. Nous avons étudié les fonctions biologiques des N-glycannes de TgGAP50 qui appartient au glidéosome, un moteur impliqué dans la motilité du parasite. Nous avons déterminé que TgGAP50 porte des N-glycannes hétérogènes riches en mannoses. Nous avons montré que la N-glycosylation de TgGAP50 est impliquée dans le trafic de la protéine et dans l’interaction avec les partenaires du glidéosome. Nos travaux démontrent que T. gondii est capable de synthétiser des N-glycoprotéines et que les N-glycannes sont potentiellement impliqués dans le trafic des protéines et dans les interactions moléculaires importantes pour la motilité et l’invasion des cellules hôtes par le parasite.The apicomplexan parasite Toxoplasma gondii penetrates virtually any kind of mammalian cell using proteins released from late secretory organelles and a unique form of gliding motility. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. Here, we report comprehensive proteomics and glycomics analyses showing that several key components required for interactions between T. gondii and host cells are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic and paucimannosidic sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography identified components involved in gliding motility, moving junction, and other additional functions. Importantly, tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. In addition, we show that all three potential N-glycosylated sites of GAP50 are occupied by unusual N-glycan structures with terminal glucoses. Using site-directed mutagenesis, we demonstrate that N-glycosylation is a prerequisite for GAP50 transport into the inner membrane complex. Assembly of key partners into gliding complex by unglycosylated GAP50 and parasite motility are severely impaired. Collectively, these results provide the first molecular description of T. gondii N-glycosylation functions that are vital for parasite motility and host cell entry