Regulation of surface coat exchange by differentiating African trypanosomes

Abstract

African trypanosomes (Trypanosoma brucei) have a digenetic lifecycle that alternates between the mammalian bloodstream and the tsetse fly vector. In the bloodstream, replicating long slender parasites transform into non-dividing short stumpy forms. Upon transmission into the fly midgut, short stumpy cells differentiate into actively dividing procyclics. A hallmark of this process is the replacement of the bloodstream-stage surface coat composed of variant surface glycoprotein (VSG) with a new coat composed of procyclin. Pre-existing VSG is shed by a zinc metalloprotease activity (MSP-B) and glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC). We now provide a detailed analysis of the coordinate and inverse regulation of these activities during synchronous differentiation. MSP-B mRNA and protein levels are upregulated during differentiation at the same time as proteolysis whereas GPI-PLC levels decrease. When transcription or translation is inhibited, VSG release is incomplete and a substantial amount of protein stays cell-associated. Both modes of release are still evident under these conditions, but GPI hydrolysis plays a quantitatively minor role during normal differentiation. Nevertheless, GPI biosynthesis shifts early in differentiation from a GPI-PLC sensitive structure to a resistant procyclic-type anchor. Translation inhibition also results in a marked increase in the mRNA levels of both MSP-B and GPI-PLC, consistent with negative regulation by labile protein factors. The relegation of short stumpy surface GPI-PLC to a secondary role in differentiation suggests that it may play a more important role as a virulence factor within the mammalian host.

Introduction

African trypanosomes are digenetic parasites whose lifecycle alternates between the midgut of the tsetse fly vector and the bloodstream of mammalian hosts. Native and recently isolated stocks of trypanosomes are pleomorphic in the mammalian bloodstream, transforming from a replicating long slender form into a non-dividing short stumpy form pre-adapted for transmission into the tsetse fly. Once in the fly midgut, the short stumpy form differentiates into a replicating procyclic form. Although laboratory-adapted monomorphic strains are also capable of differentiating into procyclics in vitro [1], it is most likely that the short stumpy form is responsible for natural transmission to the fly in vivo [2], [3], [4].

The most prominent marker for differentiation of bloodstream parasites into procyclic forms is the exchange of the main surface antigens: variant surface glycoprotein (VSG) in the bloodstream stage [5] and procyclin in the procyclic stage [6]. Both proteins are attached to the cell surface of their respective lifecycle stages by a glycosylphosphatidylinositol (GPI)-anchor [7]. VSG, a homodimer constituting ∼10% of total cell protein in the bloodstream stage, enshrouds the cell and forms a dense monolayer impenetrable to host serum macromolecules, e.g. host immunoglobulins. Up to 1000 VSG genes encode a superfamily of potential coat proteins, but only one is expressed at a time. It is through the regulated expression of distinct VSG genes, or antigenic variation, that the parasite is able to evade the host immune response [8]. In contrast to VSG, procyclin genes encode a more restricted family composed of two basic isoforms, EP and GPEET, defined by the amino acid sequences of C-terminal repeat domains [6]. Procyclin is also very abundant and, because its repeat domains are protease resistant, is believed to provide a protective glycocalyx in the hydrolytic environment of the tsetse fly midgut.

Differentiation of bloodstream stage cells and subsequent coat exchange can be induced in vitro by the addition of cis-aconitate to the culture media along with a temperature reduction from 37 °C to 27 °C [9]. This process is synchronous when initiated with a predominantly short stumpy population and consequently, has been shown to consist of a series of temporally regulated events [2], [3], [10]. Almost immediately, VSG synthesis is repressed and procyclin expression is induced. By 12 h, surface coat exchange is complete and differentiating trypanosomes enter into their first cell cycle as fully transformed procyclics.

Because differentiating cells are non-dividing, the pre-existing VSG coat cannot be eliminated by dilution and is actively removed from the cell surface by two demonstrated modes, GPI hydrolysis and endoproteolysis [11], [12], [13]. GPI hydrolysis is mediated by an endogenous GPI-specific phospholipase C (GPI-PLC) found exclusively in the bloodstream stage of the parasite [14], [15]. GPI-PLC has been shown to localize to the cytoplasmic face of intracellular vesicles [16], but can also be detected on the surface of short stumpy trypanosomes [13]. Furthermore, VSG GPI-anchor hydrolysis is present at the very beginning of the differentiation process in the starting short stumpy population. The other mode of release, endoproteolysis, is mediated by a zinc metalloprotease activity that is upregulated during differentiation [13]. Fully differentiated procyclic trypanosomes also possess a robust cell surface zinc metalloprotease activity capable of releasing transgenic VSGs [17], [18]. Selective metalloprotease inhibitors are capable of blocking proteolytic VSG release from both transgenic procyclics and differentiating bloodstream forms suggesting the same or similar enzymes mediate both processes [13], [18]. A family of zinc metalloprotease genes related to the well-characterized major surface protease (MSP) genes of Leishmania has been discovered in African trypanosomes by genomic sequencing [19]. Based on sequence and developmental expression, T. brucei MSP genes can be placed into three distinct classes: MSP-A, -B and -C. While MSP-A and MSP-C mRNAs are exclusively expressed in bloodstream stage trypanosomes, MSP-B mRNA is more abundant in procyclics than bloodstream stage cells [20]. Moreover, RNAi analysis indicates that MSP-B activity is responsible for release of VSG from transgenic procyclics [20]. Thus MSP-B has the characteristics required for proteolytic release of VSG during differentiation, and indeed, ablation of expression reduces release of VSG in differentiating monomorphic bloodstream trypanosomes (John Donelson, personal communication). Several factors must come into play for effective coat remodelling during short stumpy to procyclic differentiation. Short-stumpy parasites are ‘primed’ by activation of GPI-PLC prior to initiation of differentiation, but once differentiation begins GPI-PLC must be downregulated while MSP-B activity is upregulated. In addition, to prevent loss of the newly synthesized procyclin coat the structure of the GPI anchor precursor that is attached to surface proteins must change from that found in bloodstream cells, which is susceptible to GPI-PLC activity [21], [22], to that found in procyclic parasites, which is resistant [23].

In this work, we evaluate the relative contribution of the two modes of VSG release, GPI hydrolysis and endoproteolysis, as well as the precise timing of the shift to synthesis of a procyclic-type GPI anchor. We find that GPI hydrolysis plays only a supporting role in VSG release. Nevertheless, GPI synthesis switches early in the differentiation process. We also examine the interplay of these processes using inhibitors of transcription and translation to modulate MSP-B and GPI-PLC expression during the synchronous differentiation of short stumpy cells into procyclic forms. Our results suggest that MSP-B and GPI-PLC expression are coordinately and inversely regulated, most likely under the negative control of labile trans-acting factors.