Crosstalk between Trypanosoma brucei and the tsetse fly

Abstract

Trypanosoma brucei cause the fatal disease sleeping sickness in humans and the morbid disease nagana in animals. About 36 sub-Saharan African countries are affected by these diseases. The parasites are transmitted by tsetse flies (Glossina spp.) exclusively where they colonise the alimentary tract and the salivary glands. The trypanosomes establish first in the midgut as procyclic forms from where they colonise then the proventriculus (that connects the mid- with the foregut) and migrate later as epimastigote forms into the salivary glands via the foregut and proboscis. In the salivary glands epimastigote forms attach to the epithelium and give rise to the mammalian infective forms, the metacyclics. During transmission through the fly, trypanosomes are frequently severely reduced when they invade a new compartment. Trypanosomes either recover and develop an infection or fail to establish an infection and are eliminated by the tsetse fly’s defence. This complex interaction between vector and parasite shows that both counterparts specifically regulate genes. In this thesis we wanted to shed light into this complex crosstalk in three projects: We established a model to analyse how the severe reductions during the life cycle influence the diversity of trypanosomes. Short variable DNA sequences were integrated into the trypanosome’s genome to establish an artificial diversity. These transfected trypanosomes were cyclically transmitted through flies and mice. Tag DNA was isolated from infected flies and/ or mice and identified by sequencing. This allowed us to monitor diversity of the trypanosomes throughout their life cycle. We found that diversity was moderately reduced in the tsetse fly’s midgut but that migration into the salivary glands decreases the diversity. This decrease is mainly due to a shift in relative frequency which leads to a very uneven distribution of the tags. The diversity constantly decreased during mouse infection due to the constant gain of trypanosomes bearing the dominant tag. Surprisingly, the number of different tags was not reduced during the whole life cycle of the trypanosomes. The two anti-microbial peptides (AMPs), attacin and defensin, of tsetse flies were reported to play an important role in eliminating trypanosomes in the midgut. The mRNA of these AMPs was shown to be up-regulated upon trypanosome infection and it was hypothesised that procyclins might specifically induce its activation. We wanted to test this with different trypanosome strains as well with trypanosomes with incomplete or deleted procyclin coats. Tsetse flies were infected and mRNA isolated after various times of trypanosome exposition. None of the flies showed an up-regulated level of attacin and defensin mRNA. This result is in strong contradiction to some publications dealing with AMP regulation in infected tsetse flies. The tsetse flies, from the colony in Bratislava (Slovakia), show a high level of attacin and defensin mRNA in teneral flies (what not all G. m. morsitans do), show a midgut infection rate of about 50% (which is high compared to the infection rate in other laboratories), and are infected sometimes with the salivary gland hypertrophy virus (SGHV). It is very possible that attacin and defensin are not always up-regulated and that its activation is dependent on tsetse colony and origin. During the establishment in the midgut trypanosomes express procyclins, a stage specific surface protein coat that was suggested to protect against proteolytic enzymes or to be important to direct the parasite in the host. To test this hypothesis all procyclin genes were deleted and tsetse fly infection experiments were carried out. Interestingly, the null-mutant (Δprocyclin) was able to infect the midgut comparable to wild type trypanosomes, disclosing that procyclins are not needed for the establishment in the midgut and that probably free glycosylphosphatidylinositol (GPI) anchors, which are loaded with procyclins in wild type trypanosomes, overtook their function. Surprisingly, Δprocyclin was able to infect the salivary glands even though at very low rates, which reflects difficulties of trypanosomes to re-load the free GPIs with epimastigote specific surface proteins (e.g. BARP) for efficient migration. In competition, Δprocyclin was completely overgrown by wild type trypanosomes in the tsetse midgut, reflecting the selective advantage of a procyclin coat.