A new asymmetric division contributes to the continuous production of infective trypanosomes in the tsetse fly

Abstract

SUMMARYAfrican trypanosomes are flagellated protozoan parasites that cause sleeping sickness and are transmitted by the bite of the tsetse fly. To complete their life cycle in the insect, trypanosomes reach the salivary glands and transform into the metacyclic infective form. The latter are expelled with the saliva at each blood meal during the whole life of the insect. Here, we reveal a means by which the continuous production of infective parasites could be ensured. Dividing trypanosomes present in the salivary glands of infected tsetse flies were monitored by live video-microscopy and by quantitative immunofluorescence analysis using molecular markers for the cytoskeleton and for surface antigens. This revealed the existence of two distinct modes of trypanosome proliferation occurring simultaneously in the salivary glands. The first cycle produces two equivalent cells that are not competent for infection and are attached to the epithelium. This mode of proliferation is predominant at the early steps of infection, ensuring a rapid colonization of the glands. The second mode is more frequent at later stages of infection and involves an asymmetric division. It produces a daughter cell that matures into the infective metacyclic form that is released in the saliva, as demonstrated by the expression of specific molecular markers -the calflagins. The levels of these calcium-binding proteins increase exclusively in the new flagellum during the asymmetric division, showing the commitment of the future daughter cell to differentiation. The coordination of these two alternative cell cycles contributes to the continuous production of infective parasites, turning the tsetse fly into an efficient and long-lasting vector for African trypanosomes.

Forward motility is essential for trypanosome infection in the tsetse fly

“…The posterior and anterior midgut, the proventriculus and foregut were checked for the presence of parasites under the microscope. Similar to our previous studies 2012;Subota et al, 2011), parasites were found in 45% of the posterior midguts, 11% of the anterior midguts, and 4% of the foreguts of flies fed with WT trypanosomes (Fig. 3A).…”

“…Trypanosomes are transmitted by the bite of the tsetse flies, which become vectors when they consume a blood meal from an infected mammal. To complete their life cycle, trypanosomes initially present in the posterior midgut of the fly have to reach the tsetse salivary glands in order to transform into infective parasites (Vickerman, 1985;Peacock et al, 2007;Oberle et al, 2010;Rotureau et al, 2012). This is not direct and requires several proliferation, differentiation and migration steps that take place for 2-3 weeks in a strictly defined chronological order in five distinct fly tissues (Vickerman, 1985;Van Den Abbeele et al, 1999;Sharma et al, 2009;Oberle et al, 2010).…”

Intraflagellar transport is required for the maintenance of the trypanosome flagellum composition but not length

“…IFT activity and association to various cargoes therefore could allow the generation of molecular diversity inside cilia by concentrating (or excluding) sensors, structural elements or other motors. This is relevant for multicellular organisms that produce several types of cilia but also in protists that assemble different types of flagella either in the same cell (Dawson and House, 2010) or in different life cycle stages (Gluenz et al, 2010;Rotureau et al, 2012). In addition to protein distribution, IFT could contribute to the transport of other molecules.…”

Flagellar adhesion inTrypanosoma bruceirelies on interactions between different skeletal structures present in the flagellum and in the cell body

“…Intact cells were treated for immunofluorescence after paraformaldehyde (PFA) or methanol fixation as described previously (Rotureau et al, 2011;Rotureau et al, 2012). Cytoskeletons were detergent-extracted with 1% Triton X-100 or 1% Nonidet-P40 in PEM buffer (100 mM PIPES, pH 6.9, 1 mM EGTA, 1 mM MgSO 4 ) for 10 minutes, washed in PBS and fixed in methanol as described elsewhere (Sherwin et al, 1987;Sherwin and Gull, 1989a).…”