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Title: The role of tsetse serpins in the establishment of trypanosomes in the fly midgut
Author: Ooi, Cher Pheng
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2011
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Abstract:
African trypanosomiasis (AT) affects both man and his domesticated animals and this neglected disease has seen a resurgence in recent years due to laxness in the enforcement of tsetse control measures. An overlap in existence occurs within the tsetse midgut between the three parties representing the transmission cycle of African trypanosomiasis: the vertebrate host (more specifically its blood), the trypanosome parasite and tsetse physiological factors. It is thus interesting to investigate how the interplay between these three parties affect trypanosome establishment within the fly midgut. Procyclic form T. b. bruce! trypanosomes are highly susceptible to vertebrate serum. This holds true with horse serum and this susceptibility is not an artefact of culturing as tsetse midgut infected trypanosomes show the same degree of susceptibility as procyclic culture forms (PCFs). This susceptibility to horse serum was no longer observed when Naja naja kaouthia cobra venom factor (CVF) was used to inactivate the complement activity of horse serum. Therefore T. b. brucei killing by horse serum is complement related. This complement related killing of procyclics is a phenomenon relevant within the fly midgut, as horse serum maintains its ability to lyse trypanosome procyclics up to 1 hour post ingestion and the majority of trypanosomes would have transformed into procyclics within that period. Due to the lack of complement activity when Ca2+ ions were selectively chelated using EGTA, the pathway with which trypanosome killing is initiated by either the lectin or classical pathways. As it is unlikely that anti-trypanosome antibodies are present in naive horse serum, it is most likely that the lectin pathway is responsible. The presence of mannan binding lectins (MBLs) within the pallet fraction of procyclic trypanosomes assayed with horse serum, as detected via Western blotting, supports this. Five G. m. morsitans midgut specific serpins were identified from the Glossina midgut expressed sequence tag (EST) library and analysed using bioinformatics. Four of the five serpins (Serp-1, Serp-2, Serp-3 and Serp-4) had a serpin domain homologous to that of human complement C1-inh, while one (Serp-Kaz) was homologous to a Kazal inhibitor. No discernable pattern could be gleaned by comparing the homology of the tsetse midgut serpins and other insect serpins, blood feeders or otherwise. Using RNA interference (RNAi) techniques it was possible to discern that the knockdown of the four C1-inh homologs resulted in a decreased number of infected midguts. This suggests that the four C1-inh homologs impart an advantage to procyclics when they attempt to establish themselves within the fly midgut. A recombinant version of one of the Glossina midgut serpins was expressed and used to characterise the properties of the C1-inh homologs. The identity of rSerp-2 was confirmed by mass spectrometry and it was determined that rSerp-2 has serine protease inhibiting properties. rSerp-2 was likewise capable of inhibiting the killing of PCFs in vitro in horse serum assays and this inhibition was dependent upon the concentration of rSerp-2. Since it was shown that complement is the major killing factor of T. b. bruce! PCFs in horse serum, it is reasonable to conclude that this suppression of trypanolytic activity by rSerp-2 (and potentially the other G. m. morsitans midgut serpins) is due to the inactivation of the complement cascade. The remaining question is where within the cascade this occurs, or if this inhibitory function is specific to any given serine protease activated step within the cascade at all.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.548773  DOI: Not available
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