A Function for a Specific Zinc Metalloprotease of African Trypanosomes

Abstract

The Trypanosoma brucei genome encodes three groups of zinc metalloproteases, each of which contains ∼30% amino acid identity with the major surface protease (MSP, also called GP63) of Leishmania. One of these proteases, TbMSP-B, is encoded by four nearly identical, tandem genes transcribed in both bloodstream and procyclic trypanosomes. Earlier work showed that RNA interference against TbMSP-B prevents release of a recombinant variant surface glycoprotein (VSG) from procyclic trypanosomes. Here, we used gene deletions to show that TbMSP-B and a phospholipase C (GPI-PLC) act in concert to remove native VSG during differentiation of bloodstream trypanosomes to procyclic form. When the four tandem TbMSP-B genes were deleted from both chromosomal alleles, bloodstream B −/− trypanosomes could still differentiate to procyclic form, but VSG was removed more slowly and in a non-truncated form compared to differentiation of wild-type organisms. Similarly, when both alleles of the single-copy GPI-PLC gene were deleted, bloodstream PLC −/− cells could still differentiate. However, when all the genes for both TbMSP-B and GPI-PLC were deleted from the diploid genome, the bloodstream B −/− PLC −/− trypanosomes did not proliferate in the differentiation medium, and 60% of the VSG remained on the cell surface. Inhibitors of cysteine proteases did not affect this result. These findings demonstrate that removal of 60% of the VSG during differentiation from bloodstream to procyclic form is due to the synergistic activities of GPI-PLC and TbMSP-B.

A Glycosylation Mutant of Trypanosoma brucei Links Social Motility Defects In Vitro to Impaired Colonization of Tsetse Flies In Vivo

“…However, both adenylate cyclases that regulate SoMo are glycosylated (14). Moreover, there are a number of surface-associated enzymes (10,11), nutrient transporters (8), and flagellar components (32,33) that are (potentially) N-glycosylated and might play a role in SoMo, as well as additional adenylate cyclases that are differentially expressed between early and late procyclic forms (12).…”

“…The late procyclic forms found in the ectoperitrophic space are negative for GPEET but continue to express EP1 and EP3 procyclin, both of which are glycosylated (7). In addition to these major surface glycoproteins, trypanosomes express other, less abundant membrane proteins, many of which have the capacity to be modified by carbohydrates (8)(9)(10)(11).…”

Motility and more: the flagellum of Trypanosoma brucei

“…Although the precise biological function of GPI-PLC-dependent VSG release is not known, the enzyme is required for full virulence of pleomorphic trypanosomes, as mice infected with GPI-PLC null mutants have extended survival and control parasitemia better than mice infected with control trypanosomes 109 . GPI-PLC also participates in VSG shedding during differentiation of bloodstream forms to procyclic forms 110 .…”

Major Surface Protease of Trypanosomatids: One Size Fits All?

“…In contrast, TbMSP-B Ϫ/Ϫ or TbPLC Ϫ/Ϫ single mutants differentiate successfully, with VSG being released into the extracellular medium in a nontruncated form reminiscent of wild-type controls (41). A further detailed analysis provides evidence for a coordinated and inverse regulation of the activities of MSP-B and PLC during synchronous differentiation.…”