Studies on the Flagellar Attachment of African Trypanosomes

Attachment of trypanosomes by the flagellum to a host internal surface is believed to represent an important stage in the development of these parasites in their vectors. This thesis is concerned with an investigation into various aspects of the flagellar attachment of Trypanosoma congolense epimastigotes. The morphology of the attachment is described in both the tsetse fly labrum and in vitro where the epimastigotes attach to the plastic wall of the culture flask. Epimastigotes attach to the substratum at one or more sites along the length of the flagellum. At the attachment site the flagellar membrane is extended from the axis and an electron dense, hemidesmosome-like plaque (approx. 35nm wide) forms on the cytoplasmic face of the membrane. 5nm fiIaiments converge on the plaque and associate with similar filaments emanating from flagellum-cell body attachment plaques. Filaments form at the attachment site soon after contact with the substratum. Plaques form subsequently and become more compact with advancing epimastigote age. An extracellular gap (15nm) is present between flagellum and substratum. Ruthenium red binding in this gap indicates that glycoproteins are situated there. Differentiated attachment plaques appear to be specific to attached epimastigotes and are absent from detached epimastigotes. They are absent from the flagella of bloodstream trypomastigotes of T. congolense even though the flagellar tip of this stage in the tryp-anosome's life cycle attaches the parasite to the endothelium of bloodvessel walls. The necessity of attachment for epimastigote proliferation and for differentiation of the mammal-infecting metacyclic stage from the epimastigote has been examined. Prevention of epimastigote attachment by shaking cultures or growing trypanosomes on a polypropylene substratum does not affect the rate of epimastigote division. Metacyclogenesis does not occur in unattached trypanosomes, however, suggesting that, in this species, attachment is a necessary prerequisite for this developmental step. Interference reflection microscopy has been used to visualize the effect on extracellular gap width of agents added to the culture medium. An increase in gap width following trypsinization on addition of Concanavalin A, Lentil lectin and Wheatgerm Agglutinin indicate that protein, D-mannose and N-acetyl-D-glucosamine are present in this location. External serum concentration, monovalent ion concentrations and the presence of tunicamycin have little effect on the interfer-ometry pattern. Tunicamycin insensitivity reflects a lack of glycoprotein turnover at the site. Divalent cation removal decreases the gap width. The binding of epimastigotes to positively charged Sephadex beads indicates a net negative surface charge. This charge maybe less negative in the region of the attachment as binding to positively charged beads is reduced at the flagellar tip. Indirect immunofluorescence and indirect immunogold electron microscopical cytochemistry indicate that the proteins actin, vinculin, filamin, intermediate filament proteins and desmoplakins (found associated with morphologically similar metazoan cell attachment structures - hemidesmosomes and focal adhesions) are absent from the epimastigote attachment. Immunofluorescence reactions suggest that actin and intermediate filaments are present in these cells but not localized in their distribution. Confirmation of the presence of these proteins can be obtained with SDS-PAGE and associated Western blotting experiments of both whole cell and cytoskeleton preparations. Comparative 2D-gel electrophoresis of attached and unattached whole epimastigotes and cytoskeletons shows the presence of a novel group of proteins (approx. Mr=70kd) in attached preparations. It is suggested that these proteins maybe involved in the attachment complex