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Experimental Parasitology 117 (2007) 111–114 Trypanosoma brucei 29–13 strain is inducible in but not permissive for the tsetse fly vector Ste´phane Herder a,1, Jan Voty´pka b,c,1, Milan Jirku˚ c,d, Jana Ra´drova´ b, Christian J. Janzen e, Julius Lukesˇ c,d,* a UMR 177 IRD CIRAD, Campus International de Baillarguet, 34398 Montpellier Cedex 5, France b Faculty of Sciences, Charles University, Vinicˇna´ 7, 12844 Prague, Czech Republic c Biology Centre, Institute of Parasitology, Czech Academy of Sciences, Branisˇovska´ 31, 37005 Cˇeske´ Budeˇjovice (Budweis), Czech Republic d Faculty of Biology, University of South Bohemia, Branisˇovska´ 31, 37005 Cˇeske´ Budeˇjovice (Budweis), Czech Republic e Department of Biology, University of Munich, Maria-Ward-Str. 1a, D-80638 Munich, Germany Received 16 April 2007; received in revised form 25 May 2007; accepted 25 May 2007 Using green fluorescent protein as a reporter, we have shown that the strain 29–13 of Trypanosoma brucei, widely used for inducible down-regulation of mRNA, is inducible in, but not permissive for the tsetse flies Glossina palpalis gambiensis and Glossina morsitansmorsitans. Within two weeks post-infection, 42% males and females of teneral and non-teneral tsetse flies harboured intestinal infections,yet not a single infection progressed into the salivary glands.
Ó 2007 Elsevier Inc. All rights reserved.
Index Descriptors and Abbreviations: Trypanosoma brucei; Tsetse; Glossina; GFP; Transmission; Midgut infection; Tetracycline induction Trypanosoma brucei, the causative agent of African The synthesis of double stranded RNA can be efficiently sleeping sickness in humans and nagana in animals, be- induced in both procyclic and bloodstream stages of T. bru- came one of the first organisms in which RNA interference cei under cultivation conditions and most functional stud- (RNAi) was shown to be effective ). More- ies have thus been performed in the culture media. Such over, thanks to a tight and efficient inducible system conditions enable biochemical and molecular studies of ), the target transcripts can be down-regulated gene functions, but at the same time are artificial and can by the addition of tetracycline (Tet) to the growth medium.
hardly replace experiments with these parasitic stages in Unexpectedly, Trypanosoma cruzi and Leishmania major, their respective hosts. It was shown that in the blood of the other two medically important kinetoplastid flagellates mice fed with doxycycline, an analogue of Tet, RNAi can for which the whole genome sequence is available be triggered enabling the study of ensuing phenotypic effects (). Moreover, Tet-inducible gene expression has been recently demonstrated also within the T. brucei became the favourite flagellate for studies of gene tsetse fly vector. The expression of green fluorescent pro- function, with hundreds of genes already studied by means tein (GFP) was successfully induced in the T. brucei strain K11 by feeding 1 lg/ml Tet to tsetse flies Glossina morsi-tans 12 days after they have been infected with the blood- Corresponding author. Fax: +420 38 5310388.
T. brucei strain AnTat 1.1 with genes knocked out by 1 Both authors contributed equally to this work.
homologous recombination has been tested for possible 0014-4894/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.exppara.2007.05.011 S. Herder et al. / Experimental Parasitology 117 (2007) 111–114 from flies field-collected in Burkina Faso and Zimbabwe, Lately, genetically modified cell lines de- rived from the strain Lister 427 have been assayed for phe- Out of a total of 1211 infected tsetse flies, 423 died in the notypes confined to the midgut infection course of the 2-month long experiment, which is in agree- ment with a mortality predicted from literature for these in- strain may not be able to produce mature infection in fected insects (Surviving 788 flies have been microdissected in two different periods. Within 2–15 Most functional analyses are being performed via days post-infection, 41.8% of a total of 287 males and fe- RNAi, all making use of the T. brucei strain 29–13 strain.
males of teneral G. p. gambiensis and G. m. morsitans and This is because only this strain, derived from the strain Lis- non-teneral G. m. morsitans harboured intestinal infections (Both species and sexes have been susceptible for tem and viral T7 RNA polymerase and can be efficiently the 29–13 cells, with the infections varying from very strong transformed with a number of different RNAi vectors tion with the particular host or sex was observed (data not study with the aim of testing the inducibility and permissi- shown). The non-teneral males and females of G. m. morsi- bility of the 29–13 strain for Glossina spp.
tans were not refractory to the early midgut infection, but Our preliminary experiments confirmed previous obser- the infection rate for non-teneral flies was lower (p < 0.05) than in their teneral counterparts As expected, with blood containing 1 lg/ml Tet does not compromise cells containing constructs with constitutively expressed their viability and longevity (data not shown). The drug- GFP fluoresced in the insect midgut at an equal rate and resistance plasmids CJ12 and CJ17 were constructed based intensity as in the medium. With the inducible strain, induc- tion was initiated either in parallel with the infection, or dur- CJ12 was prepared by replacing the reporter cassette of ing the 2nd or 3rd feeding of infected flies within one week of pbRn6 with an inducible T7 promoter-driven GFP cassette infection. In all cases the majority of cells started to fluoresce, using the SpeI and StuI restriction sites. A PCR-amplified proving that the induction can be postponed in the course of blasticidin cassette was introduced downstream of the GFP reporter using the NheI sites generating plasmid CJ11.
The next step was to show whether the midgut infection Next, a rRNA promoter, released from pbRn6 as a SfiI– by the T. brucei strain 29–13 can progress into the salivary EcoRI fragment and blunt-ended with T4 DNA polymer- glands and thus complete its life cycle in tsetse. For this, ase, was inserted in front of the blasticidin cassette using 501 flies of both sexes and species have been dissected the StuI site. To generate plasmid CJ17, constitutively and carefully examined 60 days post-infection. Regretta- expressing GFP, the Tet-operator downstream of the T7 bly, none of them harboured trypanosomes in the salivary promoter was removed by BglII digest of CJ11 followed glands. After two months, the percentage of surviving flies by religation of the plasmid. Plasmids CJ12 and CJ17 con- with a late midgut infection dropped to 4.4% (), taining GFP under either Tet-inducible or constitutive pro- however, the persisting infections were high (). At this moter, respectively, have been electroporated into the 29– stage none of the flagellates was found to express GFP at 13 strain cultured in the SDM-79 medium under standard the level visible under fluorescent light. This is not surpris- conditions, and cells resistant to 10 lg/ml of blasticidin ing in the case of trypanosomes containing the inducible have been cloned out. Selected clonal cell lines containing construct, since they were growing in the absence of Tet the constitutive GFP vector showed medium to strong fluo- for a long period of time. However, the reasons for an rescence in about 85% cells, whereas virtually all cells con- eventual shutdown of the constitutive promoter regulating taining the inducible GFP started to strongly fluoresce 12– the expression of GFP remain unclear. More than 99% of 24 h upon the addition of 1 lg/ml Tet to the medium. Next, the non-teneral G. m. morsitans flies were able to eliminate 1 · 109 cultured procyclics of both cell lines were mixed the infection during two months, whereas in average 7.5% with 100 ml of fresh defibrillated inactivated bovine blood of infections survived in the teneral males and females of and immediately fed through a silicon membrane to teneral the same species (). Once the infection is established or non-teneral flies of both sexes. Engorged flies were in a susceptible female fly, it will remain there without placed in cages in small mono-specific and mono-sexed shortening the life span of its host. The situation seems groups of up to 30 specimens and maintained by feeding to be different for males, where the incubation time in mid- on fresh defibrillated inactivated bovine blood (first three gut is short, proceeds rapidly into salivary glands, and feedings) and on uninfected rabbits throughout the rest eventually increases the mortality of the infected specimen of the experiment (see below). Because of their high permis- sibility for T. brucei, two species of Glossina vectors were However, our results show that the midgut infection selected. Populations of Glossina palpalis gambiensis and caused by the strain 29–13 does not interfere with the life Glossina morsitans morsitans were maintained in a level-2 span of either females or males, and is within two months containment insectary (CIRAD, Montpellier, France) at eliminated probably by the failure of trypanosomes to re- 23 °C and 80% relative humidity. These colonies originate sist the immune defence of the fly.
S. Herder et al. / Experimental Parasitology 117 (2007) 111–114 Table 1Intestinal infections of teneral (T) and non-teneral (NT) flies of Glossina palpalis gambiensis and G. morsitans morsitans in two different periods ofdissection protozoan pathogens, repeated transmission of T. brucei via its mammalian host will increase its pathogenicity ( ), whereas an opposing effect oflong term cultivation in a simplified environment of the culture medium can be anticipated, as demonstrated for the related flagellate Leishmania tarentolae ( Our experiments showed the limits of phenotypic tests of the 29–13 strain in tsetse that can be focused mainly on the early midgut infection. The cells can be induced by RNAiduring or shortly after the infection, but they fail to pro- Fig. 1. Percentage of tsetse fly midguts containing trypanosomes within ceed into the salivary glands. The improper development in its insect vector of the most widely used strain and theflagship of RNAi-based functional genomics of T. brucei is an impetus to the scientific community to prepare an-other strain, in which genetically interfered procyclic stages can be studied in their natural environment.
We thank George A.M. Cross for useful comments on the manuscript. This work was supported by the Czech Ministry of Education (Grants 2B06129, 0021620828 and LC06009) and the exchange grant Barrande 2-06-28.
Fig. 2. Intensity of midgut infection within first 2 weeks after infection and 2 months after infection. At least 10 flies were dissected each day.
Black bar: heavy infections (>1000 parasites per midgut); grey bar: El-Sayed, N.N. et al., 2005. Comparative genomics of trypanosomatid medium infections (100–1000 parasites per midgut); white bar: light parasitic protozoa. Science 309, 404–409.
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