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Major surface glycoproteins of insect forms of Trypanosoma brucei are not essential for cyclical transmission by tsetse.

Vassella E, Oberle M, Urwyler S, Renggli CK, Studer E, Hemphill A, Fragoso C, Bütikofer P, Brun R, Roditi I - PLoS ONE (2009)

Bottom Line: To determine if procyclins are essential for survival and transmission of T. brucei, all four genes were deleted and parasite fitness was compared in vitro and in vivo.In contrast, when flies were infected with the same mixture, the mutant was rapidly overgrown in the midgut, reflecting a reduction in fitness in vivo.The procyclic form of T. brucei thus differs strikingly from the bloodstream form, which does not tolerate any perturbation of its variant surface glycoprotein coat, and from other parasites such as Plasmodium berghei, which requires the circumsporozoite protein for successful transmission to a new host.

View Article: PubMed Central - PubMed

Affiliation: Institut für Zellbiologie, Universität Bern, Bern, Switzerland.

ABSTRACT
Procyclic forms of Trypanosoma brucei reside in the midgut of tsetse flies where they are covered by several million copies of glycosylphosphatidylinositol-anchored proteins known as procyclins. It has been proposed that procyclins protect parasites against proteases and/or participate in tropism, directing them from the midgut to the salivary glands. There are four different procyclin genes, each subject to elaborate levels of regulation. To determine if procyclins are essential for survival and transmission of T. brucei, all four genes were deleted and parasite fitness was compared in vitro and in vivo. When co-cultured in vitro, the mutant and wild type trypanosomes (tagged with cyan fluorescent protein) maintained a near-constant equilibrium. In contrast, when flies were infected with the same mixture, the mutant was rapidly overgrown in the midgut, reflecting a reduction in fitness in vivo. Although the mutant is patently defective in competition with procyclin-positive parasites, on its own it can complete the life cycle and generate infectious metacyclic forms. The procyclic form of T. brucei thus differs strikingly from the bloodstream form, which does not tolerate any perturbation of its variant surface glycoprotein coat, and from other parasites such as Plasmodium berghei, which requires the circumsporozoite protein for successful transmission to a new host.

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A. Competition with CFP-tagged trypanosomes in culture. Stumpy forms of wild type AnTat 1.1 (wt), ΔGPEET or Δprocyclin (Δproc) were mixed with an equal number of stumpy forms of CFP#5, which expresses cyan fluorescence protein from the procyclin promoter. The mixed cultures were triggered to differentiate by the addition of cis-aconitate and incubated at 27°C in SDM-79 supplemented with 10% FBS and 20 mM glycerol. The percentage of CFP-positive cells was determined by observing the cells under a fluorescence microscope. B. Mixed infections with CFP#5. Stumpy forms of the cell mixtures described above were used to infect tsetse flies and the percentage of CFP-positive cells was determined from cells isolated from the midgut of infected flies. As a control for CFP expression, a pure culture of CFP#5 was analysed in parallel.
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pone-0004493-g005: A. Competition with CFP-tagged trypanosomes in culture. Stumpy forms of wild type AnTat 1.1 (wt), ΔGPEET or Δprocyclin (Δproc) were mixed with an equal number of stumpy forms of CFP#5, which expresses cyan fluorescence protein from the procyclin promoter. The mixed cultures were triggered to differentiate by the addition of cis-aconitate and incubated at 27°C in SDM-79 supplemented with 10% FBS and 20 mM glycerol. The percentage of CFP-positive cells was determined by observing the cells under a fluorescence microscope. B. Mixed infections with CFP#5. Stumpy forms of the cell mixtures described above were used to infect tsetse flies and the percentage of CFP-positive cells was determined from cells isolated from the midgut of infected flies. As a control for CFP expression, a pure culture of CFP#5 was analysed in parallel.

Mentions: The experiments described above clearly show that, even without procyclins, trypanosomes can establish midgut infections reasonably successfully. In the long term, however, variants with even a slight competitive disadvantage would be lost from the population. In order to gauge the influence of procyclin genes on fitness, a tagged form of AnTat 1.1 expressing cyan fluorescent protein (CFP#5) was used as a reference in competition experiments [25]. Stumpy bloodstream forms of CFP#5 were mixed with equal numbers of wild type AnTat 1.1, ΔGPEET or Δprocyclin and induced to differentiate by the addition of cis-aconitate. The percentage of CFP-positive cells in each culture was monitored over a period of two weeks (Figure 5A). A control culture containing CFP#5 alone became 100% positive within two days of triggering differentiation and remained so throughout the experiment. When CFP#5 was combined with Δprocyclin, the ratio of the two cell types remained reasonably constant over 14 days. In contrast, CFP#5 was overgrown by the wild type and ΔGPEET with the same kinetics and was barely detectable at the last time point. These experiments indicate that Δprocyclin has a similar level of fitness to CFP#5 in culture, but both are less robust than the wild type and ΔGPEET.


Major surface glycoproteins of insect forms of Trypanosoma brucei are not essential for cyclical transmission by tsetse.

Vassella E, Oberle M, Urwyler S, Renggli CK, Studer E, Hemphill A, Fragoso C, Bütikofer P, Brun R, Roditi I - PLoS ONE (2009)

A. Competition with CFP-tagged trypanosomes in culture. Stumpy forms of wild type AnTat 1.1 (wt), ΔGPEET or Δprocyclin (Δproc) were mixed with an equal number of stumpy forms of CFP#5, which expresses cyan fluorescence protein from the procyclin promoter. The mixed cultures were triggered to differentiate by the addition of cis-aconitate and incubated at 27°C in SDM-79 supplemented with 10% FBS and 20 mM glycerol. The percentage of CFP-positive cells was determined by observing the cells under a fluorescence microscope. B. Mixed infections with CFP#5. Stumpy forms of the cell mixtures described above were used to infect tsetse flies and the percentage of CFP-positive cells was determined from cells isolated from the midgut of infected flies. As a control for CFP expression, a pure culture of CFP#5 was analysed in parallel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2637416&req=5

pone-0004493-g005: A. Competition with CFP-tagged trypanosomes in culture. Stumpy forms of wild type AnTat 1.1 (wt), ΔGPEET or Δprocyclin (Δproc) were mixed with an equal number of stumpy forms of CFP#5, which expresses cyan fluorescence protein from the procyclin promoter. The mixed cultures were triggered to differentiate by the addition of cis-aconitate and incubated at 27°C in SDM-79 supplemented with 10% FBS and 20 mM glycerol. The percentage of CFP-positive cells was determined by observing the cells under a fluorescence microscope. B. Mixed infections with CFP#5. Stumpy forms of the cell mixtures described above were used to infect tsetse flies and the percentage of CFP-positive cells was determined from cells isolated from the midgut of infected flies. As a control for CFP expression, a pure culture of CFP#5 was analysed in parallel.
Mentions: The experiments described above clearly show that, even without procyclins, trypanosomes can establish midgut infections reasonably successfully. In the long term, however, variants with even a slight competitive disadvantage would be lost from the population. In order to gauge the influence of procyclin genes on fitness, a tagged form of AnTat 1.1 expressing cyan fluorescent protein (CFP#5) was used as a reference in competition experiments [25]. Stumpy bloodstream forms of CFP#5 were mixed with equal numbers of wild type AnTat 1.1, ΔGPEET or Δprocyclin and induced to differentiate by the addition of cis-aconitate. The percentage of CFP-positive cells in each culture was monitored over a period of two weeks (Figure 5A). A control culture containing CFP#5 alone became 100% positive within two days of triggering differentiation and remained so throughout the experiment. When CFP#5 was combined with Δprocyclin, the ratio of the two cell types remained reasonably constant over 14 days. In contrast, CFP#5 was overgrown by the wild type and ΔGPEET with the same kinetics and was barely detectable at the last time point. These experiments indicate that Δprocyclin has a similar level of fitness to CFP#5 in culture, but both are less robust than the wild type and ΔGPEET.

Bottom Line: To determine if procyclins are essential for survival and transmission of T. brucei, all four genes were deleted and parasite fitness was compared in vitro and in vivo.In contrast, when flies were infected with the same mixture, the mutant was rapidly overgrown in the midgut, reflecting a reduction in fitness in vivo.The procyclic form of T. brucei thus differs strikingly from the bloodstream form, which does not tolerate any perturbation of its variant surface glycoprotein coat, and from other parasites such as Plasmodium berghei, which requires the circumsporozoite protein for successful transmission to a new host.

View Article: PubMed Central - PubMed

Affiliation: Institut für Zellbiologie, Universität Bern, Bern, Switzerland.

ABSTRACT
Procyclic forms of Trypanosoma brucei reside in the midgut of tsetse flies where they are covered by several million copies of glycosylphosphatidylinositol-anchored proteins known as procyclins. It has been proposed that procyclins protect parasites against proteases and/or participate in tropism, directing them from the midgut to the salivary glands. There are four different procyclin genes, each subject to elaborate levels of regulation. To determine if procyclins are essential for survival and transmission of T. brucei, all four genes were deleted and parasite fitness was compared in vitro and in vivo. When co-cultured in vitro, the mutant and wild type trypanosomes (tagged with cyan fluorescent protein) maintained a near-constant equilibrium. In contrast, when flies were infected with the same mixture, the mutant was rapidly overgrown in the midgut, reflecting a reduction in fitness in vivo. Although the mutant is patently defective in competition with procyclin-positive parasites, on its own it can complete the life cycle and generate infectious metacyclic forms. The procyclic form of T. brucei thus differs strikingly from the bloodstream form, which does not tolerate any perturbation of its variant surface glycoprotein coat, and from other parasites such as Plasmodium berghei, which requires the circumsporozoite protein for successful transmission to a new host.

Show MeSH
Related in: MedlinePlus