A new MAP kinase protein involved in estradiol-stimulated reproduction of the helminth parasite Taenia crassiceps

Abstract

MAP kinases (MAPK) are involved in the regulation of cellular processes such as reproduction and growth. In parasites, the role of MAPK has been scarcely studied. Here, we describe the participation of an ERK-like protein in estrogen-dependent reproduction of the helminth parasite Taenia crassiceps. Our results show that 17beta-estradiol induces a concentration-dependent increase in the bud number of in vitro cultured cysticerci. If parasites are also incubated in presence of an ERK-inhibitor, the stimulatory effect of estrogen is blocked. The expression of ERK-like mRNA and its corresponding protein was detected in the parasite. The ERK-like protein was over-expressed by all treatments. Nevertheless, a strong induction of phosphorylation of this protein was observed only in response to 17beta-estradiol. Cross-contamination by host cells was discarded by flow cytometry analysis. Parasite cells expressing the ERK-like protein were exclusively located at the subtegument tissue by confocal microscopy. Finally, the ERK-like protein was separated by bidimensional electrophoresis and then sequenced, showing the conserved TEY activation motif, typical of all known ERK 1/2 proteins. Our results show that an ERK-like protein is involved in the molecular signalling during the interaction between the host and T. crassiceps, and may be considered as target for anti-helminth drugs design.

Figure 1

Concentration-response and response-time curves. 17β-estradiol augments the number of buds in Taenia crassiceps under physiologic concentrations (0.1 μM). Specific inhibition of ERK blocks the 17β-estradiol effect on the parasite, resulting in a similar number of buds between control and ERK inhibitor-treated cysticerci; (a) 17β-estradiol effect was progressively increasing during the five days of in vitro culture, whereas ERK inhibitor 0.5 μM did not modify parasite reproduction when compared to control cysticerci, even in presence of 17β-estradiol 0.1 μM, and (b) Control = Parasites treated with the vehicle where hormone and inhibitors were dissolved; **P < .05.

Figure 2

ERK-like expression in Taenia crassiceps. A single band of approximately 112 bp corresponding to ERK was detected in all in vitro treated cysticerci. Unexpectedly, either 17β-estradiol or ERK inhibitor treatment significantly induced ERK-like expression, related to control cysticerci. β-actin was used as control gene of constitutive expression. Optical densitometry was performed on 2% agarose gel from six independent experiments. Control = Parasites treated with the vehicle where hormone and inhibitors were dissolved; **P < .05.

Figure 3

ERK-like protein detection in Taenia crassiceps. Total ERK-like protein in T. crassiceps was not modified by 17β-estradiol or ERK inhibitor treatments (a), contrary to that observed in ERK-like gene expression. Nevertheless, 17β-estradiol specifically induced phosphorylation of ERK-like by four-fold respect to control groups (b). Optical densitometry was performed on 10% acrylamide gel from six independent experiments. Control = Parasites treated with the vehicle where hormone and inhibitors were dissolved; **P < .05.

Figure 4

Specific detection of ERK-like in Taenia crassiceps. FACS assays showed that ERK-like detected in T. crassiceps was not a contamination product from host immune cells CD3+, CD4+, CD8+, and CD19+. As expected, ERK-like was specifically detected in paramyosin positive cells, a cytoskeleton protein only present in cestodes and some insects (a). By using the Mean of Fluorescence Intensity (MFI), no differences in total ERK-like and pERK were observed between control and 17β-estradiol cysticerci (b). Control = Parasites treated with the vehicle where hormone and inhibitors were dissolved. Red = unstained cells; green=control parasites; blue = E₂-treated parasites; brown = ERK inhibitor-treated parasites; SSC = Side Scatter; FSC = Forward Scatter; pERK = ERK phosphorylated form; MFI = Fluorescence average.

Figure 5

ERK-like location in Taenia crassiceps by immunocytochemistry. (a) Transversal section of one T. crassiceps cysticercus where tegument, subtegument, and cells are observed with no staining. (b) Parasite cross-section incubated with FITC-conjugated goat anti-rabbit antibody, as staining control. Nonreacting cells are shown in red at 10× and 40×. Unspecific detection of ERK-like was not observed during our experiments. (c) Specific detection of ERK-like (green fluorescent) was located in parasite cells mainly disposed along subtegument tissue, 40×. (d) A magnification of 100× shows in detail the T. crassiceps cells expressing ERK-like exclusively on subtegument tissue. T = Tegument tissue; St = Subtegument tissue.

Figure 6

ERK-like detection by 2DE and protein sequencing. Total protein of in vitro cultured T. crassiceps was resolved in a double dimension gel (a). A well-defined dot (~55 KDa, pH = 6.8) was detected in T. crassiceps and then purified from the gel for sequencing (b). MW = Molecular weight marker.