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

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.

Figure 1. Life cycle, growth kinetics and peanut lectin agglutination of C. fasciculata choanomastigotes.

(A) The monogenetic life cycle of C. fasciculata involves a culicid host, where amastigotes attached to the gut epithelium and voided in faeces are disseminated in the environment and orally passed to other hosts at any of the developmental stages. Choanomastigotes are the motile stage that allows the colonization of the gut of the host. GE: gut epithelium. Adapted from Olsen, 1974. (B) Average growth curve of three C. fasciculata choanomastigote cultures (three biological replicates). Total proteins were extracted every day until the culture reached the stationary phase. N is the average cell density. (C) and (D) 10% Giemsa staining of the PNA⁺ and PNA^- C. fasciculata choanomastigote subpopulations within the stationary phase of axenic culture, respectively.

Figure 2. 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 Cf_p (E and F) preceding the spot numbers (see Tables 1–3).

Figure 3. Differentially expressed proteins related with carbohydrate metabolic processes in C. fasciculata choanomastigotes.

Functional connection of differentially expressed enzymes involved in glucid metabolic processes. Legend: proteins/protein variants in blue are constitutively expressed throughout the growth curve; proteins/protein variants in orange are up-regulated at day 1 or 2 (logarithmic phase); proteins/variants in green are up-regulated at day 3 or 4 (late logarithmic/stationary phase); proteins in red are up-regulated in PNA⁺ choanomastigotes; proteins in purple are up-regulated in PNA^- choanomastigotes.

Figure 4. 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).

Figure 5. Differentially expressed proteins involved in redox homeostasis and translation in C. fasciculata choanomastigotes.

(A) The redox control system. Legend: proteins/protein variants in blue are constitutively expressed throughout the growth curve; proteins/protein variants in red are up-regulated at day 1 or 2 (logarithmic phase); proteins in purple are up-regulated in PNA^- choanomastigotes. (B) Summary of differential abundance of translation factors and enzymes involved in redox homeostasis throughout the growth curve and in the PNA^- subpopulation of choanomastigotes in stationary phase.