No description available.

No description available.

No description available.

This work consists of a study of the general biology, morphology and ultrastructure, feeding and digestion of a species of Collinia.

No description available.

Pyrimidine uptake has previously been investigated in Trypanosoma brucei procyclics and partly investigated in promastigotes of Leishmania major; however, no such study has been performed using bloodstream forms of Trypanosoma or promastigotes of Leishmania. Here we report a comprehensive study of pyrimidine salvage and metabolism in bloodstream forms of Trypanosoma and promastigotes of Leishmania species. In T. b. brucei bloodstream forms, the uptake of 3H-uracil and 3H-tymidine each appeared to be mediated by a single transporter, designated TbU3 and TbT1, respectively. The procyclic uracil transporter,TbU1, has a high affinity for uracil, with a Km value of 0.46 ± 0.09 μM and Vmax of 0.65 ± 0.008 pmol (107cell)-1 s-1. These values were similar for TbU3 (Km = 0.54 ± 0.11 µM; Vmax = 0.14 ± 0.03), but the main differences between TbU1 and TbU3 are their sensitivity to uridine and 4-thiouracil. Thymidine uptake is detectable at 10 μM over a period from 5 to 30 minutes. This uptake was not inhibited by uracil which indicates that TbT1 is a novel thymidine transporter. The uptake of other pyrimidines, including uridine and 2’-deoxyuridine, by BSF are investigated here but these substrates were also transported by TbU3, and no additional pyrimidine transport activities were found. In L. mexicana and L. major, the uptake of 3H-uracil and 3H-uridine was mediated by separate transporters, designated as follows; for uracil uptake LmexU1, LmajU1; and for uridine uptake LmexNT1, LmajNT1 and LmajNT2, respectively. LmexU1 is a uracil transporter with high affinity to uridine and 2’deoxyuridine, and the LmexNT1 is a nucleoside transporter with broad specificity for purine and pyrimidine nucleosides. L. major uracil transporter (LmajU1) has already been reported by others; and here we report that there are also two distinct uridine transporters expressed in L. major. LmajNT1 is a high affinity uridine transporter which is also inhibited by uracil, inosine and adenine; LmajNT2 is low affinity uridine transporter, with very poor affinity for uracil, inosine and adenine. However, both transporters are inhibited by 2’-deoxyuridine, thymidine and adenosine. Several fluorinated pyrimidine analogues were assessed against kinetoplastid cells, the most effective compounds, which displayed EC50 values at micromolar level, are 5-FU, 5F-2’dUrd, 5-FOA (only against T. brucei BSF) and 5F-Urd (only against L. major). We induced resistance to 5-FU, 5-F2’dUrd and 5-FOA by in vitro exposure of Tbb-BSF and promastigotes of L. mexicana and L. major. The resistance was performed by stepwise increase concentration of the drugs. For T. b. brucei BSF, the resistance factors of clonal lines were 131, 825, and 83-fold, respectively. For L. mexicana and L. major, the resistance factor for 5-FU were 147 and 17-fold, and for 5F-2’dUrd were >3500 and 381-fold, respectively. We also measured 3H–pyrimidine uptake in these cell lines; the resistant bloodstream form strains showed no changes in pyrimidine uptake, with one exception, which is a 76% reduction in 5-FU uptake. In contrast, each resistant strain of Leishmania spp had lost its natural pyrimidine transporter. For example, Leishmania cells resistant to 5-FU had lost uracil transport activity, and cells that were resistant to 5F-2’dUrd had lost uridine transport activity. In addition, we identified kinetoplastid genes that appeared to be associated with resistance to fluorinated pyrimidines. Based on these findings, metabolomic analysis of fluorinated pyrimidines in T. b. brucei resistant cell lines was performed in comparison with parental wild-type; for Leishmania species we only investigated the metabolism of fluorinated pyrimidine in wild type cells, as the fluorinated analogues were simply not taken up in the resistant clones. The metabolomic analysis data showed that, in T. b. brucei, 5-fluorouracil and 5-fluoro orotate are incorporated into a large number of metabolites, but likely act through incorporation into RNA. 5F-2’dUrd and 5F-2’dCtd are not incorporated into nucleic acids but act as prodrugs by inhibiting thymidylate synthase after conversion to 5F-dUMP. Cells treated with 5-fluoro-2’deoxyuridine showed an increase of dUMP, which suggest a block in thymidylate synthase or possibly thymidylate kinase. We also present the most complete model of pyrimidine salvage in T. brucei to date, supported by genome-wide profiling of the predicted pyrimidine biosynthesis and conversion enzymes. The effect of fluorinated pyrimidine analogues in the two Leishmania species was almost identical. Each of the tested drugs (5-FU, 5F-2’dUrd and 5F-Urd) produced a limited number of fluorinated metabolites, and their common mode of action was inhibition in thymidylate synthase by 5F-dUMP and thymidine kinase by 5F-2’dUrd. Interestingly, we found that the cause of L. mexicana resistance to 5F-Urd was due to the absence of the 5F-2’dUrd metabolite, as a result of the rapid conversion of 5F-2’dUrd to 5F-dUMP; also we suggest that, in L. mexicana, but not in L. major the high affinity salvage of thymidine is sufficient to provide the cells with thymidine deoxynucleotides. It has been found that pyrimidine salvage is not an essential function for Leishmania cells in vitro conditions. However, it is not known whether either, pyrimidine salvage or biosynthesis, or both of these systems are essential to the trypanosomes in vitro and in vivo study. As T. b. brucei bloodstream forms grew unimpeded in vitro in the complete absence of pyrimidines, uptake is clearly not essential. Disruption of the pyrimidine biosynthesis pathway by deletion of the OMPDCase/OPRTase gene resulted in pyrimidine auxotrophic trypanosomes that were unable to grow in the absence of added pyrimidines. The phenotype was rescued by addition of uracil, and to a lesser extent by some pyrimidine nucleosides. Pyrimidine starvation led rapidly to DNA fragmentation. Adaptations to low pyrimidine availability included upregulation of uracil transport capacity and of uridine phosphorylase expression. However, pyrimidine auxotrophic T. brucei were able to establish a high parasitemia in mice. We therefore conclude that pyrimidine salvage was not an essential function for bloodstream T. b. brucei. However, trypanosomes lacking de novo pyrimidine biosynthesis are completely dependent on an extracellular pyrimidine source, strongly preferring uracil, and display reduced infectivity and strongly increased sensitivity to fluorinated pyrimidines. As T. brucei are able to salvage sufficient pyrimidines from the host environment, the pyrimidine biosynthesis pathway is not a viable drug target, although any interruption of pyrimidine supply was lethal.

La phase érythrocytaire du cycle de Plasmodium est caractérisée par une hémolyse importante et la libération d’hème chez l’hôte. Grâce à ses activités pro-oxydantes et immunomodulatrices, l’hème contribuerait au dysfonctionnement de la réponse immune observée durant le paludisme grave. Ainsi, ce travail de thèse avait pour but de clarifier le rôle de l’hème sur la réponse des lymphocytes T et des cellules gliales au cours du neuropaludisme et de l’anémie palustre sévère dans des modèles murins de paludisme. De plus, nous avons étendu nos études au paludisme chez des patients infectés par P. falciparum manifestant différents phénotypes cliniques. D‘après nos résultats l’hème diminue la réponse délétère de type 1 des lymphocytes T CD4 et des microglies chez les modèles murins. Alors que cette réponse est essentielle à la clairance du parasite, le prétraitement des souris BALB/c avec l’hème induit une parasitémie plus faible, en comparaison au groupe non traité, qui est également associée à une anémie liée à une dysérythropoïèse. Cependant, l’injection d’hème chez des souris C57BL/6 pendant l’infection les protège du neuropaludisme en diminuant le recrutement des lymphocytes T, la réponse de type M1 des microglies et la production de cytokines pro-inflammatoires dans le cerveau. Par l’étude réalisée dans l’Odisha en Inde nous avons corroboré l’ensemble de ces résultats avec des données cliniques, et montrons que les niveaux plasmatiques d’hème augmentent chez les patients atteints de neuropaludisme et corrèlent positivement avec les cytokines IP-10, IL-10, TNF-alpha et MCP-1. Nous soulignons dans notre étude la complexité de la physiopathologie du paludisme grave. / During the erythrocytic phase of the parasite Plasmodium, large amounts of heme are release in circulation. Due to its pro-oxidant, and immunomodulating activities, heme might be responsible for the alterations of the immune response that were reported during severe malaria. Thus, the objectives of this work were to clarify the impact of heme on the response of T lymphocytes and glial cells in rodent models of severe malarial anemia, and cerebral malaria. In addition, we have also looked at the relationship between heme levels and malaria severity in a human cohort of P. falciparum-infected patients manifesting mild and severe malaria. Heme decreased the pro-inflammatory type 1 response of CD4 T lymphocytes and microglia cells in rodent models. Despite the essential role of this response for the elimination of the parasite, BALB/c mice that were preconditioned with heme before infection had lower parasitemia compared to the untreated group. However, heme induced stronger anemia during the infection that was concomitant with alterations in erythropoiesis. In addition, heme injection during infection protected partly C57BL/6 mice from developing cerebral malaria by preventing T cell recruitment to the brain, the M1 response of microglia cells, and the production of pro-inflammatory cytokines by brain cells. Interestingly, we show from our field study in Odisha, India, that plasma heme levels increased with the severity of malaria, and more specifically during cerebral malaria, and correlated positively with the plasma levels of four cytokines: IP-10, IL-10, TNF-alpha and MCP-1. Herein we underscore the complexity of the pathophysiology of severe malaria.

The present study investigates Acanthamoeba-bacteria interaction and how this relation can influence human health aiming at the influence of bacteria on Acanthamoeba in terms of their isolation and diversity, and the effect of Acanthamoeba on bacteria focusing on two emerging human bacterial pathogens Arcobacter butzleri and Rhodococcus equi. To first objective was investigated by the test question “can the presence of a particular type of bacteria play role in the diversity of Acanthamoeba by masking and/or favouring certain genotypes of Acanthamoeba?” To answer this, two different bacteria the Gram+ve Enterococcus the Gram-ve Arcobacter were used as food source for isolation of Acanthamoeba from 102 soil samples while E. coli was used as control. It was found that the presence of different bacteria could affect the isolation of genotypes specially the subgroups and subtypes of Acanthamoeba as manifested by greater diversity of 18S rRNA sequences of Acanthamoeba isolated from environmental samples on Arcobacter (Arc) and Enterococcus (Ent) than those isolated on E. coli (Eco). The Eco isolates consisted of only T4 > T11=T13 compared to Ent isolates with T4 > T16 > T13/16 and the Arc isolates which comprised of T4 > T2 > T2/6 = T13 > T13/16. The T13/16 were the intermediate sequence types with no match to any T types. There were also considerable differences among the T4 subgroups; the Eco isolates consisted of T4-A > T4-B > T4-N > T4- E > T4-D > T4-C while Ent isolates comprised of T4-A > T4-C=T4-D=T4-E=T4-N > T4-B and the Arc isolates had only T4-E > T4-A > T4-B > T4-N. In both Eco and Ent isolates 11 subtypes were recovered with T4-36 being the most abundant, however, in Arc isolates eight subtypes were recovered with T4-12 as the most abundant. The non-Eco isolates were also different in their bacterial endosymiotic profile from Eco isolates with Arc isolates having the greatest proportion of bacterial endosymbionts (15.7%) as compared to 7.8% of Eco and 12.9% of Ent isolates. Together these results indicate a prominent role of prey bacteria on favouring certain genotypes and thus compelling consideration for use of different types of bacteria for isolation of Acanthamoeba to help surface the masked populations as well for more realistic prevalence that will help in better designing of prevention and control strategies. The influence of Acanthamoeba on bacteria was investigated for A. butzleri and R. equi both of which appeared to exploit the former as an environmental reservoir and for modulation of their pathogenic potential. A. butzleri which are closely related to Campylobacter, appeared to have a smooth interaction with Acanthamoeba. They were shown to be easily located through chemotaxis, readily attached and internalized using monosaccharide receptors and a complex phagocytic process, and could survive/proliferate in Acanthamoeba by defying the intra-vacuolar killing processes. Intracellular survival in Acanthamoeba did play a role in promoting the pathogenicity of these bacteria enabling them to survive more than three times longer. Co-culturing of the two organisms also seemed to benefit the bacteria but not Acanthamoeba. A. butzleri were found to be able to sense the environmental changes and thus modulate their virulence, a feature that together with selection pressure for intracellular survival in Acanthamoeba can cause rapid adaptation to intra-amoebal environment and enhance the pathogenic potential of these bacteria for humans and animals. Exploitation of Acanthamoeba for survival was also found to be exhibited by the Mycobacterium-resembling Gram+ve R. equi by utilizing similar strategies for survival/proliferation as used for macrophages, which involved the definite presence of virulence plasmid and its activation at higher temperatures. Moreover, similar genes (vapA, vapC and vapF) were found to play role in intracellular survival in both the macrophages and amoeba cells. The intra-amoebal survival/proliferation capabilities of A. butzleri and R. equi appear to support the notion that free living protists like Acanthamoeba act as environmental reservoirs/virulence trait selectors and are strong candidates for the “missing link” between the ecology and pathology of these emerging pathogenic pathogens. Overall, the observations made in this study explore the vital role of Acanthamoeba-bacteria interaction not only mutually on each other but as a consequence the impact on human health either as a result of masked genotypes in clinical diagnosis of Acanthamoeba or due to environmental reservoir role of Acanthamoeba in selecting virulence traits of bacteria, can pose serious challenges leaving ample opportunities for more emerging bacterial pathogens. These observations call for revising the protocols for Acanthamoeba prevalence, eradication and control strategies.

The energetics of, the holotrich ciliate Colpidium campylum fed on the bacterium Moraxella sp. at 10°C, 15°C and 20°C were investigated. The parameter used for ascertaining growth. was the volume of protoplasm produced measured by means of a Coulter Counter with a mean cell volume converter attachment. Growth and consumption were measured in relation to food availability as indicated by the ratio of bacteria: protozoan. Mean cell volume variation and reproduction were also measured in relation to food availability and energy consumed. Protozoan and bacterial material were harvested by centrifugation and freeze-dried for dried weight determinations and calorimetry studies. The energy content of Colpidium and its food source was determined with a Phillipson microbomb calorimeter. Respiration was measured in a Warburg respirometer. Oxygen uptake in relation to population density and cell size was considered as well as the production of information concerning the heat lost during respiration for incorporation into energy budgets. Energy budgets of. two types were constructed: a 24-hour energy budget for an individual and the life-span or generation energy budget for an individual. Gross growth efficiencies, net growth efficiencies and assimilation efficiencies were considered in detail. In addition to laboratory work the vertical distribution of Ciliophora in the mud of Loch Leven, Kinross, Scotland, was also considered; three sites, two shallow and one deep, being sampled with a core sampler over a 12 month period.

Apusozoa (Protozoa) is a phylum of heterotrophic gliding zooflagellates of unknown taxonomic affiliation, commonly observed in environmental samples. Almost nothing was previously known about the diversity and ecology of apusozoan species though, as bacterivores, they are probably important functional constituents within microbial assemblages. We explored apusozoan morphological and genetic diversity, ecology, and related methodological questions. By culturing environmental material from a range of habitats, we isolated and maintained monocultures of both previously described apusozoan orders, Apusomonadida (apusomonads) and Planomonadida (planomonads). For planomonads, we present a revised taxonomy based on morphology, ultrastructure, and 18S rDNA genetic differences. We describe nine new species and new genera Nutomonas and Fabomonas, and demonstrate ITS2 rDNA secondary structure analysis for species delineation. During our culturing effort, we also isolated two genotypes of a previously unknown flagellate group, shown here to belong to a novel third apusozoan order, Mantamonadida. We designed molecular probes specific to all three orders and applied them to environmental DNA, detecting novel 18S and ITS1 rDNA lineages in a range of habitats. We mined publically available metagenomic and metatranscriptomic sequence databases using 18S rDNA of described species as seeds, identifying hundreds of sequences with affinities to all three orders. Phylogenies featuring newly retrieved lineages with previously described species suggest that direct sequencing of transcriptomic material is more effective than amplification-dependent methods at detecting rare cells in mixed microbial assemblages. Finally, to test potential future applications of our newly isolated strains, we ran microcosm experiments examining the effect of protozoan (Cercozoa) grazing on the structure of bacterial assemblages, demonstrating that closely related and morphologically similar species can have different impacts on their prey base. Taken together, by combining traditional culturing and modern molecular methods, this thesis drastically improves our understanding of apusozoan diversity and sets the scene for future work using next-generation sequencing and ecologically driven functional experiments.