Giardia intestinalis induces alterations on structural and functional components, in a cultured human enterocyte-like Caco-2/TC7 cells model.

HUMEN, M. .A.; LIÉVIN-LE MOAL V.; SERVIN A.; PÉREZ P.F.

Congreso; XII Congreso Argentino de Microbiología.; 2010

Giardia intestinalis is the etiological agent of giardiasis, one of the most frequent intestinal diseases in both developing and developed countries, leading to severe and protracted diarrhea. The mechanisms used by G. intestinalis to induce structural and functional alterations in host epithelia still remain unknown.   In the present work, we sought to gain insight in the specific and directed alterations on structural and functional components induced by G. intestinalis in host epithelia, and the activation of relevant signaling pathways.   In order to evaluate the cellular damage of infected enterocytes, we assessed different markers related to cellular architecture (F-actin cytoskeleton and intermediate filaments, cytokeratin 18), distribution of brush border associated enzymes (sucrase-isomaltase (SI), dipeptidylpeptidase IV (DPP IV) and the fructose transporter GLUT 5), as well as distribution of intercellular junctional proteins and adhesion molecules (occludin, claudin-1 and E-cadherin). Transferrin receptor and focal adhesion kinases (FAK) were also analyzed. Immunofluorescence labeling of fixed post-confluent Caco-2/TC7 cells was conducted and samples were examined by confocal microscopy. In addition, we assessed, by a pharmacological approach, phosphorilation processes and cell signaling pathways activated upon G. intestinalis infection. Cells incubated in the presence or absence of different inhibitors of signaling pathways, were lysated and analysed by SDS-PAGE.. Detection of occludin, mitogen-activated protein kinases (ERK1/2, p38 and JNK) and Src-kinases was carried out by western blot.   Parasite-epithelia interaction promoted the disorganization of the apical F-actin cytoskeleton, and leads to an uneaven distribution of the brush border-associated  sucrase-isomaltase and fructose transporter, GLUT5. Nevertheless, DDPIV and the transferrin receptor did not modify their distribution in the surface of the enterocyte. Furthermore, the epithelial barrier was altered as the result of dramatic rearrangements in the pattern of functional tight junction-associated proteins, occludin and claudin-1. In addition, G. intestinalis infection promoted the phosphorylation of occludin in Caco-2/TC7 cells. Cytokeratin 18, FAK and E-cadherins were not modified after infection. G. intestinalis induced a strong activation of the mitogen-activated protein kinase Erk1/2 in a PI3-kinase dependent manner, and a moderate activation of JNK and p38. No activation of Src kinase was observed.     Our findings are relevant to understand the mechanisms associated with Giardia-induced intestinal disorders. Noteworthy a generalized cellular damage did not occurred and only specific signalling cascades are activated. These results disclose novel aspects of the pathogenesis of Giardia infection that could contribute to improve our understanding of this parasite in the context of preventive and therapeutic strategies.