Ablation of an Ovarian Tumor Family Deubiquitinase Exposes the Underlying Regulation Governing the Plasticity of Cell Cycle Progression in Toxoplasma gondii.

The Toxoplasma genome encodes the capacity for distinct architectures underlying cell cycle progression in a life cycle stage-dependent manner. Replication in intermediate hosts occurs by endodyogeny, whereas a hybrid of schizogony and endopolygeny occurs in the gut of the definitive feline host. Here, we characterize the consequence of the loss of a cell cycle-regulated ovarian tumor (OTU family) deubiquitinase, OTUD3A of Toxoplasma gondii (TgOTUD3A; TGGT1_258780), in T. gondii tachyzoites. Rather than the mutation being detrimental, mutant parasites exhibited a fitness advantage, outcompeting the wild type. This phenotype was due to roughly one-third of TgOTUD3A-knockout (TgOTUD3A-KO) tachyzoites exhibiting deviations from endodyogeny by employing replication strategies that produced 3, 4, or 5 viable progeny within a gravid mother instead of the usual 2. We established the mechanistic basis underlying these altered replication strategies to be a dysregulation of centrosome duplication, causing a transient loss of stoichiometry between the inner and outer cores that resulted in a failure to terminate S phase at the attainment of 2N ploidy and/or the decoupling of mitosis and cytokinesis. The resulting dysregulation manifested as deviations in the normal transitions from S phase to mitosis (S/M) (endopolygeny-like) or M phase to cytokinesis (M/C) (schizogony-like). Notably, these imbalances are corrected prior to cytokinesis, resulting in the generation of normal progeny. Our findings suggest that decisions regarding the utilization of specific cell cycle architectures are controlled by a ubiquitin-mediated mechanism that is dependent on the absolute threshold levels of an as-yet-unknown target(s). Analysis of the TgOTUD3A-KO mutant provides new insights into mechanisms underlying the plasticity of apicomplexan cell cycle architecture.IMPORTANCE Replication by Toxoplasma gondii can occur by 3 distinct cell cycle architectures. Endodyogeny is used by asexual stages, while a hybrid of schizogony and endopolygeny is used by merozoites in the definitive feline host. Here, we establish that the disruption of an ovarian-tumor (OTU) family deubiquitinase, TgOTUD3A, in tachyzoites results in dysregulation of the mechanism controlling the selection of replication strategy in a subset of parasites. The mechanistic basis for these altered cell cycles lies in the unique biology of the bipartite centrosome that is associated with the transient loss of stoichiometry between the inner and outer centrosome cores in the TgOTUD3A-KO mutant. This highlights the importance of ubiquitin-mediated regulation in the transition from the nuclear to the budding phases of the cell cycle and provides new mechanistic insights into the regulation of the organization of the apicomplexan cell cycle.

Integrative analysis associates monocytes with insufficient erythropoiesis during acute Plasmodium cynomolgi malaria in rhesus macaques.

BACKGROUND: Mild to severe anaemia is a common complication of malaria that is caused in part by insufficient erythropoiesis in the bone marrow. This study used systems biology to evaluate the transcriptional and alterations in cell populations in the bone marrow during Plasmodium cynomolgi infection of rhesus macaques (a model of Plasmodium vivax malaria) that may affect erythropoiesis. RESULTS: An appropriate erythropoietic response did not occur to compensate for anaemia during acute cynomolgi malaria despite an increase in erythropoietin levels. During this period, there were significant perturbations in the bone marrow transcriptome. In contrast, relapses did not induce anaemia and minimal changes in the bone marrow transcriptome were detected. The differentially expressed genes during acute infection were primarily related to ongoing inflammatory responses with significant contributions from Type I and Type II Interferon transcriptional signatures. These were associated with increased frequency of intermediate and non-classical monocytes. Recruitment and/or expansion of these populations was correlated with a decrease in the erythroid progenitor population during acute infection, suggesting that monocyte-associated inflammation may have contributed to anaemia. The decrease in erythroid progenitors was associated with downregulation of genes regulated by GATA1 and GATA2, two master regulators of erythropoiesis, providing a potential molecular basis for these findings. CONCLUSIONS: These data suggest the possibility that malarial anaemia may be driven by monocyte-associated disruption of GATA1/GATA2 function in erythroid progenitors resulting in insufficient erythropoiesis during acute infection.

Systems-based analysis of the Sarcocystis neurona genome identifies pathways that contribute to a heteroxenous life cycle.

UNLABELLED: Sarcocystis neurona is a member of the coccidia, a clade of single-celled parasites of medical and veterinary importance including Eimeria, Sarcocystis, Neospora, and Toxoplasma. Unlike Eimeria, a single-host enteric pathogen, Sarcocystis, Neospora, and Toxoplasma are two-host parasites that infect and produce infectious tissue cysts in a wide range of intermediate hosts. As a genus, Sarcocystis is one of the most successful protozoan parasites; all vertebrates, including birds, reptiles, fish, and mammals are hosts to at least one Sarcocystis species. Here we sequenced Sarcocystis neurona, the causal agent of fatal equine protozoal myeloencephalitis. The S. neurona genome is 127 Mbp, more than twice the size of other sequenced coccidian genomes. Comparative analyses identified conservation of the invasion machinery among the coccidia. However, many dense-granule and rhoptry kinase genes, responsible for altering host effector pathways in Toxoplasma and Neospora, are absent from S. neurona. Further, S. neurona has a divergent repertoire of SRS proteins, previously implicated in tissue cyst formation in Toxoplasma. Systems-based analyses identified a series of metabolic innovations, including the ability to exploit alternative sources of energy. Finally, we present an S. neurona model detailing conserved molecular innovations that promote the transition from a purely enteric lifestyle (Eimeria) to a heteroxenous parasite capable of infecting a wide range of intermediate hosts. IMPORTANCE: Sarcocystis neurona is a member of the coccidia, a clade of single-celled apicomplexan parasites responsible for major economic and health care burdens worldwide. A cousin of Plasmodium, Cryptosporidium, Theileria, and Eimeria, Sarcocystis is one of the most successful parasite genera; it is capable of infecting all vertebrates (fish, reptiles, birds, and mammals-including humans). The past decade has witnessed an increasing number of human outbreaks of clinical significance associated with acute sarcocystosis. Among Sarcocystis species, S. neurona has a wide host range and causes fatal encephalitis in horses, marine mammals, and several other mammals. To provide insights into the transition from a purely enteric parasite (e.g., Eimeria) to one that forms tissue cysts (Toxoplasma), we present the first genome sequence of S. neurona. Comparisons with other coccidian genomes highlight the molecular innovations that drive its distinct life cycle strategies.

Metabolomics in the fight against malaria

Metabolomics uses high-resolution mass spectrometry to provide a chemical fingerprint of thousands of metabolites present in cells, tissues or body fluids. Such metabolic phenotyping has been successfully used to study various biologic processes and disease states. High-resolution metabolomics can shed new light on the intricacies of host-parasite interactions in each stage of the Plasmodium life cycle and the downstream ramifications on the host’s metabolism, pathogenesis and disease. Such data can become integrated with other large datasets generated using top-down systems biology approaches and be utilised by computational biologists to develop and enhance models of malaria pathogenesis relevant for identifying new drug targets or intervention strategies. Here, we focus on the promise of metabolomics to complement systems biology approaches in the quest for novel interventions in the fight against malaria. We introduce the Malaria Host-Pathogen Interaction Center (MaHPIC), a new systems biology research coalition. A primary goal of the MaHPIC is to generate systems biology datasets relating to human and non-human primate (NHP) malaria parasites and their hosts making these openly available from an online relational database. Metabolomic data from NHP infections and clinical malaria infections from around the world will comprise a unique global resource.

Many gene and domain families have convergent fates following independent whole-genome duplication events in Arabidopsis, Oryza, Saccharomyces and Tetraodon.

Genome duplication is potentially a good source of new genes, but such genes take time to evolve. We have found a group of "duplication-resistant" genes, which have undergone convergent restoration to singleton status following several independent genome duplications. Restoration of duplication-resistant genes to singleton status could be important to long-term survival of a polyploid lineage. Angiosperms show more frequent polyploidization and a higher degree of duplicate gene preservation than other paleopolyploids, making them well-suited to further study of duplication-resistant genes.

O-glycosylation in Toxoplasma gondii: identification and analysis of a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases.

The initiation of mucin-type O-glycosylation is catalysed by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (EC 2.4.1.41). These enzymes are responsible for the transfer of N-acetylgalactosamine from the nucleotide sugar donor, UDP-GalNAc, to the hydroxyl group on specific serine or threonine residues in acceptor proteins. By screening a Toxoplasma gondii cDNA library, three distinct isoforms of the ppGalNAc-T gene family were cloned. Two additional isoforms were identified and partially cloned following analysis of the T. gondii genome sequence database. All of the cloned and identified ppGalNAc-T's are type II membrane proteins that share up to 50% amino acid sequence identity within the conserved catalytic domain. They each contain an N-terminal cytoplasmic domain, a hydrophobic transmembrane domain, and a lumenal domain; the latter consists of stem, catalytic, and lectin-like domains. Moreover, each of this ppGalNAc-T's contains important sequence motifs that are typical for this class of glycosyltransferases. These include a glycosyltransferase 1 motif containing the DXH sequence, a Gal/GalNAc-T motif, and the CLD and QXW sequence motifs located in alpha-, beta-, and gamma-repeats present within the lectin-like domain. The coding regions of T. gondii ppGalNAc-T1, -T2, and -T3 reside in multiple exons ranging in number from 6 to 10. Our results demonstrate that mucin-type O-glycosylation in T. gondii is catalysed by a multimember gene family, which is evolutionarily conserved from single-celled eukaryotes through nematodes and insects up to mammals. Taken together, this information creates the basis for future studies of the function of the ppGalNAc-T gene family in the pathobiology of this apicomplexan parasite.

Analysis of the first and second internal transcribed spacer sequences of the ribosomal DNA in Biomphalaria tenagophila complex (Mollusca: Planorbidae)

The first and second internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal DNA of Biomphalaria tenagophila complex (B. tenagophila, B. occidentalis, and B. t. guaibensis) were sequenced and compared. The alignment lengths of these regions were about 655 bp and 481 bp, respectively. Phylogenetic relationships among the Biomphalaria species were inferred by Maximum Parsimony and Neighbor-joining methods. The phylogenetic trees produced, in most of the cases, were in accordance with morphological systematics and other molecular data previously obtained by polymerase chain reaction and restriction fragment length polymorphism analysis. The present results provide support for the proposal that B. tenagophila represents a complex comprising B. tenagophila, B. occidentalis and B. t. guaibensis.

Gene transfer in the evolution of parasite nucleotide biosynthesis.

Nucleotide metabolic pathways provide numerous successful targets for antiparasitic chemotherapy, but the human pathogen Cryptosporidium parvum thus far has proved extraordinarily refractory to classical treatments. Given the importance of this protist as an opportunistic pathogen afflicting immunosuppressed individuals, effective treatments are urgently needed. The genome sequence of C. parvum is approaching completion, and we have used this resource to critically assess nucleotide biosynthesis as a target in C. parvum. Genomic analysis indicates that this parasite is entirely dependent on salvage from the host for its purines and pyrimidines. Metabolic pathway reconstruction and experimental validation in the laboratory further suggest that the loss of pyrimidine de novo synthesis is compensated for by possession of three salvage enzymes. Two of these, uridine kinase-uracil phosphoribosyltransferase and thymidine kinase, are unique to C. parvum within the phylum Apicomplexa. Phylogenetic analysis suggests horizontal gene transfer of thymidine kinase from a proteobacterium. We further show that the purine metabolism in C. parvum follows a highly streamlined pathway. Salvage of adenosine provides C. parvum's sole source of purines. This renders the parasite susceptible to inhibition of inosine monophosphate dehydrogenase, the rate-limiting enzyme in the multistep conversion of AMP to GMP. The inosine 5' monophosphate dehydrogenase inhibitors ribavirin and mycophenolic acid, which are already in clinical use, show pronounced anticryptosporidial activity. Taken together, these data help to explain why widely used drugs fail in the treatment of cryptosporidiosis and suggest more promising targets.