Estudo das alterações morfológicas induzidas pela Toxina A, do Clostridium difficile em células epiteliais intestinais e do efeito protetor da Glutamina e Alanil-glutamina

Abstract

Clostridium difficile is the major cause of antibiotic-associated colitis, a disease with significant morbidity and mortality. Glutamine (Gln), a non-essential aminoacid, is a major fuel for the dynamic intestinal cell population. Alanyl-glutamine (Ala-Gln) is a dipeptide that is highly soluble and well tolerated. The aim of this study was to analyze the changes induced by Clostridium difficile toxin A (TcdA) in intestinal epithelial cell morphology and cytoskeletal element and the effect of Gln and Ala-Gln treatment, using advanced microscopic techniques. Twelve well cell culture plates, with 13 mm diameter glass coverlids, were seeded with 5x105 IEC-6 cells and grown for 24h in DMEM media. Afterwards, the wells were incubated for 24h as follow: control, TcdA (10 ng/mL), TcdA + Gln (10 mM) and TcdA + Ala-Gln (10 mM). The cells were than fixed in 4% formaldehyde for 14 h and afterwards they were examined by atomic force microscopy (AFM), scanning electronic microscopy (SEM) and Fluorescent microscopy. For the SEM the samples were fixed to samples holders with carbon adhesive tape and covered with a 15 mm gold film for conductivity by sputter. To fluorescent microscopy the cells were permeabilizided with PBS/Triton after that they were marked with stained with FITC-RhoA, Rodhamine-phallodin and DAPI performed using an inverted fluorescence microscope. An immunoblotting was realized with the same groups. The PVDF membrane was incubated with RhoA antibody overnight and afterwards activated by Amershan kit. The proteic control was made by α- tubulin. Also performed experiments of cellular proliferation and oxidative stress. As observed by AFM, SEM and Fluorescent microscopy TcdA caused intense cell shrinkage with multiple extensions. This change in shape was associated with collapse of the F-actin cytoskeleton demonstrated by fluorescent microscopy. An increase of RhoA production was detected in the groups treated with Gln e Ala-Gln. We demonstrated that TcdA dramatically altered the intestinal cell morphology and cytoskeleton organization and that Ala-Gln and Gln supplementation consistently prevented the intestinal epithelial cell damage induced by TcdA probably by increasing RhoA expression. The TcdA induced a reduction of 8.4% in cell proliferation while the Ala-Gln and Gln increased by 13.2% and 12.7%, respectively. The TcdA induced cells to oxidative damage, which was reversed by the use of Gln and Ala-Gln.. Our findings provide rationale for the potential use of Ala-Gln and Gln as adjuvant therapy in Clostridium difficile disease. Investigation of morphological and cytoskeleton changes using advanced microscopic techniques may aid in the evaluation of the protective or therapeutic activity of drugs against TcdA effects.