Force spectroscopy, a variant of atomic force microscopy (AFM), was used to characterize the adhesive properties of the glycocalyx of the single cell (protozoan) parasite, Leishmania, in order to understand the structural features of attachment in its sand fly vector. In the case of Leishmania mexicana and Leishmania major, parasitic protozoa responsible for cutaneous leishmaniasis, an important adhesin is lipophosphoglycan (LPG), an outer membrane glycoprotein containing carbohydrates that adhere to the sand fly midgut epithelium. Adhesion of Leishmania promastigotes, the flagellated insect-borne forms, to the sand fly midgut is crucial for colonization of the vector and transmission, since parasite–midgut attachment must be of sufficient strength to resist the peristaltic action of the sand fly gut during defecation of digested bloodmeal. Here, the interaction of an analog of N-acetyl-D-galactosamine, a carbohydrate present on the surface of permissive sand fly vectors, with L. mexicana LPG was investigated. AFM tips were first functionalized with grafted layers (brushes) of poly(methacrylic acid), grown using atom-transfer radical polymerization. Immersion in aqueous solutions of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide readied the brush for functionalization with 2-aminoethyl-β-D-galactopyranoside, producing AFM tips coated with a poly[N-2-(β-D-galactosyloxy)ethyl methacrylamide] brush. These carbohydrate-functionalized AFM tips revealed that nectomonad promastigotes had adhesive molecules distributed across their surface, but that these were largely absent from the infective, metacyclic stage of the parasite, and from LPG-deficient parasites (mutants). Consequently adhesion to infective parasites was substantially less than those to the nectomonad promastigotes. Furthermore, up to half of the wild-type promastigotes analyzed displayed a line of high adhesive force along the cell body, indicating a highly adhesive subpopulation. The selectivity of the N-acetyl-D-galactosamine analog to the parasites was compared with an antibody (anti-LPG) adsorbed to an AFM tip, which did not effectively differentiate between the different lifecycle stages of the parasite. Again, in LPG-deficient mutants, antibody adhesion was not observed. The molecular specificity afforded by coating AFM tips with polymer brushes enables key molecular roles to be identified, and the technique can be extended to other biological problems.

Force spectroscopy measurement of the adhesion of galactose to the human pathogen Leishmania mexicana

2019 Fall National Meeting

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.