P15 trypanosome microtubule associated protein : structure/function analysis and vaccine development for the prevention of African sleeping sickness.
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Date
2001
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Abstract
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.
Description
Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2001.
Keywords
Parasitic vaccines., Microtubules., Trypanosoma brucei., Trypanosomiasis--Control., Trypanosomiasis--Africa., Theses--Microbiology.