Activation of endocytosis as an adaptation to the mammalian host by trypanosomes

Abstract

Immune evasion in African trypanosomes is principally mediated by antigenic variation, but rapid internalization of surface-bound immune factors may contribute to survival. Endocytosis is upregulated approximately 10-fold in bloodstream compared to procyclic forms, and surface coat remodeling accompanies transition between these life stages. Here we examined expression of endocytosis markers in tsetse fly stages in vivo and monitored modulation during transition from bloodstream to procyclic forms in vitro. Among bloodstream stages nonproliferative stumpy forms have endocytic activity similar to that seen with rapidly dividing slender forms, while differentiation of stumpy forms to procyclic forms is accompanied by rapid down-regulation of Rab11 and clathrin, suggesting that modulation of endocytic and recycling systems accompanies this differentiation event. Significantly, rapid down-regulation of endocytic markers occurs upon entering the insect midgut and expression of Rab11 and clathrin remains low throughout subsequent development, which suggests that high endocytic activity is not required for remodeling the parasite surface or for survival within the fly. However, salivary gland metacyclic forms dramatically increase expression of clathrin and Rab11, indicating that emergence of mammalian infective forms is coupled to reacquisition of a high-activity endocytic-recycling system. These data suggest that high-level endocytosis in Trypanosoma brucei is an adaptation required for viability in the mammalian host.

FIG. 1.

Life cycle of Trypanosoma brucei. Designations of life stages that can be cultured in vitro are boxed, and those of stages expressing the variant surface glycoprotein are in bold. Following asymmetric division, the short epimastigote is believed to give rise to salivary gland infection; the subsequent fate of the postdivisional long epimastigote is not known at this time, as indicated by the question mark.

FIG. 2.

Expression of clathrin and Rab11 is high in all mammalian stages. (A) Western blots of T. brucei analyzed using antibodies to clathrin, Rab5A, Rab11, and BiP. Note that expression of Rab5A is essentially constant for all life stages but that clathrin and Rab11 expression levels decrease specifically in the PCF. (B) Location of clathrin, Rab11, p67, and BiP in cultured stages of T. brucei. Immunofluorescence images of parasites stained with antibody to clathrin, Rab11, BiP (red), and p67 (green) are shown. Cells were counterstained with DAPI (4′,6′-diamidino-2-phenylindole) (blue) for DNA. Phase-contrast images are shown below the respective fluorescence images. The relative positions of the antigens remain similar in all life stages. Scale bar, 2 μm.

FIG. 3.

In vitro-cultured BSF and long slender and stumpy cells have similar rates of endocytosis. (A) Accumulation of ConA and transferrin. Trypanosomes were incubated with fluorescein-conjugated ConA or Alexa Fluor-conjugated transferrin. At 0 min neither ConA nor transferrin is internalized, but by 10 min an internal pool of ConA and transferrin is present. Raw data for ConA are shown. (B) Quantification of internalized ConA and transferrin. Approximately 40 individual BSF or SS cells and approximately 25 LS cells were analyzed at each time point. Mean values for total fluorescence intensity are plotted against time; error bars represent standard deviations. Abbreviations are as introduced in the text.

FIG. 4.

Clathrin and Rab11 levels are similar in the cultured and midgut (MG) procyclic forms of T. brucei. (A) Clathrin, Rab11, p67, and BiP in midgut procyclic forms of T. brucei after 24 h of tsetse fly infection. Immunofluorescence of midgut PCFs, showing parasites stained with antibody to clathrin, Rab11, BiP (red), and p67 (green), is presented. Cells were counterstained with DAPI (blue) for DNA. Phase-contrast images are shown below the respective fluorescence images. Scale bar, 2 μm. Note that locations of antigens are highly similar to those of cultured PCFs (Fig. 2B). (B) Quantitation of fluorescence for clathrin, Rab11, p67, and BiP in midgut PCFs 24 h after a blood meal. The levels of clathrin and Rab11 in midgut cells are similar to cultured PCF levels. Numbers in parentheses indicate the numbers of cells analyzed for each life cycle stage. Values show the mean and standard deviations. **, statistically significant difference compared to bloodstream levels at P < 0.001.

FIG. 5.

Kinetics of clathrin and Rab11 down-regulation during in vitro differentiation of T. brucei. Trypanosomes, containing >80% stumpy forms (see Materials and Methods), were placed under in vitro differentiation conditions. (A) Protein lysates stained with Coomassie blue; VSG (molecular weight, ∼62,000) is indicated. (B) Cell density during differentiation. (C) Levels of clathrin and Rab11 diminish whereas CAP5.5 levels increase during differentiation of BSF to PCF forms in culture as detected by Western blotting. Note that the regions of panels B and C between 14 and 48 h are shaded to indicate a change in the time base.

FIG. 6.

Differential turnover of clathrin and Rab11. (A) Half-life of clathrin. Clathrin levels were determined in BSF and PCF cells by radioimmunoprecipitation following a 1-h pulse with ³⁵S-Met. Raw data at top and the lower graph show data from two replicate experiments together with standard errors. (B) Half-life of Rab11 in BSF trypanosomes. BSF parasites were treated with 100 μg/ml cycloheximide, and residual levels of Rab11 were determined by Western blotting, using BiP as a loading control. Note that BiP is known to be highly stable (2). The graph shows the mean results of two determinations with standard errors; the inset shows a representative blot.

FIG. 7.

Expression of clathrin, Rab11, BiP, and p67 in T. brucei during development in tsetse flies. Localization of antigens in insect stages of T. brucei after 30 days of infection in flies. Immunofluorescence of PCF in midgut, LTs, AsDEs, PdLE, and PdSE in the proventriculus, and epimastigotes (Epi) and metacyclic forms (Meta) in salivary glands. Phase-contrast images are shown below the respective fluorescence images. Representative images shown here are false colored and exposure enhanced for presentation purposes. Scale bar, 2 μm. (A) Clathrin and Rab11. Parasites are stained for CLH and Rab11 (red) and costained with DAPI (blue) for DNA. (B) BiP and p67. Parasites were stained for BiP (red) or (green) and costained with DAPI (blue) for DNA. In both panels, arrowheads indicate the kinetoplast and the scale bar represents 2 μm. (C) Quantitation of clathrin, Rab11, BiP, and p67 fluorescence. Numbers in parentheses indicate the numbers of cells analyzed for each life cycle stage; values show means and standard deviations. * and **, statistically significant difference compared to BSF levels (P < 0.01 and P < 0.001, respectively).

FIG. 8.

Activity of endocytic and recycling systems through the trypanosome life cycle. Endocytic and recycling activity is high in BSF stages; this activity is retained following differentiation to the nonproliferative stumpy form. Both systems are rapidly down-regulated following differentiation to the insect form within 24 h. Both recycling and endocytosis remain at low levels throughout development in the tsetse fly except for the latter stages. In the metacyclic form, expression of Rab11 and clathrin increases, probably as a component of preadaptation to the mammalian host. By contrast, levels of p67, a lysosomal marker, are similar through the entire life cycle. Levels of BiP, an endoplasmic reticulum marker, are higher in the mammalian bloodstream forms and slightly reduced in the insect stages; the lower level of BiP, particularly in the proventricular forms, is due to the smaller size of the parasites. The model assumes that recycling activity is proportional to expression levels of Rab11 and that endocytosis is similarly proportional to clathrin protein levels, an assumption that is supported by published work (29, 32, 33). Shading is used to indicate separate life cycles, with the transition of BSF to PCF arbitrarily considered to be the start or end of a cycle. Clathrin is shown in blue, Rab11 in yellow, p67 in green, and BiP in red. Epi, epimastigote; Meta, metacyclic form.