CSGID Solves Structures and Identifies Phenotypes for Five Enzymes in Toxoplasma gondii

The subcellular distribution of enzymes of the oxidative pentose phosphate pathway was studied in plants. Root and leaf tissues from several species were separated by differential centrifugation into plastidic and cytosolic fractions. In all tissues studied, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were found in both plastidic and cytosolic compartments. In maize and pea root, and spinach and pea leaf, the non-oxidative enzymes of the pentose phosphate pathway (transaldolase, transketolase, ribose 5-phosphate isomerase, ribulose 5-phosphate 3-epimerase) appear to be restricted to the plastid. In tobacco root and leaf, however, the non-oxidative enzymes were found in the cytosolic as well as the plastidic compartments. In the absence of ribose 5-phosphate isomerase and ribulose 5-phosphate 3-epimerase in the cytosol, the product of the oxidative limb of the pathway (ribulose 5-phosphate) must be transported into a compartment capable of utilizing it. Ribulose 5-phosphate was supplied to isolated intact pea root plastids and was shown to be capable of supporting nitrite reduction. The kinetics of ribulose 5-phosphate-driven nitrite reduction in isolated pea root plastids suggested that the metabolite was translocated across the plastid envelope in a carrier-mediated transport process, indicating the presence of a translocator capable of transporting pentose phosphates.

The diagnostic and therapeutic approach to respiratory disease in the immunocompromised host remains a challenge for several reasons: (1) the current increase in both the number of immunocompromised hosts and...

Methods for the quantitative determination of ribose 5-phosphate isomerase, ribulose 5-phosphate 3-epimerase, transketolase and transaldolase in tissue extracts are described. The determinations depend on the measurement of glyceraldehyde 3-phosphate by using the coupled system triose phosphate isomerase, alpha-glycero-phosphate dehydrogenase and NADH. By using additional purified enzymes transketolase, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase conditions could be arranged so that each enzyme in turn was made rate-limiting in the overall system. Transaldolase was measured with fructose 6-phosphate and erythrose 4-phosphate as substrates, and again glyceraldehyde 3-phosphate was measured by using the same coupled system. Measurements of the activities of the non-oxidative reactions of the pentose phosphate pathway were made in a variety of tissues and the values compared with those of the two oxidative steps catalysed by glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase.

The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man(5-8)(GlcNAc)2) and paucimannosidic (Man(3-4)(GlcNAc)2) 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 followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii.

Apicomplexan parasites, such as Toxoplasma gondii, have specific adaptations that enable invasion and exit from the host cell. Owing to the phylogenetic distance between apicomplexan parasites and model organisms, comparative genomics has limited capacity to infer gene functions. Further, although CRISPR/Cas9-based screens have assigned roles to some Toxoplasma genes, the functions of encoded proteins have proven difficult to assign. To overcome this problem, we devised a conditional Cas9-system in T. gondii that enables phenotypic screens. Using an indicator strain for F-actin dynamics and apicoplast segregation, we screened 320 genes to identify those required for defined steps in the asexual life cycle. The detailed characterization of two genes identified in our screen, through the generation of conditional knockout parasites using the DiCre-system, revealed that signalling linking factor (SLF) is an integral part of a signalling complex required for early induction of egress, and a novel conoid protein (conoid gliding protein, CGP) functions late during egress and is required for the activation of gliding motility. Establishing different indicator lines and applying our conditional Cas9 screen could enable the identification of genes involved in organellar biogenesis, parasite replication or maintenance of the endosymbiotic organelles in the future.

Post-translational modifications of microtubules regulate their stability and dynamics. Acetylation of tubulin at lysine 40 (K40) by -acetyltransferase (TAT) occurs on the luminal side of microtubules, stabilizes their structure, and plays essential roles in various cellular processes across eukaryotes. Apicomplexan parasites include the malaria-causing Plasmodium species and Toxoplasma gondii, both of which possess unusually stable subpellicular microtubules, a set of cytoskeletal filaments underlying the parasite’s inner membrane complex. Interestingly, while Toxoplasma gondii and human-infecting Plasmodium species retain both K40 and TAT, rodent-infecting Plasmodium species have lost TAT, and K40 has been replaced by glutamine (Q40), a residue that can mimic acetylated lysine. Here, we investigate the role of microtubule acetylation in apicomplexan parasites by generating and characterizing genetic mutants in Plasmodium berghei and Toxoplasma gondii. In Plasmodium berghei, introduction of a Q40K mutation in 1 tubulin did not affect parasite development or infectivity, suggesting that the absence of K40 acetylation is not detrimental. In Toxoplasma gondii, we confirmed that TAT is responsible for microtubule acetylation but, contrary to previous reports, its deletion had no impact on parasite growth in vitro. Together, these results indicate that luminal K40 acetylation is not essential for microtubule function in either species, pointing to functional redundancy and highlighting the plasticity of cytoskeletal regulation in apicomplexan parasites.

Apicomplexan parasites like Toxoplasma gondii are distinctive in their utilization of para site encoded motor systems to invade cells. Invasion results in the establishment of the parasitophorous vacuole (PV) within the infected cell. Most apicomplexans complete their intracellular tenure within the infected cell in the PV that is demarcated from the host cytoplasm by the parasitophorous vacuole membrane (PVM). In this chapter I focus on the events surrounding the formation of the PVM and selected activities attributed to it. Its central role as the interface between the parasite and its immediate environment, the host cytoplasm, is validated by the diversity of functions attributed to it. While functions in structural organization, nutrient acquisitions and signaling have been defined their molecular bases remain largely unknown. Several recent studies and the decoding of the Toxoplasma genome have set the stage for a rapid expansion in our understanding of the role of the PVM in parasite biology. Toxoplasma gondii, like all apicomplexan parasites are obligate intracellular pathogens. This family of parasites utilize their own actin-myosin based motor systems to gain entry into susceptible cells establishing themselves, in some cases transiently (e.g., Theileria spp) in specialized vacuolar compartment, the parasitophorous vacuole (PV). The T. gondii PV is highly dynamic compartment defining the replication permissive niche for the parasite. The delimiting membrane defining the parasitophorous vacuole, the parasitophorous vacuole membrane or PVM is increasingly being recognized as a specialized "organelle" that in the context of the infected cell is extracorporeal to the parent organism, the parasite. A systematic study of this enigmatic organelle has been severely limited by several issues. Primary among these is the fact that it is formed only in the context of the infected cell thereby limiting the amount of material. Secondly, unlike other cellular organelles that can often be purified by conventional approaches, the PVM, cannot be purified away from host cell organelles (see below). In spite of these significant obstacles considerable progress has been made in recent years toward understanding the biogenesis of the PVM, identification of its protein complement and the characterization of activities within it. These studies demonstrate that the PVM, on its own and by virtue of its interactions with cellular components, plays critical functions in the structural integrity of the vacuole, nutrient acquisition and the manipulation of cellular functions. In addition it appears that the repertoire of activities at the PVM is likely to be plastic reflecting temporal changes associated with the replicative phase of parasite growth. Finally, the PVM likely forms the foundation for the cyst wall as the parasite differentiates in the establishment of latent infection. As the critical border crossing between the parasite and invaded cell the study of the PVM provides a fertile area for new investigation aided by the recent decoding of the Toxoplasma genome (available at wwww.ToxoDB.org) and the application of proteomic analyses to basic questions in parasite biology.

Bovine neosporosis is a disease of concern due to its global distribution and significant economic impact through massive losses in the dairy and meat industries. To date, there is no effective chemotherapeutic drug or vaccine to prevent neosporosis. Control of this disease is therefore dependent on efficient detection tests that may affect treatment management strategies. This study was conducted to identify the specific immunoreactive proteins of Neospora caninum tachyzoites recognised by sera from cattle infected with N. caninum, Toxoplasma gondii, Cryptosporidium parvum, Babesia bovis and B. bigemina, and by sera from uninfected cattle using two-DE dimensional gel electrophoresis (2-DE) combined with immunoblot and mass spectrometry (LC-MS/MS). Among 70 protein spots that reacted with all infected sera, 20 specific antigenic spots corresponding to 14 different antigenic proteins were recognised by N. caninum-positive sera. Of these immunoreactive antigens, proteins involved in cell proliferation and invasion process were highly immunogenic, including HSP90-like protein, putative microneme 4 (MIC4), actin, elongation factor 1-alpha and armadillo/beta-catenin-like repeat-containing protein. Interestingly, we discovered an unnamed protein product, rhoptry protein (ROP1), possessing strong immunoreactivity against N. caninum but with no data on function available. Moreover, we identified cross-reactive antigens among these apicomplexan parasites, especially N. caninum, T. gondii and C. parvum. Neospora caninum-specific immunodominant proteins were identified for immunodiagnosis and vaccine development. The cross-reactive antigens could be evaluated as potential common vaccine candidates or drug targets to control the diseases caused by these apicomplexan protozoan parasites.

The apicomplexan parasite Cryptosporidium represents a major public health problem in humans and animals by causing self-limited diarrhea in immunocompetent individuals and life-threatening disease in immunocompromised hosts [...].

We have used drugs to examine the role(s) of the actin and microtubule cytoskeletons in the intracellular growth and replication of the intracellular protozoan parasite, Toxoplasma gondii. By using a 5 minute infection period and adding the drugs shortly after entry we can treat parasites at the start of intracellular development and 6-8 hours prior to the onset of daughter cell budding. Using this approach we found, somewhat surprisingly, that reagents that perturb the actin cytoskeleton in different ways (cytochalasin D, latrunculin A and jasplakinolide) had little effect on parasite replication although they had the expected effects on the host cells. These actin inhibitors did, however, disrupt the orderly turnover of the mother cell organelles leading to the formation of a large residual body at the posterior end of each pair of budding parasites. Treating established parasite cultures with the actin inhibitors blocked ionophore-induced egression of tachyzoites from the host cells, demonstrating that intracellular parasites were susceptible to the effects of these inhibitors. In contrast, the anti-microtubule drugs oryzalin and taxol, and to a much lesser extent nocodazole, which affect microtubule dynamics in different ways, blocked parasite replication by disrupting the normal assembly of the apical conoid and the microtubule inner membrane complex (IMC) in the budding daughter parasites. Centrosome replication and assembly of intranuclear spindles, however, occurred normally. Thus, daughter cell budding per se is dependent primarily on the parasite microtubule system and does not require a dynamic actin cytoskeleton, although disruption of actin dynamics causes problems in the turnover of parasite organelles.

Limonene, a terpenoid compound found in various plants such as oranges, lemons, and mint, has numerous applications in different industrial fields, including as a fragrance, flavoring agent, and biofuel. Conventional limonene production relies on agricultural outputs vulnerable to fluctuations caused by diseases or climate change. Therefore, metabolic engineering using microorganisms offers an interesting alternative for more efficient and sustainable limonene production. This review article aims to summarize the procedures for limonene production through the modification of the pentose phosphate (PP) and methylerythritol 4-phosphate (MEP) biosynthetic pathways in the cyanobacterium Synechocystis sp. PCC 6803 to produce limonene. Limonene synthase (lims) obtained from C. limon and M. spicata plants was cloned and transformed into cyanobacteria to enhance limonene production. Experimental results showed that genes associated with the limonene biosynthesis pathway, including ribose 5-phosphate isomerase (rpi), ribulose 5-phosphate 3-epimerase (rpe), and geranyl diphosphate synthase (gpps), were successfully expressed in Synechocystis. This study demonstrates that Synechocystis can be an efficient microbial system for limonene and other isoprene compound production, offering a more stable and environmentally friendly alternative than agricultural-based production.

Apicomplexan parasites, including the human pathogens Toxoplasma gondii and Plasmodium falciparum, employ specialized secretory organelles (micronemes, rhoptries, dense granules) to invade and survive within host cells. Because molecules secreted from these organelles function at the host/parasite interface, their identification is important for understanding invasion mechanisms, and central to the development of therapeutic strategies. Using a computational approach based on predicted functional domains, we have identified more than 600 candidate secretory organelle proteins in twelve apicomplexan parasites. Expression in transgenic T. gondii of eight proteins identified in silico confirms that all enter into the secretory pathway, and seven target to apical organelles associated with invasion. An in silico approach intended to identify possible host interacting proteins yields a dataset enriched in secretory/transmembrane proteins, including most of the antigens known to be engaged by apicomplexan parasites during infection. These domain pattern and projected interactome approaches significantly expand the repertoire of proteins that may be involved in host parasite interactions.

In biology, models are experimental systems meant to recreate aspects of diseases or human tissue with the goal of generating inferences and approximations that can contribute to the resolution of specific biological problems. Although there are many models for studying intracellular parasites, their data have produced critical contradictions, especially in immunological assays. Peripheral blood mononuclear cells (PBMCs) represent an attractive tissue source in pharmacogenomics and in molecular and immunologic studies, as these cells are easily collected from patients and can serve as sentinel tissue for monitoring physiological perturbations due to disease. However, these cells are a very sensitive model due to variables such as temperature, type of stimulus and time of collection as part of posterior processes. PBMCs have been used to study Toxoplasma gondii and other apicomplexan parasites. For instance, this model is frequently used in new therapies or vaccines that use peptides or recombinant proteins derived from the parasite. The immune response to T. gondii is highly variable, so it may be necessary to refine this cellular model. This mini review highlights the major approaches in which PBMCs are used as a model of study for T. gondii and other apicomplexan parasites. The variables related to this model have significant implications for data interpretation and conclusions related to host-parasite interaction.

Plasmodium spp. and Toxoplasma gondii are important members of the apicomplexan phylum, collectively they pose a considerable threat to human health and result in worldwide economic losses. Apicomplexan parasites have evolved an array of specialised organelles which they exploit to enable their parasitic lifestyle. The apical micronemes are critical for motility, invasion and egress. In this thesis, we identify and investigate multiple proteins contributing to microneme secretion, acting at the level of signalling for exocytosis or towards the apical replenishment of micronemes. Apicomplexan integral membrane proteins of the Major Facilitator Superfamily (MFS) have been found to be critically involved in nutrient uptake, organelle biogenesis and maturation. In light of these results, we have performed a restricted screen of MFSs in T. gondii. Combining both conditional genetic depletion with untargeted metabolomic analysis we investigate the proteins role and the nature of the metabolites transported.

The aim of this study was to utilise wild rabbits (Oryctolagus cuniculus) as a sentinel species to study levels of environmental contamination with N. caninum and T. gondii in South Australia, and to examine associations with rainfall, climate and land use. Toxoplasma gondii (T. gondii), an apicomplexan parasite, causes the clinical disease toxoplasmosis, which can affect sheep as well as humans and many other animals. Neosporosis, the clinical disease caused by closely related Neospora caninum (N. caninum), causes abortions in cattle, with large economic impacts to cattle industries. Aliquots of wild rabbit (Oryctolagus cuniculus) serum were obtained from twelve sites across South Australia over a period of eighteen years, with a total of 2114 samples. An in-house Modified Agglutination Test (MAT) was developed, and samples were screened for the specific antibodies against both T.gondii and N. caninum. Overall, 9.9% of samples screened for T. gondii tested positive and 6.1% of samples screened for N. caninum tested positive. There was no difference observed in seroprevalence of T.gondii specific antibodies between samples collected throughout summer, autumn, winter or spring. By contrast, a significantly higher (p=0.030) seroprevalence of N. caninum specific antibodies was observed in spring than any other season. T. gondii and N. caninum antibodies were discovered at sites across a broad area of South Australia, indicating these environments maybe infected with both parasites. Results provide evidence that suggests N. caninum oocysts may have different survival characteristics, such as varying tolerances to low relative humidity, than T. gondii oocysts.