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Social motility of African trypanosomes is a property of a distinct life-cycle stage that occurs early in tsetse fly transmission.

Imhof S, Knüsel S, Gunasekera K, Vu XL, Roditi I - PLoS Pathog. (2014)

Bottom Line: These findings link the phenomenon of SoMo in vitro to the parasite forms found during the first 4-7 days of a midgut infection.Moreover, the process can be uncoupled from colonisation of the salivary glands.Although they are the major surface proteins of procyclic forms, EP and GPEET are not essential for SoMo, nor, as shown previously, are they required for near normal colonisation of the fly midgut.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology, University of Bern, Bern, Switzerland; Graduate School of Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.

ABSTRACT
The protozoan pathogen Trypanosoma brucei is transmitted between mammals by tsetse flies. The first compartment colonised by trypanosomes after a blood meal is the fly midgut lumen. Trypanosomes present in the lumen-designated as early procyclic forms-express the stage-specific surface glycoproteins EP and GPEET procyclin. When the trypanosomes establish a mature infection and colonise the ectoperitrophic space, GPEET is down-regulated, and EP becomes the major surface protein of late procyclic forms. A few years ago, it was discovered that procyclic form trypanosomes exhibit social motility (SoMo) when inoculated on a semi-solid surface. We demonstrate that SoMo is a feature of early procyclic forms, and that late procyclic forms are invariably SoMo-negative. In addition, we show that, apart from GPEET, other markers are differentially expressed in these two life-cycle stages, both in culture and in tsetse flies, indicating that they have different biological properties and should be considered distinct stages of the life cycle. Differentially expressed genes include two closely related adenylate cyclases, both hexokinases and calflagins. These findings link the phenomenon of SoMo in vitro to the parasite forms found during the first 4-7 days of a midgut infection. We postulate that ordered group movement on plates reflects the migration of parasites from the midgut lumen into the ectoperitrophic space within the tsetse fly. Moreover, the process can be uncoupled from colonisation of the salivary glands. Although they are the major surface proteins of procyclic forms, EP and GPEET are not essential for SoMo, nor, as shown previously, are they required for near normal colonisation of the fly midgut.

No MeSH data available.


Related in: MedlinePlus

Proteins up-regulated in late procyclic forms.Yellow: proteins showing ≥2-fold increase in abundance. Grey: ≤2-fold change in abundance. N.D. not detected. Biological replicates (exp1 and exp2) were performed.
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ppat-1004493-g005: Proteins up-regulated in late procyclic forms.Yellow: proteins showing ≥2-fold increase in abundance. Grey: ≤2-fold change in abundance. N.D. not detected. Biological replicates (exp1 and exp2) were performed.

Mentions: In culture, early and late procyclic forms are morphologically indistinguishable. Since GPEET was the only known marker for early procyclic forms at the beginning of this study, we used SILAC to identify additional proteins that were differentially expressed between GPEET-positive and GPEET-negative cells. Two independent experiments identified a limited number of candidates that were significantly different in at least one experiment (≥2-fold; Figures 4 and 5; Table S2). Of the differentially regulated proteins, three examples were encoded by related genes. These were the calflagins (Tb-44, Tb-24 and Tb-17), the two hexokinases (HK1 and HK2) and three adenylate cyclases. The members of a protein family could not be identified unequivocally as they contained identical peptides that are randomly assigned during mapping. Lacking antibodies that discriminated between isoforms, we analysed the transcripts for unique signatures. In the case of the adenylate cyclases (Tb927.5.285b, Tb927.5.320 and Tb 927.5.330 - here designated AC330, AC320 and AC285b) differences in their 3′ untranslated regions, allowed AC330 to be distinguished from AC320/285b by Northern blot analysis (Figure 6A). Both were differentially expressed, with AC330 up 9-fold in early procyclic forms and AC320/285b up 6.25-fold in late procyclic forms. Thus, the changes in protein levels detected by SILAC are probably an under-estimate for the individual proteins. Since an antiserum was available against calflagins, we monitored expression of these proteins by immunofluorescence. This revealed that calflagin-positive cells were always also positive for GPEET (Figure 6B). In addition, we performed quantitative RT-PCR to measure transcript levels in early and late procyclic forms (Figure 6C). This confirmed the differential expression at the level of mRNA for the adenylate cyclases and HK1/HK2. HK1 mRNA was expressed 4-fold more in early procyclic forms while HK2 was up-regulated 2-fold in late procyclic forms. In contrast to what was observed by immunofluorescence and SILAC, calflagin transcripts were down-regulated only 2-fold in late procyclic forms, suggesting that there is an additional level of regulation. Finally, we tested the mRNA levels of a set of putative pteridine transporters (PPT: Tb927.1.2850, Tb927.1.2880), which we have observed to be up-regulated (at least transiently) during differentiation of early to late procyclic forms; these were increased 4.6-fold in late procyclic forms. In summary, although no other gene is as tightly regulated as GPEET, we have identified several additional differentially regulated transcripts/proteins in early and late procyclic forms.


Social motility of African trypanosomes is a property of a distinct life-cycle stage that occurs early in tsetse fly transmission.

Imhof S, Knüsel S, Gunasekera K, Vu XL, Roditi I - PLoS Pathog. (2014)

Proteins up-regulated in late procyclic forms.Yellow: proteins showing ≥2-fold increase in abundance. Grey: ≤2-fold change in abundance. N.D. not detected. Biological replicates (exp1 and exp2) were performed.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214818&req=5

ppat-1004493-g005: Proteins up-regulated in late procyclic forms.Yellow: proteins showing ≥2-fold increase in abundance. Grey: ≤2-fold change in abundance. N.D. not detected. Biological replicates (exp1 and exp2) were performed.
Mentions: In culture, early and late procyclic forms are morphologically indistinguishable. Since GPEET was the only known marker for early procyclic forms at the beginning of this study, we used SILAC to identify additional proteins that were differentially expressed between GPEET-positive and GPEET-negative cells. Two independent experiments identified a limited number of candidates that were significantly different in at least one experiment (≥2-fold; Figures 4 and 5; Table S2). Of the differentially regulated proteins, three examples were encoded by related genes. These were the calflagins (Tb-44, Tb-24 and Tb-17), the two hexokinases (HK1 and HK2) and three adenylate cyclases. The members of a protein family could not be identified unequivocally as they contained identical peptides that are randomly assigned during mapping. Lacking antibodies that discriminated between isoforms, we analysed the transcripts for unique signatures. In the case of the adenylate cyclases (Tb927.5.285b, Tb927.5.320 and Tb 927.5.330 - here designated AC330, AC320 and AC285b) differences in their 3′ untranslated regions, allowed AC330 to be distinguished from AC320/285b by Northern blot analysis (Figure 6A). Both were differentially expressed, with AC330 up 9-fold in early procyclic forms and AC320/285b up 6.25-fold in late procyclic forms. Thus, the changes in protein levels detected by SILAC are probably an under-estimate for the individual proteins. Since an antiserum was available against calflagins, we monitored expression of these proteins by immunofluorescence. This revealed that calflagin-positive cells were always also positive for GPEET (Figure 6B). In addition, we performed quantitative RT-PCR to measure transcript levels in early and late procyclic forms (Figure 6C). This confirmed the differential expression at the level of mRNA for the adenylate cyclases and HK1/HK2. HK1 mRNA was expressed 4-fold more in early procyclic forms while HK2 was up-regulated 2-fold in late procyclic forms. In contrast to what was observed by immunofluorescence and SILAC, calflagin transcripts were down-regulated only 2-fold in late procyclic forms, suggesting that there is an additional level of regulation. Finally, we tested the mRNA levels of a set of putative pteridine transporters (PPT: Tb927.1.2850, Tb927.1.2880), which we have observed to be up-regulated (at least transiently) during differentiation of early to late procyclic forms; these were increased 4.6-fold in late procyclic forms. In summary, although no other gene is as tightly regulated as GPEET, we have identified several additional differentially regulated transcripts/proteins in early and late procyclic forms.

Bottom Line: These findings link the phenomenon of SoMo in vitro to the parasite forms found during the first 4-7 days of a midgut infection.Moreover, the process can be uncoupled from colonisation of the salivary glands.Although they are the major surface proteins of procyclic forms, EP and GPEET are not essential for SoMo, nor, as shown previously, are they required for near normal colonisation of the fly midgut.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology, University of Bern, Bern, Switzerland; Graduate School of Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.

ABSTRACT
The protozoan pathogen Trypanosoma brucei is transmitted between mammals by tsetse flies. The first compartment colonised by trypanosomes after a blood meal is the fly midgut lumen. Trypanosomes present in the lumen-designated as early procyclic forms-express the stage-specific surface glycoproteins EP and GPEET procyclin. When the trypanosomes establish a mature infection and colonise the ectoperitrophic space, GPEET is down-regulated, and EP becomes the major surface protein of late procyclic forms. A few years ago, it was discovered that procyclic form trypanosomes exhibit social motility (SoMo) when inoculated on a semi-solid surface. We demonstrate that SoMo is a feature of early procyclic forms, and that late procyclic forms are invariably SoMo-negative. In addition, we show that, apart from GPEET, other markers are differentially expressed in these two life-cycle stages, both in culture and in tsetse flies, indicating that they have different biological properties and should be considered distinct stages of the life cycle. Differentially expressed genes include two closely related adenylate cyclases, both hexokinases and calflagins. These findings link the phenomenon of SoMo in vitro to the parasite forms found during the first 4-7 days of a midgut infection. We postulate that ordered group movement on plates reflects the migration of parasites from the midgut lumen into the ectoperitrophic space within the tsetse fly. Moreover, the process can be uncoupled from colonisation of the salivary glands. Although they are the major surface proteins of procyclic forms, EP and GPEET are not essential for SoMo, nor, as shown previously, are they required for near normal colonisation of the fly midgut.

No MeSH data available.


Related in: MedlinePlus