The lysosomal/endosomal system of African trypanosomes is developmentally regulated and is important in the pathogenesis associated with infection of the mammalian bloodstream. Long considered to be a target for drug development, the internal pH of the lysosome has been variously reported to range from <5.0 to >6.0. We have refined a flow cytometric technique using a pH-sensitive probe that specifically targets the lysosome, tomato lectin:Oregon Green 488 conjugate. The probe is delivered to the lysosome with fidelity, where it is shielded against external pH. Measurement of fluorescent output in the presence and absence of lysomotropic agent (NH 4 Cl) then allows precise titration of steady state lysosomal pH (4.84 ± 0.23). Using bafilomycin A1 to inhibit acidification we demonstrate that this method is responsive to pharmacological perturbation of lysosomal physiology. This work should facilitate future studies of the lysosomal function in African trypanosomiasis, as well as other parasitic protozoa.
Keywordstrypanosome; lysosome; flow cytometry; pHThe eukaryotic lysosome has been traditionally defined as a discrete terminal degradative organelle of the endocytic pathway with a high density (r = 1.10 g/ml) and a low internal pH (≤ 5.0), [1]. It contains a unique complement of structural membrane glycoprotein markers (lysosome-associated membrane proteins; LAMPs), acid hydrolases (lipases, proteases, phosphatases and glycosidases), and a membrane proton pump (V-ATPase) that is responsible for lumenal acidification. In African trypanosomes the lysosome is a single discrete organelle located at a perinuclear position in the posterior end of the cell. Morphologically it is indistinguishable between the procyclic insect and mammalian bloodstream stages. It too is defined by a LAMP-like membrane glycoprotein called p67 [2,3], acidic hydrolases such as cathepsin-L and -B orthologues [4][5][6][7], and a recent survey of the genome data base revealed a full complement of V-ATPase subunit orthologues [8]. It is well established that endocytosis is greatly upregulated in the bloodstream stage relative to procyclic forms [9][10][11], and this is mirrored by upregulation of lysosomal hydrolytic activities [6,7,9,12]. This difference at least in part reflects issues of nutrient acquisition for each life cycle stage. Procyclic cells can rely on the hydrolytic environment of the tsetse midgut to provide solutes for transport, while the bloodstream stage must aggressively take up and digest host serum macromolecules. A secondary consideration is immune evasion; potentially lytic or opsonic immune complexes # Corresponding Author: J.D. Bangs, email: jdbangs@wisc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the...