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Studies on a multicatalytic, protease complex from Trypanosoma brucei brucei.Lomo, Peter Onyimbo. 20 December 2013 (has links)
Subcellular fractionation (together with immunocytochemical localisation studies) showed that
the parasite Trypanosoma brucei brucei possesses a multicatalytic protease complex (MCPTb).
This complex is predominantly cytosolic but some activity is also present in the nuclear
fraction. MCP-Tb was isolated from T. b. brucei and compared to the properties of other
proteasomes reported in the literature and to the 20S MCP isolated from bovine red blood cells
(MCP-rbc). The isolation procedure employed four-steps: anion exchange chromatography on
Q-Sepharose, adsorption chromatography on HA-Ultrogel, molecular exclusion
chromatography on Sephacryl S-300 and glycerol density gradient sedimentation.
The molecular mass of intact MCP-Tb was shown to be smaller than that of MCP-rbc.
Separation of the different proteasome subunits by 2D-PAGE showed that MCP-Tb has 12
different polypeptide components compared to the 28 different polypeptide components of
MCP-rbc. The N-terminal sequence of an MCP-Tb subunit showed that this subunit did not
have any obvious sequence homology with the subunits of proteasomes from other cells.
Furthermore, anti-MCP-Tb antibodies (which exhibited the in vitro inhibitory activity of
MCP-Tb) did not cross-react with MCP-rbc showing that MCP-Tb and MCP-rbc are antigenically distinct.
The basic enzymatic properties of MCP-Tb were fairly typical of other 20S proteasomes.
MCP-Tb had multiple peptidase activities (identified as chymotrypsin-like, trypsin-like and
peptidyl glutamylpeptide hydrolase activities) that are characteristic of proteasomes.
Furthermore, the characteristics of inhibition by a variety of inhibitors were similar to those of
other proteasomes, including MCP-rbc. The activities of 20S proteasomes from most cell
types are activated by endogenous high molecular mass complexes such as the bovine 19S
complex called PA700. These complexes form end-on associations with the 20S proteasome.
However, no endogenous MCP-activator was found in T. b. brucei. Nevertheless, MCP-Tb
was activated in an ATP-dependent manner by bovine PA700. Inhibition of the intrinsic
phosphatase activity of PA700 inhibited the protease enhancing effect of PA700. Electron microscopic examination of negatively stained MCP-Tb and MCP-rbc showed
particles that were morphologically indistinguishable. However, the MCP-Tb also exhibited
unique end-on associations between individual units forming long (up to 200 nm) ribbon-like
chains. Since access to the active sites of proteasomes occurs through the pores at the end of the complexes, this end-on association, when coupled to our observation of an apparent lack of an endogenous activator, suggests that T. b. brucei may have evolved an alternate mechanism
for controlling their proteasome activity. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1999.
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Trypanopain : a possible target for anti-trypanosomal agents?Troeberg, Linda. January 1997 (has links)
The protozoan parasite Trypanosoma brucei brucei causes nagana in cattle and is a widely used
model for human sleeping sickness. The major lysosomal cysteine proteinases (trypanopains) of
African trypanosomes may contribute to pathogenesis by degrading proteins in the mammalian
bloodstream and also appear to be essential for the viability of T. cruzi and T. congolense. This
study describes the first purification to electrophoretic homogeneity of trypanopain-Tb from
T. b. brucei and the first reported characterisation of its enzymatic properties. Trypanopain-Tb
was purified from bloodstream forms of T. b. brucei by a combination of three phase
partitioning (between ammonium sulfate and tertiary butanol), and chromatography on
quaternary amine or pepstatin A-Sepharose resins.
Trypanopain-Tb was found to be a typical cysteine proteinase, in that it is inhibited by typical
cysteine proteinase inhibitors and requires reducing agents for full activity. Trypanopain has
cathepsin L-like specificity for synthetic substrates and readily degrades various proteins.
In vitro analysis of the kinetics of trypanopain interaction with cystatins suggested that these are
likely to inhibit any trypanopain released into the mammalian bloodstream. Furthermore, no
trypanopain-like activity was detectable in the blood of infected hosts, so it appears that
trypanopain is unlikely to contribute directly to pathogenesis by degrading bloodstream host
proteins.
Antibodies against a peptide corresponding to a region of the trypanopain active site were
produced in rabbits and chickens. Both enzyme activity-enhancing and enzyme activity inhibiting
antibodies were produced and these effects varied with the substrate tested. Thus, the
in vivo effects of anti-trypanopain antibodies will only become clearly understood once the
physiological substrates of trypanopain have been identified.
Various cysteine proteinase inhibitors, including peptidyl diazomethylketones, killed cultured
bloodstream forms of T. b. brucei. Use of biotinylated derivatives of peptidyl
diazomethylketone and fluoromethylketone inhibitors suggested that trypanopain is the likely
intracellular target of these inhibitors, indicating that the enzyme is essential for parasite
viability. Furthermore, chalcones (a class of reversible cysteine proteinase inhibitors) killed in
vitro cultured parasites and also prolonged the life of T. b. brucei-infected mice. Thus,
trypanopain-Tb seems to be a possible target for new anti-trypanosomal drugs. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1997.
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P15 trypanosome microtubule associated protein : structure/function analysis and vaccine development for the prevention of African sleeping sickness.Rasooly, Reuven. January 2001 (has links)
Trypanosomes are hemoflagellated protozoan parasites causing chagas disease in South
America, Leishmaniasis throughout the world, and African sleeping sickness in humans
and nagana in animals in Africa. About 55 million people and 25 million cattle have been
estimated to be at risk of contracting African sleeping sickness or nagana respectively.
Once injected into the blood stream via the bite of a tsetse fly, the parasite evades the
host's immune response by repeatedly changing its surface antigens, thus making the
development of a vaccine seem impossible. Furthermore, chemotherapy existing today can
be toxic, suggesting that novel methods to prevent diseases caused by trypanosomes are
essential.
All parasites of the Trypanosomatidae family contain unique microtubular structures called
the subpellicular microtubules. Microtubules are made of tubulin and of microtubule
associated proteins (MAPs). Unlike other microtubules, the subpellicular microtubules are
crosslinked to one another and to the plasma membrane. The unique structure of the
subpellicular microtubules has been attributed to unique trypanosome subpellicular MAPs
which stabilize the microtubule polymers and crosslink them to one another.
Three unique types of subpellicular MAPs have been identified: MARP, which is a high
molecular mass MAP that stabilizes microtubules, p52 that is a 52kDa MAP which
crosslinks microtubules, and pI5, which is a I5kDa protein which bundles microtubules.
Because trypanosome MAPs have been shown to be unique to these parasites, these
molecules could serve as useful target sites for therapy. In this study pI5 was cloned and
sequenced and shown to contain highly organized, nearly identical tandem repeats with a
periodicity of 10 amino acids, rich in positively charged and in hydrophobic amino acids.
It was shown that pI5 can also bind phospholipids, suggesting that it may not only
bundle the microtubule polymer through its positively charged amino acids but may also
crosslink the microtubules to the plasma membrane through its hydrophobic regions, thus
contributing to the stable structure of the subpellicular microtubules.
To test for the efficiency of pI5 as a vaccine candidate, the recombinant pI5 was cloned
into an adenovirus, which was used as a vaccine delivery system for pI5. Mice were
vaccinated with the native purified pI5, with the expressed recombinant pI5 and with the
adenovirus containing the recombinant pI5 gene (Ad-pI5). The results indicated that pI5
protected 100% of the animals vaccinated with the recombinant molecule (8/8), and 87%
of the animals vaccinated with the native protein (7/S), while none of the control animals
were protected. Animals that were vaccinated with the Ad-pI5 were protected but so were
the control animals vaccinated with an adenovirus containing the lacZ gene. We have
shown that vaccination with the adenovirus is associated with an elevated CDS+ T cell
response which is known to be trypanostatic (S6), suggesting that animals vaccinated with
Ad-pIS may have been protected not only by the specific anti-plS response but also by
non specific immunity that was induced by the adenovirus itself.
The source of the native and recombinant pI5 was from a different strain of T. brucei that
was used for challenge. Since the subpellicular microtubules are common to all members
of the Trypanosomatidae family, pI5 may ultimately serve as a common target for therapy
to all types of diseases caused by trypanosomes. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2001.
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Epitope mapping of a trypanosomal cysteine proteinase.Mkhize, Pamela Phumelele. 28 November 2013 (has links)
Trypanosomosis is a parasitic disease in man, domestic and wild animals and is of major
economic importance in many parts of the world, particularly in Sub-Saharan Africa.
Trypanosoma congolense, T vivax and T brucei brucei are the major pathogenic
trypanosomes infecting cattle in sub-Saharan Africa. The parasite itself is not directly
responsible for the disease, but rather causes illness through the release of pathogenic factors.
One of the major pathogenic factors released by trypanosomes is proteinases.
Trypanotolerant cattle produce antibodies against a trypanosomal proteinase, congopain, that
inhibit congopain activity. Congopain thus has vaccine potential. This study describes the
mapping of immunogenic epitopes of congopain to identify peptide regions of the protein that
induce enzyme inhibitory antibodies for inclusion in a trypanosome vaccine. This vaccine
approach targets the disease, rather than the parasite by focusing on a pathogenic factor. These
peptides also have potential for use in diagnostic assays. Peptides from the catalytic domain of
a trypanosomal cysteine proteinase, congopain, were selected using an epitope prediction
program. Peptides selected were from the two forms of congopain called CP1 and CP2.
Antibodies against peptide-carrier conjugates were produced in chickens. The antibodies
recognised native congopain, recombinant CP2 and the recombinant catalytic domain (C2).
This suggests that the peptides selected have promise for use in vaccines.
The peptides were also used to determine whether they are natural immunogenic epitopes of
CP2 and thus have potential for use in diagnostic assays. Antibodies in the sera from T.
congolense infected cattle recognised all the peptides in an ELISA. Antibodies in the sera
from C2-immunised, non-infected cattle recognised most of the peptides in an ELISA. In
order to distinguish between T. congolense and T vivax infection, two different peptides from
the C-terminal extensions of CP2 and vivapain were used in ELISA tests with sera from
infected cattle. Although anti-peptide antibodies produced against the two C-terminal
extension peptides were specific for their respective peptides, thereby indicating the
discriminatory power of the peptides selected, there was cross-reactivity by the sera from T.
congolense and T. vivax infected cattle. Optimal antibody binding peptide sequences of these
two peptides need to be identified by testing modified sequences of these two peptides to improve the sensitivity of this assay.
In addition to attempting to define the epitopes of congopain, preliminary studies to increase
the immunogenicity of congopain were also undertaken. Alpha 2-macroglobulin is a natural
host inhibitor of proteinases. Inhibition occurs by entrapment of an active proteinase within
the alpha 2-macroglobulin cage. In addition, it has been demonstrated that antigen complexed
with alpha 2-macroglobulin becomes more immunogenic, resulting in enhanced antigenic
presentation of an entrapped antigen. This study reports the interaction between congopain and
alpha 2-macroglobulin. The preliminary results of this study showing congopain-alpha 2-macroglobulin
interaction could be used to explore the possibility of increasing the
immunogenicity of congopain and congopain epitopes by complexing these to alpha 2-macroglobulin.
Congopain epitopes complexed with alpha 2-macroglobulin could be used to
form a peptide-based vaccine. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.
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Protein discovery in African Trypanosomes: studying differential protein expression throughout the parasite life cycle and identification of candidate biomarkers for diagnosing Trypanosome infectionsEyford, Brett Alexander 22 February 2013 (has links)
Research was undertaken to discover and study trypanosome proteins that may play important roles in host-parasite or vector-parasite interactions. The methods used mass spectrometry based proteomics ideally suited for analysis of low abundance molecules. First, isobaric tags were used to monitor changes in proteins expression throughout the life cycle of Trypanosoma congolense, an economically important livestock pathogen. This was the first large scale survey of protein expression in trypanosomes. In addition to generating protein expression data for approximately 2000 different parasite proteins, 6 previously undescribed T. congolense proteins were discovered. Several of the proteins with interesting expression trends were selected for molecular characterization and monoclonal antibody derivation. Second, immunoenrichment and mass spectrometry were used to identify the cognate antigen recognized by a T. congolense-specific monoclonal antibody. The antigen, a flagellar calcium binding protein, was expressed as a recombinant protein and used to test its utility as a potential serodiagnostic antigen for diagnosis of T. congolense infections. Third, a “deep-mining” protein discovery mass spectrometric method was used to identify trypanosome proteins present in the plasma of late-stage African sleeping sickness patients. A total of 254 trypanosome proteins were unequivocally identified by tandem mass spectrometry. These findings are unprecedented since never before have such a large number of pathogen proteins been discovered in human blood using a non-biased approach (i.e. without using a targeted assay). The proteins discovered provide insights into host-parasite interactions and are strong candidates as targets for new diagnostic assays. / Graduate
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Parasite signalling and host responses in experimental and human African trypanosomiasis /Hamadien, Maha, January 2002 (has links)
Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.
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Kinetoplastid RNA editing : analysis of the mechanism of guide RNA directed uridylate insertion into precursor messenger RNA /Kable, Moffett Lee. January 1998 (has links)
Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [87]-96).
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Parasitological and molecular characterisation of isometamidium-sensitive and -resistant Trypanosoma congolense and T. brucei brucei isolates from cattle in East and West AfricaMebratu, Yohannes Afework January 1900 (has links)
Berlin : Freie Univ., Diss., 2005 / Dateiformat: zip, Dateien im PDF-Format. - Erscheinungsjahr an der Haupttitelstelle: 2005
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cAMP signaling and regulation by phosphodiesterases in trypanosomes /Laxman, Sunil. January 2006 (has links)
Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 132-145).
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Kinetoplastids biology, from the group phylogeny and evolution into the secrets of the mitochondrion of one representative: \kur{Trypanosoma brucei}, the model organism in which new roles of the evolutionary conserved genes can be explored / Kinetoplastids biology, from the group phylogeny and evolution into the secrets of the mitochondrion of one representative: \kur{Trypanosoma brucei}, the model organism in which new roles of the evolutionary conserved genes can be exploredTÝČ, Jiří January 2015 (has links)
This thesis is composed of two topics, for which trypanosomatids and evolution are common denominators. First part deals with phylogenetic relationships among monoxenous trypanosomatids, with emphasis on flagellates parasitizing dipteran hosts, analyzed mainly from biogeographical and evolutionary perspectives. Second part focuses on the trypanosomatid Trypanosoma brucei, causative agent of severe diseases, which serves as a model organism for functional studies of evolutionary conserved mitochondrial proteins, in particular those involved in replication, maintenance and expression of the mitochondrial genome, also termed the kinetoplast. This thesis identified the mtHsp70/mtHsp40 chaperone machinery as an essential component of replication and maintenance of the kinetoplast, and also identified numerous conditions under which mtHsp70 has a tendency to aggregate. Moreover, several conserved proteins, previously identified to be part of the mitochondrial ribosome, were shown to be important for translation of the mitochondrial transcripts.
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