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An Insight into the proteome of Crithidia fasciculata choanomastigotes as a comparative approach to axenic growth, peanut lectin agglutination and differentiation of Leishmania spp. promastigotes.

Alcolea PJ, Alonso A, García-Tabares F, Toraño A, Larraga V - PLoS ONE (2014)

Bottom Line: A ground-breaking analysis of differential protein abundance in Crithidia fasciculata is reported herein.The comparison of the outcome with previous gene expression profiling studies developed in the related human pathogens of the genus Leishmania has revealed substantial differences between the motile stages of these closely related organisms in abundance of proteins involved in catabolism, redox homeostasis, intracellular signalling, and gene expression regulation.The result is that choanomastigotes are able to agglutinate with peanut lectin and a non-agglutinating subpopulation can be also isolated.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Biology of Infections and Service of Proteomics and Genomics, Centro de Investigaciones Biológicas (Consejo Superior de Investigaciones Científicas), Madrid, Spain.

ABSTRACT
The life cycle of the trypanosomatid Crithidia fasciculata is monogenetic, as the unique hosts of these parasites are different species of culicids. The comparison of these non-pathogenic microorganisms evolutionary close to other species of trypanosomatids that develop digenetic life cycles and cause chronic severe sickness to millions of people worldwide is of outstanding interest. A ground-breaking analysis of differential protein abundance in Crithidia fasciculata is reported herein. The comparison of the outcome with previous gene expression profiling studies developed in the related human pathogens of the genus Leishmania has revealed substantial differences between the motile stages of these closely related organisms in abundance of proteins involved in catabolism, redox homeostasis, intracellular signalling, and gene expression regulation. As L. major and L. infantum agglutinate with peanut lectin and non-agglutinating parasites are more infective, the agglutination properties were evaluated in C. fasciculata. The result is that choanomastigotes are able to agglutinate with peanut lectin and a non-agglutinating subpopulation can be also isolated. As a difference with L. infantum, the non-agglutinating subpopulation over-expresses the whole machinery for maintenance of redox homeostasis and the translation factors eIF5a, EF1α and EF2, what suggests a relationship between the lack of agglutination and a differentiation process.

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2DE of total protein extracts throughout the growth curve and of the PNA+ and PNA- subpopulations of C. fasciculata choanomastigotes.2DE of 50 µg of total protein extracts of C. fasciculata choanomastigotes at (A) early logarithmic, (B) mid logarithmic, (C) late logarithmic and (D) stationary phase. (E) PNA- and (F) PNA+ subpopulations. One out of three replicates is shown for each phase/subpopulation. IEF was performed in a non-linear 3–10 pH interval. Complete spot names include Cf (A–D) or Cfp (E and F) preceding the spot numbers (see Tables 1–3).
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pone-0113837-g002: 2DE of total protein extracts throughout the growth curve and of the PNA+ and PNA- subpopulations of C. fasciculata choanomastigotes.2DE of 50 µg of total protein extracts of C. fasciculata choanomastigotes at (A) early logarithmic, (B) mid logarithmic, (C) late logarithmic and (D) stationary phase. (E) PNA- and (F) PNA+ subpopulations. One out of three replicates is shown for each phase/subpopulation. IEF was performed in a non-linear 3–10 pH interval. Complete spot names include Cf (A–D) or Cfp (E and F) preceding the spot numbers (see Tables 1–3).

Mentions: Choanomastigote cultures reached the stationary phase within 3 days (Fig. 1B), twice as fast as Leishmania spp. promastigotes. Similar growth kinetics of C. fasciculata choanomastigote clones has been reported [20]. Total protein of 5×108 choanomastigotes was extracted at early logarithmic (day 1), mid-logarithmic (day 2), late logarithmic/early stationary (day 3) and stationary phase (day 4). In addition, protein extracts were successfully obtained from the PNA+ and PNA- subpopulations within the cultures in stationary phase. Protein concentrations were comprised between 4 and 9 µg/µl and this was confirmed by PAGE-SDS as described [14]. After 2DE separations, normalization with the Total Quantity in Valid Spots algorithm and manual check of all the spots, 136 changes in abundance of proteins were detected throughout the four time points of the growth curve. Some proteins showed significant differences in abundance in more than one time point of the choanomastigote growth curve. The cut-off values were: ratio to day 1, R≥1.7 or≤0.6 within the significance level inferred with Student's t test (p<0.05). Of these, 63 spots that correspond to 83 differences in abundance (Fig. 2A–D, Table 1) were excised from the 2DE gels as they were suitable to be identified by MALDI-TOF/TOF. Therefore, 10 proteins are differentially expressed at two of the time points compared. We also analyzed 43 spots containing constantly expressed proteins (Table 2), as well as 67 spots differentially expressed between the PNA- and PNA+ subpopulations within the stationary phase culture (Fig. 2E and F, Table 3). All proteins could be identified when MASCOT searches were performed against the reference genome sequence of C. fasciculata (Tables 1–3) when there was sufficient amount for identification, whereas a total of 20 constantly expressed proteins (Table S2 in S1 File), 41 differentially expressed proteins in the growth curve (Table S1 in S1 File) and 30 proteins with different abundance between PNA+ and PNA- choanomastigotes (Table S3 in S1 File) could be identified against the NCBInr database, which is 53.4% of the proteins analyzed by MALDI-TOF/TOF mass spectometry. Most of the identifications (73.9%) were consistent between the two databases and most of those successfully performed with the NCBInr database (63.9%) had the highest MASCOT scores for orthologue proteins of the genus Leishmania, whereas only 9.3% of them matched with a Trypanosoma spp. orthologue (Tables S1–S3 in S1 File). This is additional evidence for the hypothesis supporting very close evolutionary relationship between Leishmania spp. and Crithidia spp [6], [7], [8], [9]. Also, only 9.3% matched with Crithidia spp., as very few genes had been identified in this organism prior to the release of the reference genome sequence (Tables S1–S3 in S1 File). Sometimes, different spots represent the same type of protein. This may be due to the presence of different isoforms, post-translational modifications or protein aggregation at the conditions assayed. In the next sections, we refer to these possibilities using the term variant of a given protein.


An Insight into the proteome of Crithidia fasciculata choanomastigotes as a comparative approach to axenic growth, peanut lectin agglutination and differentiation of Leishmania spp. promastigotes.

Alcolea PJ, Alonso A, García-Tabares F, Toraño A, Larraga V - PLoS ONE (2014)

2DE of total protein extracts throughout the growth curve and of the PNA+ and PNA- subpopulations of C. fasciculata choanomastigotes.2DE of 50 µg of total protein extracts of C. fasciculata choanomastigotes at (A) early logarithmic, (B) mid logarithmic, (C) late logarithmic and (D) stationary phase. (E) PNA- and (F) PNA+ subpopulations. One out of three replicates is shown for each phase/subpopulation. IEF was performed in a non-linear 3–10 pH interval. Complete spot names include Cf (A–D) or Cfp (E and F) preceding the spot numbers (see Tables 1–3).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263474&req=5

pone-0113837-g002: 2DE of total protein extracts throughout the growth curve and of the PNA+ and PNA- subpopulations of C. fasciculata choanomastigotes.2DE of 50 µg of total protein extracts of C. fasciculata choanomastigotes at (A) early logarithmic, (B) mid logarithmic, (C) late logarithmic and (D) stationary phase. (E) PNA- and (F) PNA+ subpopulations. One out of three replicates is shown for each phase/subpopulation. IEF was performed in a non-linear 3–10 pH interval. Complete spot names include Cf (A–D) or Cfp (E and F) preceding the spot numbers (see Tables 1–3).
Mentions: Choanomastigote cultures reached the stationary phase within 3 days (Fig. 1B), twice as fast as Leishmania spp. promastigotes. Similar growth kinetics of C. fasciculata choanomastigote clones has been reported [20]. Total protein of 5×108 choanomastigotes was extracted at early logarithmic (day 1), mid-logarithmic (day 2), late logarithmic/early stationary (day 3) and stationary phase (day 4). In addition, protein extracts were successfully obtained from the PNA+ and PNA- subpopulations within the cultures in stationary phase. Protein concentrations were comprised between 4 and 9 µg/µl and this was confirmed by PAGE-SDS as described [14]. After 2DE separations, normalization with the Total Quantity in Valid Spots algorithm and manual check of all the spots, 136 changes in abundance of proteins were detected throughout the four time points of the growth curve. Some proteins showed significant differences in abundance in more than one time point of the choanomastigote growth curve. The cut-off values were: ratio to day 1, R≥1.7 or≤0.6 within the significance level inferred with Student's t test (p<0.05). Of these, 63 spots that correspond to 83 differences in abundance (Fig. 2A–D, Table 1) were excised from the 2DE gels as they were suitable to be identified by MALDI-TOF/TOF. Therefore, 10 proteins are differentially expressed at two of the time points compared. We also analyzed 43 spots containing constantly expressed proteins (Table 2), as well as 67 spots differentially expressed between the PNA- and PNA+ subpopulations within the stationary phase culture (Fig. 2E and F, Table 3). All proteins could be identified when MASCOT searches were performed against the reference genome sequence of C. fasciculata (Tables 1–3) when there was sufficient amount for identification, whereas a total of 20 constantly expressed proteins (Table S2 in S1 File), 41 differentially expressed proteins in the growth curve (Table S1 in S1 File) and 30 proteins with different abundance between PNA+ and PNA- choanomastigotes (Table S3 in S1 File) could be identified against the NCBInr database, which is 53.4% of the proteins analyzed by MALDI-TOF/TOF mass spectometry. Most of the identifications (73.9%) were consistent between the two databases and most of those successfully performed with the NCBInr database (63.9%) had the highest MASCOT scores for orthologue proteins of the genus Leishmania, whereas only 9.3% of them matched with a Trypanosoma spp. orthologue (Tables S1–S3 in S1 File). This is additional evidence for the hypothesis supporting very close evolutionary relationship between Leishmania spp. and Crithidia spp [6], [7], [8], [9]. Also, only 9.3% matched with Crithidia spp., as very few genes had been identified in this organism prior to the release of the reference genome sequence (Tables S1–S3 in S1 File). Sometimes, different spots represent the same type of protein. This may be due to the presence of different isoforms, post-translational modifications or protein aggregation at the conditions assayed. In the next sections, we refer to these possibilities using the term variant of a given protein.

Bottom Line: A ground-breaking analysis of differential protein abundance in Crithidia fasciculata is reported herein.The comparison of the outcome with previous gene expression profiling studies developed in the related human pathogens of the genus Leishmania has revealed substantial differences between the motile stages of these closely related organisms in abundance of proteins involved in catabolism, redox homeostasis, intracellular signalling, and gene expression regulation.The result is that choanomastigotes are able to agglutinate with peanut lectin and a non-agglutinating subpopulation can be also isolated.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Biology of Infections and Service of Proteomics and Genomics, Centro de Investigaciones Biológicas (Consejo Superior de Investigaciones Científicas), Madrid, Spain.

ABSTRACT
The life cycle of the trypanosomatid Crithidia fasciculata is monogenetic, as the unique hosts of these parasites are different species of culicids. The comparison of these non-pathogenic microorganisms evolutionary close to other species of trypanosomatids that develop digenetic life cycles and cause chronic severe sickness to millions of people worldwide is of outstanding interest. A ground-breaking analysis of differential protein abundance in Crithidia fasciculata is reported herein. The comparison of the outcome with previous gene expression profiling studies developed in the related human pathogens of the genus Leishmania has revealed substantial differences between the motile stages of these closely related organisms in abundance of proteins involved in catabolism, redox homeostasis, intracellular signalling, and gene expression regulation. As L. major and L. infantum agglutinate with peanut lectin and non-agglutinating parasites are more infective, the agglutination properties were evaluated in C. fasciculata. The result is that choanomastigotes are able to agglutinate with peanut lectin and a non-agglutinating subpopulation can be also isolated. As a difference with L. infantum, the non-agglutinating subpopulation over-expresses the whole machinery for maintenance of redox homeostasis and the translation factors eIF5a, EF1α and EF2, what suggests a relationship between the lack of agglutination and a differentiation process.

Show MeSH
Related in: MedlinePlus