Identification and partial characterization of molecules found within the midgut of the tsetse fly

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dc.contributor.authorHaines, Lee Rafuseen_US
dc.date.accessioned2024-08-14T16:42:18Z
dc.date.available2024-08-14T16:42:18Z
dc.date.copyright2002en_US
dc.date.issued2002
dc.degree.departmentDepartment of Biochemistry and Microbiology
dc.degree.levelMaster of Science M.Sc.en
dc.description.abstractMolecules in the midgut of the tsetse fly (Diptera: Glossinidae) are thought to play an important role in the life cycle of African trypanosomes by influencing their initial establishment in the midgut and subsequent differentiation events that ultimately affect parasite transmission. It is thus important to determine the molecular composition of the tsetse midgut to aid in understanding disease transmission by these medically important insect vectors. To my surprise, I found that the most abundant protein in midguts of teneral (unfed) Glossina morsitans morsitans was a 60 kDa molecular chaperone of bacterial origin. Since two species of symbiotic bacteria reside in the tsetse midgut, Soda/is glossinidius and Wigglesworthia glossinidia, 2-D gel electrophoresis and mass spectrometry were used to determine which of these organisms was the source of the 60 kDa molecule. To do this, peptide mass maps were compared to virtual peptide maps predicted for S. glossinidius and W glossinidia chaperone sequences. Four signature peptides were identified, revealing that the source of the chaperone was W glossinidia. Comparative two-dimensional gel electrophoresis and immunoblotting further revealed that this protein was localised to the anterior midgut containing the bacteriome and not the distal portion of the tsetse midgut. The possible function of this highly abundant endosymbiont chaperone in the tsetse midgut is discussed. Both Soda/is glossinidius and Wigglesworthia glossinidia are either required for tsetse viability or fecundity, and may influence the life cycle of African trypanosomes. S. glossinidius is thought to modulate the transmission of trypanosomes and thus is of considerable interest. To obtain probes for analysis of this symbiont, pure cultures were established and used for immunisation and derivation of monoclonal antibodies (mAbs). One mAb bound to an abundant 60 kDa protein that was highly expressed on the surface of Soda/is and was secreted as a soluble protein into the culture medium. This mAb recognised molecules only in S. glossinidius and W glossinidia and not in other members of the Enterobacteriaceae family. Using the rnAb in microimmunoadsorbent columns, the 60 kDa protein was isolated from Soda/is culture supematants and identified by mass spectrometry as a GroEL-like chaperone. Three mAbs were specific for Soda/is and did not bind the primary midgut symbiont, W glossinidia, and thus are useful probes for detection of Soda/is in tsetse. A monoclonal antibody, which recognises a repetitive EP polypeptide epitope and which was originally derived against procyclic form Trypanosoma brucei, cross-reacted with midgut lysates from both Glossina pa/pa/is pa/pa/is and Glossina morsitans morsitans. The DNA sequences of the genes encoding these EP proteins were determined and the translated protein sequences were obtained. Protein structure and function analysis using a variety of predictive algorithms showed that the EP repeat proteins contained distinct domains and antigenic regions. A putative signal peptide and post-translational modifications were predicted, supporting the idea that these molecules are expressed in the lumen of the midgut, and thus are good candidates for participation in vector-parasite interactions.
dc.format.extent144 pages
dc.identifier.urihttps://hdl.handle.net/1828/18051
dc.rightsAvailable to the World Wide Weben_US
dc.titleIdentification and partial characterization of molecules found within the midgut of the tsetse flyen_US
dc.typeThesisen_US

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