No description available.

No description available.

No description available.

No description available.

Cryptosporidium spp., Giardia lamblia and Entamoeba histolytica are the most frequently identified enteric protozoa in water-borne disease outbreaks. Many PCR assays, with satisfactory results in terms of sensitivities and specificities, have been developed for detection of the aforementioned three protozoa in faecal specimens but the majority of these assays have a limited usage in the clinical laboratories due to being more costly and more time-consuming than the conventional diagnostic methods. Based on published oligonucleotide primers, three individual uniplex PCR assays were developed, properly optimised and subsequently combined into a conventional multiplex PCR format to screen for the three protozoa in the same stool specimen. The multiplex PCR assay was clinically validated with 185 control and 212 randomly selected stool samples. The assay was optimised with DNA directly retrieved from stool samples using a modified QIAamp® DNA Stool Mini Kit (Qiagen) DNA extraction protocol subsequent amplification using single-round well-controlled PCR protocol. Like the individual PCRs, the multiplex PCR assay detected genomic DNA from control isolates matching 12, 12 and four copies of the Cryptosporidium, G. lamblia and E. histolytica genomes, respectively. Similarly, ~100 (oo)cysts per 200μl stool were successfully identified by the multiplex and the matching uniplex-PCR assays as detection limits. The diagnostic sensitivity, specificity, negative predictive value and positive predictive value of the multiplex and the individual PCR assays were comparable and equal to 97 %, 100 %, 95 % and 100 %, respectively. Furthermore, by nominating three nested PCRs as 'gold standards', the multiplex PCR demonstrated specificity and sensitivity exceeding that achieved by the combined copro-antigen immunoassay adopted for Cryptosporidium/Giardia diagnosis at the Clinical Microbiology laboratory, Leicester Royal Infirmary, University Hospital of Leicester. In conclusion, the newly developed multiplex PCR was demonstrated to be a simple, cost-effective, adequately sensitive and highly specific assay. An assay with this broad-spectrum format has a great potential to be adopted as a routine test in diagnostic laboratories especially those in resource-poor countries where parasitic protozoal infections are endemic.

Giardia intestinalis is a protozoan parasite that causes the gastrointestinal disease giardiasis, one of the most frequent parasitic infections worldwide. Giardia was for many years considered to be a primitive or early diverging eukaryote because it lacks organelles typically present in eukaryotes such as classical mitochondria, nucleoli and peroxisomes. On the other hand, it has a cytoskeleton, an endomembrane system and subcellular protein sorting functions of higher cells, which suggest it is a complex and highly developed cell. It has recently been shown that two giardial mitochondrial- related proteins, the IscU and IscS, eo-localize inside a double-membrane organelle of mitochondrial origin, the mitosome. This thesis presents evidence, from in vivo and in vitro protein import studies, that Giardia mitosomes also harbor another mitochondrion-related protein, ferredoxin, and that despite their advanced state of reductive evolution, mitosomes have retained saturable presequence-dependent and presequence-independent protein import pathways analogous to those that operate in mammalian mitochondria. Giardia also encodes a Fe-hydrogenase gene, even though it lacks recognizable hydrogenosomes. The cytosolic localization of the Fe- hydrogenase observed in this study allows the conclusion that the remnant organelles of Giardia are not hydrogenosomes, but mitosomes. The development of an in vitro protein import assay using highly enriched intact mitosomes from Giardia trophozoites and the radiolabeled precursor Fd protein is also reported. The purification method devised in this study yielded a highly enriched, intact mitosomal population competent for protein import. The mitosomal protein import assay demonstrated the proteolytic removal of the targeting peptide of Fd upon organelle import, which is a functional feature of the mitochondrial and hydrogenosomal protein import systems. These results are strong evidence that G. intestinalis possess a mitosomal protein import machinery similar to that of mitochondria and hydrogenosomes, providing further evidence that these three organelles arose from a common endosymbiont.

Metabolite transport pathways of the malaria parasite, Plasmodium falciparum, are an important area for study in order to further the understanding of the parasite's biology. Identification and characterisation of the transporters involved in these pathways may also provide potential novel drug targets or drug delivery mechanisms. This is especially valuable as chemotherapy remains one of the main management strategies in the fight against malaria and the usefulness 0 f the current range 0 f antimalarial drugs is seriously threatened by the emergence and spread of resistance . .' In this thesis the Xenopus laevis oocyte heterologous expression system was used to functionally characterise a gene-specific cDNA library of 48 putative membrane proteins and the previously annotated putative amino acid transporter PFF1430c for the uptake of several amino acids. This screening failed to identify any definite amino acid transport by the cDNA library or PFF1430c, however this could have been due to the fact that uptake of a relatively narrow range of amino acids was tested and these were used at concentrations lower than found physiologically. Inherent issues with the X laevis expression system may also have been an issue, including the expression of endogenous transporters for the substrates being investigated.

No description available.

Eukaryote cells are the products of a complex history of interspecies interactions, with some organelles now known to have arisen through the endosymbiosis of prokaryotic cells. Whilst these organelles 'evolved' from the original endosymbionts, their evolution has not stopped within the modern eukaryote. Trichomonas vaginalis is protozoan parasite, with an unusual cellular biology, this species appears to lack peroxisomes, and instead of mitochondria has divergent organelles called hydrogenosomes. The hydrogenosomes of Trichomonas represent one of a growing number of highly divergent organelles, which are present in species throughout the eukaryotic kingdom. In this investigation the hydrogenosomes of Trichomonas are investigated with respect to their preprotein membrane translocases, a multi-membraned molecular system essential for the organelle's maintenance and biogenesis. The complexity and components of such a system are unlikely to arise duplicated from a separate organellogenesis, and thus the system's architecture is expected to indicate this organelle's descent. To reveal the structure of this system two different practical approaches were used to determine the biology of the hydrogenosome. The first builds upon work to characterise translocase kinetics and probes the nature of the hydrogenosomal membrane translocon directly. The second explores the use of candidate translocases determined from the Trichomonas genome through bioinformatic analysis and their development into practical investigation through the expression of tagged proteins in transformant T. vaginalis. These transformants were used to visualise a population of membrane proteins in situ within the hydrogenosome by immunofluorescence microscopy, and further to identify their associations and interactions within the hydrogenosomal membrane using protein biochemical methods. The data produced within this study are finally brought together to present a model for the Trichomonas hydrogenosomal preprotein import system, as well as the first molecular characterization of its translocase components.

Selective delivery of drugs to parasites via plasma membrane transporters offers an effective approach to specifically target pathogens. Using biochemical techniques, we have identified and characterised a number of purine nucleobase and nucleoside transporters in protozoan parasites such as Leishmania major, Leishmania mexicana, Trypanosoma brucei and Toxoplasma gondii. In one study, the uptake of [3H]adenine, [3H]hypoxanthine, and [3H]allopurinol, and antileishmanial hypoxanthine analogue, by Leishmania major promastigotes was investigated. The results showed that these compounds were all taken up by a single high-affinity transporter, LmajNBT1, with Km values of 4.6±0.9, 0.71±0.07, and 54±3 μM and Vmax­ values of 3.2±0.3, 2.8±0.6, and 0.24±0.06 pmol (107 cells)-1s-1, respectively. [3H]adenine transport was fully inhibited by the natural purines guanine and xanthine, with Ki values of 2.8±0.7 and 23±8 μM, respectively. Using purine analogues, an extensive inhibitor profile for LmajNBT1 was obtained, which allowed the construction of a quantitative model for the interactions between the transporter binding site and the permeant. The model predicts that hypoxanthine was bound through hydrogen bonds to N(1)H, N3, N7, and N(9)H of the purine ring, with a total Gibbs free energy of -39.5 kJ/mol. The interactions with adenine were similar, except for a weak hydrogen bond to N1 (unprotonated in adenine). The predicted model of substrate binding for LmajNBT1 was almost identical to that for the Trypanosoma brucei H2 (TbH2) transporter. It is proposed that the architecture of their respective binding sites is very similar and that LmajNBT1 can be named a functional homolog of TbH2. This thesis also reports the first identification and characterization of a purine nucleobase transporter in Leishmania amastigotes. Uptake of [3H]hypoxanthine by Leishmania mexicana amastigotes was mediated by a single high-affinity transporter, LmexNBT1, with Km and Vmax values of 1.6±0.4μM and 0.092 ± 0.057 pmol (107 cells)-1s-1 respectively, with high affinity for adenine, guanine, and xanthine, with Ki values of 4.2±0.8, 1.7±0.1, and 13±2 μM respectively, but low affinity for nucleosides and pyrimidine nucleobases. Allopurinol was apparently taken up by the same transporter (Km of 33.6±6.0 μM). All evidence was compatible with a model of a single purine nucleobase transporter being expressed in amastigotes. Using various purine nucleobase analogues, a model for the interactions between hypoxanthine and the transporter's permeant binding site was constructed and compared with previously obtained models for substrate recognition by nucleobase transporters, and found to be very similar to the models of the LmajNBT1 and TbH2 transporters, but markedly different from the human FNT1 transporter.