How to detect Toxoplasma gondii oocysts in environmental samples?

Detection of Toxoplasma gondii oocysts in environmental samples is a great challenge for researchers as this coccidian parasite can be responsible for severe infections in humans and in animals via ingestion of a single oocyst from contaminated water, soil, fruits or vegetables. Despite field investigations, oocysts have been rarely recovered from the environment due to the lack of sensitive methods. Immunomagnetic separation, fluorescence-activated cell sorting, and polymerase chain reaction have recently shown promising use in detection of protozoa from complex matrices. Such procedures could be applied to T. gondii detection, if studies on the antigenic and biochemical composition of the oocyst wall are completed. Using such methods, it will be possible to assess the occurrence, prevalence, viability and virulence of T. gondii oocysts in environmental matrices and specify sources of human and animal contamination.     

Chaperoning prions: the cellular machinery for propagating an infectious protein?

Newly made polypeptide chains require the help of molecular chaperones not only to rapidly reach their final three-dimensional forms, but also to unfold and then correctly refold them back to their biologically active form should they misfold. Most prions are an unusual type of protein that can exist in one of two stable conformations, one of which leads to formation of an infectious alternatively folded form. Studies in Baker's yeast (Saccharomyces cerevisiae) have revealed that prions can exploit the molecular chaperone machinery in the cell in order to ensure stable propagation of the infectious, aggregation-prone form. The disaggregation of yeast prion aggregates by molecular chaperones generates forms of the prion protein that can seed the protein polymerisation that underlies the prion propagation cycle. In this article, we review what we have learnt about the role of molecular chaperones in yeast prion propagation, describe a model that can explain the role of various classes of molecular chaperones and their co-chaperones, and speculate on the possible involvement of chaperones in the propagation of mammalian prions.     

Toxoplasma gondii and sex: essential or optional extra?

The evolutionary and biological significance of a female-biased sex ratio within apicomplexan parasites has been the subject of much discussion. It is proposed that the sex allocation theory, as applied to inbreeding populations, can explain the sex ratios observed for this diverse group of parasites. This is based on a mathematical model, which assumes that the majority of microgametes will succeed in fertilizing macrogametes. Is this a realistic assumption? It is possible, for different reasons, that the theory may not to be applicable to either malaria parasites or Toxoplasma gondii.     

The prevalence of Toxoplasma gondii in polar bears and their marine mammal prey: evidence for a marine transmission pathway?

Little is known about the prevalence of the parasite Toxoplasma gondii in the arctic marine food chain of Svalbard, Norway. In this study, plasma samples were analyzed for T. gondii antibodies using a direct agglutination test. Antibody prevalence was 45.6% among polar bears (Ursus maritimus), 18.7% among ringed seals (Pusa hispida) and 66.7% among adult bearded seals (Erignathus barbatus) from Svalbard, but no sign of antibodies were found in bearded seal pups, harbour seals (Phoca vitulina), white whales (Delphinapterus leucas) or narwhals (Monodon monoceros) from the same area. Prevalence was significantly higher in male polar bears (52.3%) compared with females (39.3%), likely due to dietary differences between the sexes. Compared to an earlier study, T. gondii prevalence in polar bears has doubled in the past decade. Consistently, an earlier study on ringed seals did not detect T. gondii. The high recent prevalence in polar bears, ringed seals and bearded seals could be caused by an increase in the number or survivorship of oocysts being transported via the North Atlantic Current to Svalbard from southern latitudes. Warmer water temperatures have led to influxes of temperate marine invertebrate filter-feeders that could be vectors for oocysts and warmer water is also likely to favour higher survivorship of oocycts. However, a more diverse than normal array of migratory birds in the Archipelago recently, as well as a marked increase in cruise-ship and other human traffic are also potential sources of T. gondii.     

The general secretory pathway: a general misnomer?

The term general secretory pathway (GSP) has been usurped and misused in the literature over the past few years. The concept of GSP is discussed from an historical perspective, and the definitions of the general export pathway (GEP), the main terminal branch (MTB) of the GSP, the unified GSP nomenclature and the type II, IV and V secretion pathways are also described to show how they have fuelled the confusion. By putting the record straight and using novel findings within the field of bacterial protein secretion, we hope to bring clarity to this area of science and prevent further promulgation of incorrect terminologies.     

Toxin entry: how reversible is the secretory pathway?

A number of proteins produced by plants and bacteria are extremely toxic to eukaryotic cells. Their potency arises from their ability to catalyse the modification of crucial cellular components. Only a few toxin molecules are required to kill a cell, but to do so they must first reach the cytosol. How such proteins are translocated across the target cell membrane is poorly understood, but we argue here that some toxins may travel the secretory pathway in reverse, passing all the way from the cell surface to the endoplasmic reticulum (ER) before entering the cytosol.     

How old are the extant lineages of Toxoplasma gondii?

Most known isolates of Toxoplasma gondii belong to one of only three lineages, which are presumed to be clonal. Three models have been proposed for the evolutionary relationship of these lineages to the other extant lineages: Model (a) proposing that all lineages are derived from a most recent common ancestor (MRCA) in the distant past, Model (b) that all lineages are derived from a MRCA in the very recent past, and Model (c) that the clonal lineages share a recent MRCA but are related to the other lineages only in the distant past. Here, I test these models using DNA intron and coding-sequence data for loci at 14 genes, using three different methods to calculate the time of the MRCA. All of the calculations agree that the MRCA of the clonal lineages was > 70% of the age of the MRCA of all lineages, thus favouring Model (a). The MRCA may have existed approximately 150,000 years ago, with the clonal lineages expanding in prevalence approximately 10,000 years ago.     

Are residues in a protein folding nucleus evolutionarily conserved?

Protein is the working molecule of the cell, and evolution is the hallmark of life. It is important to understand how protein folding and evolution influence each other. Several studies correlating experimental measurement of residue participation in folding nucleus and sequence conservation have reached different conclusions. These studies are based on assessment of sequence conservation at folding nucleus sites using entropy or relative entropy measurement derived from multiple sequence alignment. Here we report analysis of conservation of folding nucleus using an evolutionary model alternative to entropy-based approaches. We employ a continuous time Markov model of codon substitution to distinguish mutation fixed by evolution and mutation fixed by chance. This model takes into account bias in codon frequency, bias-favoring transition over transversion, as well as explicit phylogenetic information. We measure selection pressure using the ratio omega of synonymous versus non-synonymous substitution at individual residue site. The omega-values are estimated using the PAML method, a maximum-likelihood estimator. Our results show that there is little correlation between the extent of kinetic participation in protein folding nucleus as measured by experimental phi-value and selection pressure as measured by omega-value. In addition, two randomization tests failed to show that folding nucleus residues are significantly more conserved than the whole protein, or the median omega value of all residues in the protein. These results suggest that at the level of codon substitution, there is no indication that folding nucleus residues are significantly more conserved than other residues. We further reconstruct candidate ancestral residues of the folding nucleus and suggest possible test tube mutation studies for testing folding behavior of ancient folding nucleus.     

Do neuroglobin and myoglobin protect Toxoplasma gondii from nitrosative stress?

Toxoplasma gondii is a Apicomplexa obligate intracellular protozoan parasite that infects up to a third of the world's population. In most humans infected with T. gondii, the disease toxoplasmosis is asymptomatic. However, T. gondii causes blindness, severe neurological disorders, hepatitis, and pneumonia in immunocompromised patients, and severe damage to the fetus. Here, we postulate that the colonization of the retina, heart, and skeletal muscle by T. gondii may reflect the role of neuroglobin (Ngb) and myoglobin (Mb) to protect the parasite from the toxoplasmacidal effects of nitric oxide (NO). This is based on the knowledge that Ngb and Mb catalyzes NO oxidation yielding the harmless nitrate. The postulated protective role of Ngb and Mb on the viability of T. gondii is reminiscent of that postulated for hemoglobin (Hb) and Mb in protecting intraerythrocytic Plasmodia and T. cruzi in cardiomyocytes, respectively, from the parasiticidal effect of NO. Therefore, undesirable pathogen protection by pseudo-enzymatic NO scavenging may represent a new unexpected function of members of the Hb superfamily.     

An evolving paradigm for the secretory pathway?

The paradigm that the secretory pathway consists of a stable endoplasmic reticulum and Golgi apparatus, using discrete transport vesicles to exchange their contents, gained important support from groundbreaking biochemical and genetic studies during the 1980s. However, the subsequent development of new imaging technologies with green fluorescent protein introduced data on dynamic processes not fully accounted for by the paradigm. As a result, we may be seeing an example of how a paradigm is evolving to account for the results of new technologies and their new ways of describing cellular processes.     

Do protozoa conceal a high potency of nucleoside triphosphate hydrolysis present in Toxoplasma gondii?

ATP hydrolytic activity in whole cell homogenates of some protozoa was assayed in the presence or absence of dithiothreitol. The activities in all protozoan cell homogenates, except Toxoplasma gondii, ranged from 0.6 to 32 mumol/mg protein/hr, irrespective of the presence or absence of dithiothreitol. A remarkably higher activity, 11,690 mumol/mg protein/hr, was observed for T. gondii in the presence of dithiothreitol. These results indicate that the higher ATP hydrolytic potency observed for T. gondii is not universal to protozoa, rather it is unique to T. gondii.     

The cell cycle and Toxoplasma gondii cell division: tightly knit or loosely stitched?

The flexibility displayed by apicomplexan parasites to vary their mode of replication has intrigued biologists since their discovery by electron microscopy in the 1960s and 1970s. Starting in the 1990s we began to understand the cell biology of the cytoskeleton elements driving cytokinesis. By contrast, the molecular mechanisms that regulate the various division modes and how they translate into the budding process that uniquely characterizes this parasite family are much less understood. Although growth mechanisms are a neglected area of study, it is an important pathogenic parameter as fast division rounds are associated with fulminant infection whereas slower growth attenuates virulence, as is exploited in some vaccine strains. In this review we summarize a recent body of cell biological experiments that are rapidly leading to an understanding of the events that yield successful mitosis and cytokinesis in Toxoplasma. We place these observations within a cell cycle context with comments on how these events may be regulated by known eukaryotic checkpoints active in fission and budding yeasts as well as mammalian cells. The presence of cell cycle control mechanisms in the Apicomplexa is supported by our findings that identify several cell cycle checkpoints in Toxoplasma. The progress of the cell cycle is ultimately controlled by cyclin-Cdk pair activities, which are present throughout the Apicomplexa. Although many of the known controllers of cyclin-Cdk activity are present, several key controls cannot readily be identified, suggesting that apicomplexan parasites deviate at these points from the higher eukaryotic models. Altogether, new insights in Toxoplasma replication are reciprocally applied to hypothesize how other division modes in the Toxoplasma life cycle and in other Apicomplexa species could be controlled in terms of cell cycle checkpoint regulation.     

Metabolic pathway compartmentalization: an underappreciated opportunity?

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