The process of antigenic variation in African trypanosomes allows the survival of the parasite by constantly switching the variant surface glycoprotein (VSG) expressed in their surface. There are believed to be several hundred copies of these silent VSG genes in the parasite's genome and they are expressed differentially. The majority of these genes are not capable of being transcribed in situ and must therefore be expressed from specialised transcriptional units known as bloodstream expression sites (BESs). Only one such site is active at any one time, ensuring that a single VSG is expressed in the trypanosome's surface coat. Switching the expressed VSG involves replacing the VSG in the active BES, or activating a new BES in conjunction with silencing the previously active. Differential expression of variant surface glycoprotein (VSG) genes, has a strong association with telomeres. All BESs are telomeric and differential activation involves recombination into the telomeric environment or silencing/activation of subtelomeric promoters. A number of pathogen contingency gene systems associated with immune evasion involve telomeric loci, which has prompted speculation that chromosome ends provide conditions conducive for the operation of rapid gene switching mechanisms. Ku is a protein associated with yeast telomeres that is directly involved in DNA recombination and gene silencing. The main aim of this thesis was to test the hypothesis that Ku in trypanosomes is centrally involved in differential VSG expression. In order to compare trypanosome Ku homologues with those from other organisms, it was necessary to compile homology alignments with other Ku homologues using Clustal W analysis. Subsequent experiments looked at the fate of exogenously introduced restriction enzyme target sites after transient transformation with cassettes encoding the restriction enzyme. A final analysis looked for the presence of NHEJ in homologous recombination- deficient trypanosomes. Disrupting this element of DNA repair would hopefully lead to other forms of repair becoming detectable, and even up-regulated. Rad51, in yeast a member of the Rad52 epistasis group (integral in yeast homologous recombination), had previously been demonstrated to be involved in DNA repair in trypanosomes (McCulloch & Barry, 1999). rad51 mutants were electroporated with cassettes containing noncompatible ends that would prevent their integration into the endogenous genome via conventional homologous recombination. This cassette also contained promoter DNA sequence to allow selection in the event of integration into non-transcribed regions of the genome. Study of the junctions encompassing the integration sites of the cassette allowed investigation into how the cassettes were integrated, and revealed to us the extent of the sequence homology required to catalyse integration. The method of repair detection observed indicated that classical homologous recombination is not the only pathway utilised by African trypanosomes to metabolise DNA double-strand breaks.

The unicellular eukaryote Trypanosoma brucei causes African Sleeping sickness and multiplies extracellularly in the bloodstream of the infected host. The parasite evades antibody-mediated lysis by switching its Variant Surface Glycoprotein (VSG) coat. Blocking VSG synthesis results in an abrupt growth inhibition and a precise pre-cytokinesis cell cycle arrest, with an accumulation of cells with two nuclei and two kinetoplasts. Additionally, induction of VSG RNAi triggers a global block in translation, which is not due to a general decrease in transcript levels. The mechanism behind this translation arrest was investigated. It was observed that it correlated with a decrease in polysomes, indicating that translation was blocked at the level of initiation. It was also shown that the VSG RNAi-triggered growth inhibition was reversible, which suggests that this is not a lethal phenotype. The VSG221 RNAi-induced growth arrest could be alleviated if a second different VSG (VSG117), which was not recognised by the VSG221 RNAi, was expressed immediately downstream of the promoter of the active VSG221 Expression site. Further, it was possible to delete the telomeric VSG221 in these VSG double-expressors, leaving the cells completely reliant on the second complementing VSG117 gene. VSG117 expressed from a promoter-adjacent position in the active Expression site was shown to form a functional surface coat that protected the parasites from complement-mediated lysis in vitro. Transiently transfecting cells with anti-VSG221 morpholino oligonucleotides allowed us to specifically block translation of VSG221 mRNA without degrading it. This resulted in a pre-cytokinesis cell cycle arrest similar to that induced by VSG221 RNAi. This indicates that the VSG RNAi-triggered growth inhibition was due to a lack of VSG protein or its synthesis rather than the ablation of the abundant VSG mRNA. In addition, it was shown that blocking VSG synthesis reduced the rate of surface VSG internalisation in cells that were stalled precytokinesis, but had no effect on other endocytic markers. These experiments give us further insight into the importance of the protective VSG coat for pathogenicity in T. brucei.

Trypanosoma brucei is a protozoan parasite that is the causative agent of sleeping sickness in sub-Saharan Africa. T. brucei has a complex life cycle involving passage between a mammalian host and the tsetse fly. The parasite evades the mammalian immune system via expression of Variant Surface Glycoprotein (VSG) on the cell surface. VSG genes are expressed at telomeric expression sites and at these sites are a number of Expression Site Associated Genes (ESAGs). One unusual ESAG, ESAG9, is developmentally regulated: RNA for these genes accumulates during the transition from slender to stumpy cells in the mammalian bloodstream and cellassociated protein is only detected transiently in stumpy and differentiating cells. Transgenic cell lines were generated which ectopically express one or more members of the ESAG9 gene family. Biochemical and cytological analyses using these cell lines indicated that some members of this family are glycosylated and GPI-anchored, and also that one gene, ESAG9-K69, is secreted. ESAG9-K69 is also secreted by wild-type stumpy parasites. In vivo experiments with tsetse flies did not conclusively show whether ESAG9 proteins play a role in the establishment of a tsetse fly mid-gut infection by transgenic trypanosomes. However, In vivo and ex vivo experiments using the mouse model of trypanosomiasis indicated that expression of ESAG9 proteins may alter parasitaemia in the mouse and results in a significant decrease in the proportion of CD4+ T cells in the mouse spleen.

<p><I> Trypanosoma congolense </i> is a single-cell blood parasite and an important pathogen causing African trypanosomiasis, also called ngana, in livestock. Ngana in cattle is a chronic disease associated with anemia, cachexia and increased susceptibility to secondary infections. Infection of mice can be used as an experimental model to study the host-parasite relationship. As determined by their survival time, BALB/c mice are highly susceptible to <i> T. congolense </i> infection, whereas C57BL/6 mice are relatively resistant. The surfaces of African trypanosomes are covered with a layer of a single species of glycoprotein, called variant surface glycoprotein (VSG). Production of antibodies to the VSG of African trypanosomes is one of the major immune responses leading to control of parasitemia. The reaction of antibodies with VSG of trypanosomes, for presently unknown reasons, predominantly activates the alternative complement pathway rather than the classical pathway of complement. IgM antibodies are the first and predominant class of anti-trypanosomal antibodies in infected animals. Antibody-mediated phagocytosis of <i> T. congolense </i> by macrophages is considered a major mechanism of control of parasitemia, besides antibody/complement-mediated lysis and cytotoxic effect by macrophage-derived nitric oxide (NO). The receptor(s) on macrophages that recognizes IgM antibody-coated trypanosomes and enables their phagocytosis is unknown. Interaction of antibodies with the VSG of trypanosomes not only causes phagocytosis of trypanosomes by macrophages, but also leads to the release of sVSG from the trypanosomes. sVSG has been found to modulate various functions of the host: induction of polyclonal B cell activation and modulation of macrophage functions, such as the induction of TNF-á synthesis and the inhibition of IFN-ã-induced nitric oxide production. The objectives of this thesis are:</p> <p> 1) to test whether CR3 (Mac-1; CD11b/18) is involved in IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i> by macrophages </p> <p> 2) to test the effects of anti-VSG antibody and complement on the release of soluble VSG from <i> T. congolense </i> </p> <p>1) When the trypanosomes were incubated with IgM anti-VSG antibody and fresh mouse serum, fragments of complement component C3 were found to be deposited onto <i> Trypanosoma congolense </i>. Thus, it was assessed whether complement receptor CR3 (CD11b/CD18; receptor for iC3b) might be involved in IgM anti-VSG mediated phagocytosis of <i> T. congolense </i>. In the presence of fresh mouse serum, there was significantly and markedly less phagocytosis of IgM-opsonized <i> T. congolense </i> by CD11b-deficient macrophages compared to phagocytosis by normal macrophages (78% fewer <i> T. congolense </i> were ingested per macrophage). There also was significantly less TNF-á (38% less), but significantly more NO (63% more) secreted by CD11b-deficient macrophages that had engulfed trypanosomes than by equally treated normal macrophages. It was concluded that CR3 is the major, but not the only, receptor involved in IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i> by macrophages. It was further concluded that signaling via CR3, associated with IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i>, either directly or indirectly, enhances synthesis of disease-producing TNF-á and inhibits the synthesis of parasite-controlling NO.</p> <p> 2) This investigation revealed that there was more sVSG released from <i> T. congolense </i> by interaction with IgM anti-VSG than by interaction with equal amounts of IgG2a anti-VSG. The release of sVSG occurred in an antibody dose-dependent pattern. It was also found that IgM anti-VSG, after interacting with the surface of <i> T. congolense </i>, formed soluble immune complexes with released sVSG. The results also showed that antibody-induced release of sVSG can occur without complement, but is enhanced by complement. It was further tested whether fresh sera from either relatively resistant C57BL/6 mice or highly susceptible BALB/c mice, which differ in their complement cascade, had different effects on the release of sVSG from <i> T. congolense </i>. The results showed that antibody-induced shedding of sVSG was higher in the presence of fresh C57BL/6 serum than in the presence of fresh BALB/c serum. All these data suggest that the concentration of anti-VSG antibody, antibody class and source of complement can affect the release of sVSG from <i> T. congolense </i></p>.

<p><I> Trypanosoma congolense </i> is a single-cell blood parasite and an important pathogen causing African trypanosomiasis, also called ngana, in livestock. Ngana in cattle is a chronic disease associated with anemia, cachexia and increased susceptibility to secondary infections. Infection of mice can be used as an experimental model to study the host-parasite relationship. As determined by their survival time, BALB/c mice are highly susceptible to <i> T. congolense </i> infection, whereas C57BL/6 mice are relatively resistant. The surfaces of African trypanosomes are covered with a layer of a single species of glycoprotein, called variant surface glycoprotein (VSG). Production of antibodies to the VSG of African trypanosomes is one of the major immune responses leading to control of parasitemia. The reaction of antibodies with VSG of trypanosomes, for presently unknown reasons, predominantly activates the alternative complement pathway rather than the classical pathway of complement. IgM antibodies are the first and predominant class of anti-trypanosomal antibodies in infected animals. Antibody-mediated phagocytosis of <i> T. congolense </i> by macrophages is considered a major mechanism of control of parasitemia, besides antibody/complement-mediated lysis and cytotoxic effect by macrophage-derived nitric oxide (NO). The receptor(s) on macrophages that recognizes IgM antibody-coated trypanosomes and enables their phagocytosis is unknown. Interaction of antibodies with the VSG of trypanosomes not only causes phagocytosis of trypanosomes by macrophages, but also leads to the release of sVSG from the trypanosomes. sVSG has been found to modulate various functions of the host: induction of polyclonal B cell activation and modulation of macrophage functions, such as the induction of TNF-á synthesis and the inhibition of IFN-ã-induced nitric oxide production. The objectives of this thesis are:</p> <p> 1) to test whether CR3 (Mac-1; CD11b/18) is involved in IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i> by macrophages </p> <p> 2) to test the effects of anti-VSG antibody and complement on the release of soluble VSG from <i> T. congolense </i> </p> <p>1) When the trypanosomes were incubated with IgM anti-VSG antibody and fresh mouse serum, fragments of complement component C3 were found to be deposited onto <i> Trypanosoma congolense </i>. Thus, it was assessed whether complement receptor CR3 (CD11b/CD18; receptor for iC3b) might be involved in IgM anti-VSG mediated phagocytosis of <i> T. congolense </i>. In the presence of fresh mouse serum, there was significantly and markedly less phagocytosis of IgM-opsonized <i> T. congolense </i> by CD11b-deficient macrophages compared to phagocytosis by normal macrophages (78% fewer <i> T. congolense </i> were ingested per macrophage). There also was significantly less TNF-á (38% less), but significantly more NO (63% more) secreted by CD11b-deficient macrophages that had engulfed trypanosomes than by equally treated normal macrophages. It was concluded that CR3 is the major, but not the only, receptor involved in IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i> by macrophages. It was further concluded that signaling via CR3, associated with IgM anti-VSG-mediated phagocytosis of <i> T. congolense </i>, either directly or indirectly, enhances synthesis of disease-producing TNF-á and inhibits the synthesis of parasite-controlling NO.</p> <p> 2) This investigation revealed that there was more sVSG released from <i> T. congolense </i> by interaction with IgM anti-VSG than by interaction with equal amounts of IgG2a anti-VSG. The release of sVSG occurred in an antibody dose-dependent pattern. It was also found that IgM anti-VSG, after interacting with the surface of <i> T. congolense </i>, formed soluble immune complexes with released sVSG. The results also showed that antibody-induced release of sVSG can occur without complement, but is enhanced by complement. It was further tested whether fresh sera from either relatively resistant C57BL/6 mice or highly susceptible BALB/c mice, which differ in their complement cascade, had different effects on the release of sVSG from <i> T. congolense </i>. The results showed that antibody-induced shedding of sVSG was higher in the presence of fresh C57BL/6 serum than in the presence of fresh BALB/c serum. All these data suggest that the concentration of anti-VSG antibody, antibody class and source of complement can affect the release of sVSG from <i> T. congolense </i></p>.

No description available.