Uptake of cholesterol by Tetrahymena thermophila is mainly due to phagocytosis

EUGENIA ELGUERO; MARIA LUZ SANCHEZ; MONTES, GUADALUPE; CID NICOLAS; NICOLAS O. FAVALE; B.C. NUDEL; A. D. NUSBLAT

REVISTA ARGENTINA DE MICROBIOLOGíA

ASOCIACION ARGENTINA MICROBIOLOGIA

Lugar: Buenos Aires; Año: 2017

0325-7541

Thefree-living ciliate Tetrahymenathermophilais a unicellular model organism in which landmark biologicalprocesses have beendiscovered, such as the first description oftelomerase activityand themolecular structure of telomeres, the mechanism of self-splicing RNA andribozymes, the function of histone acetylation in transcription regulation and anumber of pioneer experiments on the interference (RNAi) mechanism for programmed genomerearrangements, among others4.In contrastto most eukaryotic cells that requiresterols in their membranes, Tetrahymena satisfies its vegetativegrowth synthesizingtetrahymanol (a sterol surrogate similar to hopanoids foundin bacteria). Nevertheless, if a sterol is present, the biosynthesis oftetrahymanol is immediately repressed and the sterol is incorporated in themembrane through an unknown pathway2.The study of the uptake of cholesterol inTetrahymena contributes to the understanding pathways of sterols endocytosisthat fulfills important functions in higher eukaryotes, which still remainsunclear and share similarity with this unicellular organism used as powerfultoolkit.To date, atleast four distinct pathways of endocytic uptake have been identified:endocytosis of small vesicles from parasomal sacs, uptake of larger vesicles atthe base of oral apparatus (phagosomes), endocytosis coupledwith the exocytosis of dense-core secretory vesicles (membrane turnover) andendocytic membrane recovery upon phagosome fusion, at a cortical site(cytoproct)2.We performedan assay to identify the endocytic pathway involved in cholesterol uptake. Forthis purpose, a wild type TetrahymenathermophilaCU399 strain (WT)and a mutant obtained thereof, defective only in phagocytosis (II8G)5 were grown in rich culture medium containingthe fluorescent cholesterol analogue (Bodipy Cholesterol).The BODIPY not affect physic properties of cholesterol dissolved withvortex agitation in ethanol as primary solvent at 37ºC with a concentration of25 mm.The final ethanol concentration in these experiments did not exceed 0.1% (v/v)in culture medium.Simultaneously Escherichiacoliexpressing a red fluorescent protein (Ds Red) was added into the culturemedia and used as phagocytosis marker1.Upper panelsof the figure 1 show theWT strain with large food vacuoles containingcholesterol and E. coli fluorescentcells, whereas the mutant II8G does not display any fluorescent signal. Lowerpanels show both strains analyzed separately, to test absolute phenotype ineach population.The above result indicates that cholesterol, andprobably all sterols, are incorporated via phagocytosis and not by otherendocytic mechanisms. Conversely, in the ciliate Paramecium tetraurelia, uptake assays have indicated thatcholesterol is incorporated not only by phagocytosis but also through theplasma membrane3, suggestingdifferent endocyticpreferencesin these two evolutionary close organisms.