AMPK and calcineurin are cellular master regulators with opposite activity during reverse vesicular development in Echinococcus granulosus larval stage

LOOS, JULIA A.; NICOLAO, MARÍA CELESTE; CUMINO, ANDREA C.

La Plata

Congreso; III Congreso Panamericano de Zoonosis- VIII Congreso Argentino de Zoonosis; 2014

INTRODUCTIONCestodes are platyhelminth parasites causing chronic infections to humans and domestic animals worldwide. Echinococcus granulosus life cycle includes two mammalian hosts. The intermediate hosts (ungulates and humans) ingest eggs that develop into a hydatid cyst containing larvae or protoscoleces (PTS) both long-lived larval forms. The hydatid cyst is limited by a cyst wall (CW), consisting of an inner germinal layer of metabolically active parasite cells and an outer carbohydrate-rich laminated layer, which restricts the host contact. Infection in the definitive host (canid) arises from ingestion of PTS encysted in the viscera of the intermediate host, and the consequent development into adult worms, which eliminate eggs to the environment completing the cycle. Additionally, E. granulosus shows an alternative development. When PTS are liberated into the circulation from a ruptured cyst located on an intermediate host, these are able to differentiate asexually into secondary hydatid cysts. For the larval stage development, it is widely accepted that glucose is the major respiratory substrate and glycogen the main energy storage molecule. On the other hand, the germinal layer constituted by totipotent cells, possesses a high metabolic activity due to being involved in the synthesis of the laminated layer (towards the outside of the cyst) and the generation of brood capsules containing PTS (towards the inside). Thus, energy and intermediate metabolites are the major metabolic demand for the germinal layer. These conditions generate a controlled management of energy and a caloric restriction in the parasite, and consequently a high autophagic activity during the larval stage. In search of therapeutic solutions for the cystic echinococcosis, our research group has determined that the pharmacological modulation of two cellular master regulators such as calcineurin and AMPK showed anti-echinococcal effect against this cestode. In this study, we analyzed the expression and localization of both proteins during the development process in the parasite.MATERIALS AND METHODSEchinococcus granulosus protoscoleces were removed under aseptic conditions from hydatid cysts of infected cattle obtained from abattoirs of the province of Buenos Aires, Argentina. Incubation of protoscoleces with insulin and FBS for several days induces a progressive de-differentiation towards the metacestode stage or microcyst. Microcysts represent the phase in which the protoscolex is completely transformed into a miniature cyst (loosing suckers, rostellum and hooks, and showing a laminated layer) and a pre-microcyst, a vesicle-like structure, is a completely vesicularised protoscolex with suckers and rostellum vestiges, without a laminated layer and almost devoid of movement. In order to obtain vesicularised protoscoleces and pre-microcysts, protoscoleces (n = 1,500/25-cm2 growth area per bottle) were cultured in medium 199 supplemented with antibiotics (penicillin, streptomycin and gentamicin; 100 g/ml), glucose (4 mg/ml), insulin (2 Uml-1) and 15% FBS. Cultures were maintained at 37 °C for 50 days and the medium was changed every 5-7 days. Meanwhile, the number of developed pre-microcysts was determined by observation under an inverted light microscope at different time points. For in toto immunohistochemistry, protoscoleces and pre-microcysts developed under in vitro conditions were fixed in 4% (w/v) paraformaldehyde in 0.1 M PBS (pH 7.4) for 4 h at 4 °C, and then washed for 24 h at 4 °C in permeabilizing solution (PBS at pH 7.4 containing 0.3% v/v Triton X-100, 0.2% w/v sodium azide and 0.5% w/v BSA). Parasites were incubated for 3-5 days at 4 °C with different human primary antibodies (phospho-AMPKα rabbit monoclonal Ab -Cell Signalling #2535-, total AMPK rabbit monoclonal Ab -Cell Signalling #5832- and calcineurinα rabbit polyclonal Ab ?Sigma #C1956- at 1:200 dilutions), and washed with PBS for 24 h at 4 °C. Finally, protoscoleces were incubated with goat anti-rabbit IgG conjugated with Alexa 488 or anti-mouse IgG conjugated with FITC for 24 h at 4 ºC, washed and counterstained with 2 μg/ml propidium iodide (Molecular Probes P-3566). They were observed with an inverted confocal laser scanning microscope (Nikon, Confocal Microscope C1). Negative controls consisted of omission of primary antibody for all experiments. The intensities of fluorescence were analyzed for 10 individual parasites and images were analyzed using Image J software (http://rsb.info.nih.gov/ij/). Data within experiments were compared and significance was determined using the student?s t test and p