Volume 28, Suppl., pp. S 190-S192, 1997 Printed in Mexico Archives of Medical Research Differential Display of mRNAs from Entamoeba histolytica During Electron Dense Granules Secretion 1 ISABEL SALAZAR, ARTURO ORTEGA, PRADEEP DAS, JOSE LUIS SANCHEZ - SALAS and MARIA DE LOURDES M ~ O Z Dcpartanrcnto tie Go~dicci y Biologia Molcc~~lar. Centro tic Investigucicin y tic Estzrtfios Avanzado.r tiel Inrtituto Po1itt;cnico Nucionul, Mbico, U.F., Mexico Introduction Ainebiasis, a parasitic infection in man due to the protozoan Entarizoeha histolyticu, is an invasive enteric illness that can spread to multiple tissues. Because collagen is one of the inajor components of the extracel- lular inatrix from human tissues, collagenase from E. histolytica has been considered an important factor in the pathogenicity of this parasite. This activity is associated with electron dense granules (EDG) that are secreted to the extracellular milieu when trophozoites are in contact with collagen type I and Ca 2 ' (1). Consequently, this study aims at elucidating the molecular events triggered by the interaction of E. histolytica trophozoites with collagen, a inajor extracellular inatrix (ECM) coinpo- nent and Ca 2 +.This interaction is particularly important since it induces tyrosine kinase cascades as a key ele- ment leading to cytoskeletal organization and expres- sion of specific genes. These events will result in an increased adhesion and release of proteolytic activities allowing the parasite to invade its host (2). Emphasis was placed upon a different pattern of gene expression that results in the invasiveness of the host tissue. Recently, we have started to dessicate the biochemical cascades triggered by collagen (3). Moreover, we have identified ameba hoinologues of key inolecules that link extracel- lular signaling with regulation of gene expression (3). -- Correspondence to: Dr. Maria de Lourdes Muiioz, Department of Genetics and Molecu- lar Biology, Centro de Investigation y de Estudios Avanzados del IPN, Av. Instituto PolitPcnico Nacional 2508, Col. San Pedro Zacatenco, Mtxico D. F., C.P. 07360. Tel. (5) 747-7000 Ext. 5300; FAX: 747-7 100. ' This work was supported by a grant from CONACYT Mtxico (0252P-M9506) The knowledge generated in this study will certainly pave the way to a better understanding of the virulence mechanisms of E. histol~jticn. Materials and Methods Genes Transcribed in Response to the Stimulation of Trophozoites with Collagen Type I and CaZ+ in E.histolytica. To study differential gene expression we used differential mRNA display that has been recently described and used to identify differences in subsets of inRNA levels (4). Differential mRNADisplay. Total RNA was isolated from control and collagen-Ca 2 +treated trophozoites us- ing the guanidinium-CsC1 method and mRNA was puri- fied by means ofoligo dT chromatography. RT-PCR was performed in two independent experiments. Fragments of cDNA were separated and displayed on a sequencing gel. The highly reproducible cDNA patterns demon- strated that samples originating from control and treated cells have similar RNA composition, with some obvious differences. Some of the differentially displayed bands that are present only in samples of treated cells were eluted from the sequencing gel as described (5), and subsequently reainplified by PCR. Results and Discussion Differential display is a powerful way to isolate genes that are differentially expressed in two different states of the cell, a response to treatment, or combination of treatments, without cloning a single gene. One potential inaccuracy in these estimates is that rare transcripts are under-represented.