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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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CACK is up-regulated in logarithmic phase choanomastigotes.Detection and differential expression analysis of CACK in 20 µg total protein extracts by Western blot with 1∶500 polyclonal antibody against the LACK analogue throughout the choanomastigote growth curve. The ∼60 KDa band presumably contains dimeric CACK aggregates (González-Aseguinolaza et al., 1999). gGAPDH is the protein of reference (dilution 1∶10,000 of the monoclonal antibody).
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pone-0113837-g004: CACK is up-regulated in logarithmic phase choanomastigotes.Detection and differential expression analysis of CACK in 20 µg total protein extracts by Western blot with 1∶500 polyclonal antibody against the LACK analogue throughout the choanomastigote growth curve. The ∼60 KDa band presumably contains dimeric CACK aggregates (González-Aseguinolaza et al., 1999). gGAPDH is the protein of reference (dilution 1∶10,000 of the monoclonal antibody).

Mentions: Regarding intracellular signalling, the C. fasciculata analogue (CACK) (gi3132790; CfaC1_26_3810) of the receptor of the activated protein kinase C (RACK) is up-regulated in early logarithmic phase choanomastigotes (day 1) (Fig. 2, Table 1). This expression profile has been confirmed by Western blot (Fig. 4, Figures S1 and S2 in S2 File), which has revealed the progressive descent of CACK abundance throughout the choanomastigote growth curve. The leishmanial orthologue LACK, an antigenic protein that partially protects against canine leishmaniasis [32], [33], [34], is located in the particulate fraction of the cytoplasm near the plasma membrane. LACK is up-regulated in L. infantum amastigotes but constantly expressed in promastigotes [18], [24]. Consequently, the CACK/LACK expression patterns in the motile stages of C. fasciculata and L. infantum are different, what suggests different roles in proliferation and differentiation in their respective life cycles. PKCs are translocated by their receptor (RACK) to different intracellular sites [35] and activated via phospholipase C or Ca2+. PKCs and RACKs are involved in a variety of characterized signal transduction cascades in mammals [36]. However, their role in specific pathways is unknown in these parasites. Although the kinomes of trypanosomatids are well characterized [37], the signaling pathways may not be necessarily the same as in other organisms like yeasts and mammals. Signaling proteins are expected to regulate gene expression but most of the specific mechanisms and pathways have not been unraveled yet and may be different given the unique features of gene expression in these parasites (reviewed in [38]). The unknown specific function of this protein in signaling may be especially important in Leishmania spp. for resistance of amastigotes inside the parasitophorous vacuole of the host phagocyte cell whereas the only colonization step of the Crithidia spp. life cycle is the infection of the gut of the insect host. CACK and LACK seem to be important for growth and/or development of the motile stages of the respective species they belong to, as up-regulation in C. fasciculata logarithmic phase choanomastigotes has been found herein and its leishmanial orthologue is one of the 50 most abundant transcripts of L. major promastigotes [39].


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)

CACK is up-regulated in logarithmic phase choanomastigotes.Detection and differential expression analysis of CACK in 20 µg total protein extracts by Western blot with 1∶500 polyclonal antibody against the LACK analogue throughout the choanomastigote growth curve. The ∼60 KDa band presumably contains dimeric CACK aggregates (González-Aseguinolaza et al., 1999). gGAPDH is the protein of reference (dilution 1∶10,000 of the monoclonal antibody).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0113837-g004: CACK is up-regulated in logarithmic phase choanomastigotes.Detection and differential expression analysis of CACK in 20 µg total protein extracts by Western blot with 1∶500 polyclonal antibody against the LACK analogue throughout the choanomastigote growth curve. The ∼60 KDa band presumably contains dimeric CACK aggregates (González-Aseguinolaza et al., 1999). gGAPDH is the protein of reference (dilution 1∶10,000 of the monoclonal antibody).
Mentions: Regarding intracellular signalling, the C. fasciculata analogue (CACK) (gi3132790; CfaC1_26_3810) of the receptor of the activated protein kinase C (RACK) is up-regulated in early logarithmic phase choanomastigotes (day 1) (Fig. 2, Table 1). This expression profile has been confirmed by Western blot (Fig. 4, Figures S1 and S2 in S2 File), which has revealed the progressive descent of CACK abundance throughout the choanomastigote growth curve. The leishmanial orthologue LACK, an antigenic protein that partially protects against canine leishmaniasis [32], [33], [34], is located in the particulate fraction of the cytoplasm near the plasma membrane. LACK is up-regulated in L. infantum amastigotes but constantly expressed in promastigotes [18], [24]. Consequently, the CACK/LACK expression patterns in the motile stages of C. fasciculata and L. infantum are different, what suggests different roles in proliferation and differentiation in their respective life cycles. PKCs are translocated by their receptor (RACK) to different intracellular sites [35] and activated via phospholipase C or Ca2+. PKCs and RACKs are involved in a variety of characterized signal transduction cascades in mammals [36]. However, their role in specific pathways is unknown in these parasites. Although the kinomes of trypanosomatids are well characterized [37], the signaling pathways may not be necessarily the same as in other organisms like yeasts and mammals. Signaling proteins are expected to regulate gene expression but most of the specific mechanisms and pathways have not been unraveled yet and may be different given the unique features of gene expression in these parasites (reviewed in [38]). The unknown specific function of this protein in signaling may be especially important in Leishmania spp. for resistance of amastigotes inside the parasitophorous vacuole of the host phagocyte cell whereas the only colonization step of the Crithidia spp. life cycle is the infection of the gut of the insect host. CACK and LACK seem to be important for growth and/or development of the motile stages of the respective species they belong to, as up-regulation in C. fasciculata logarithmic phase choanomastigotes has been found herein and its leishmanial orthologue is one of the 50 most abundant transcripts of L. major promastigotes [39].

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