Meiosis and haploid gametes in the pathogen Trypanosoma brucei - PubMed (original) (raw)

Meiosis and haploid gametes in the pathogen Trypanosoma brucei

Lori Peacock et al. Curr Biol. 2014.

Abstract

In eukaryote pathogens, sex is an important driving force in spreading genes for drug resistance, pathogenicity, and virulence. For the parasitic trypanosomes that cause African sleeping sickness, mating occurs during transmission by the tsetse vector and involves meiosis, but haploid gametes have not yet been identified. Here, we show that meiosis is a normal part of development in the insect salivary glands for all subspecies of Trypanosoma brucei, including the human pathogens. By observing insect-derived trypanosomes during the window of peak expression of meiosis-specific genes, we identified promastigote-like (PL) cells that interacted with each other via their flagella and underwent fusion, as visualized by the mixing of cytoplasmic red and green fluorescent proteins. PL cells had a short, wide body, a very long anterior flagellum, and either one or two kinetoplasts, but only the anterior kinetoplast was associated with the flagellum. Measurement of nuclear DNA contents showed that PL cells were haploid relative to diploid metacyclics. Trypanosomes are among the earliest diverging eukaryotes, and our results support the hypothesis that meiosis and sexual reproduction are ubiquitous in eukaryotes and likely to have been early innovations.

Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

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Graphical abstract

Figure 1

Figure 1

Expression of Meiosis-Specific YFP Fusion Proteins in Trypanosomes from Salivary Glands of Tsetse Flies (A) Expression of YFP::DMC1 in T. b. gambiense DAL972. Scale bar, 5 μm. (B) Part of a tsetse salivary gland infected with T. b. brucei 427 var 3; live image. Many attached trypanosomes are expressing YFP::HOP1. Scale bar, 20 μm. (C) Aggregated results of expression of meiosis-specific genes over time. Flies were dissected from 14 to 38 days after infection and scored positive if the salivary glands contained a minimum of one trypanosome expressing a meiosis-specific gene (MND1, DMC1, or HOP1). The color denotes the trypanosome isolate.

Figure 2

Figure 2

Interactions and Cytoplasmic Exchange between Salivary Gland-Derived Trypanosomes Ex Vivo Mix of trypanosome clones F1G1 (green fluorescence) and F1R1 (red fluorescence). (A) Pair of interacting trypanosomes; both are 2K1N promastigote-like (PL) cells; note the intertwined flagella and close proximity of cell bodies. Scale bar, 5 μm. (B) Cluster of mating trypanosomes; two trypanosomes (arrows) have exchanged cytoplasm, demonstrated by both green and red fluorescence. Scale bar, 10 μm. (C) Diagram compiled from measurements of 1K1N and 2K1N PL cells of 1738 GFP from salivary glands dissected 20 days after infection. See also Movies S2, S3, and S4.

Figure 3

Figure 3

Morphology of 2K1N Promastigote-like Trypanosomes (A and B) Differential staining of DNA with DAPI and PI. DAPI preferentially binds to AT-rich DNA compared to PI, allowing the AT-rich kinetoplast DNA to be distinguished from nuclear DNA. Two different 2K1N PL cells are shown. (C) Localization of protein P166 from tripartite attachment complex in 2K1N PL cell of T. b. brucei J10 carrying fusion construct YFP::P166. (D) Visualization of flagellum in 2K1N PL cell of T. b. brucei J10 carrying fusion construct YFP::PFR1. The trypanosome has a single flagellum associated with the anterior kinetoplast. Scale bars, 10 μm. See also Figure S1.

Figure 4

Figure 4

Nuclear and Kinetoplast DNA Contents of Salivary Gland-Derived Trypanosomes Trypanosomes (1738 GFP) from flies dissected 17–20 days after infection were fixed and stained with DAPI and PI before imaging under uniform conditions. Total pixel intensities were measured for both DAPI and PI-stained nuclei and kinetoplasts using ImageJ (

http://rsb.info.nih.gov/ij

) and normalized relative to G1-arrested metacyclics (META, nuclear DNA content = 1.0; kinetoplast DNA content = 1.0). Other cells were categorized by morphology into promastigote-like cells (PL; see Figure 2C), procyclics (PRO; long trypomastigote with round or oval nucleus), epimastigotes (EPI; nucleus posterior to kinetoplast), cells in meiosis I (MEI; epimastigote with posterior nucleus, two anterior kinetoplasts and associated flagella). N denotes number of organelles measured in each cell category. (A) Nuclear DNA contents. Each histogram shows the frequency distribution of total normalized pixel intensities per nucleus; values on the x axis are the median for each bin (bin size = 0.25). The superimposed dotted line is a smooth curve. (B) Kinetoplast DNA contents. Each histogram shows the frequency distribution of total normalized pixel intensities per kinetoplast; values on the x axis are the median for each bin (bin size = 0.25).

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