Entamoeba histolytica: Cysteine proteinase activity and virulence. Focus on cysteine proteinase 5 expression levels Michelle A.R. Freitas a , Helen C. Fernandes a , Viviane C. Calixto b , Almir S. Martins b , Edward F. Silva a , Jorge L. Pesquero b , Maria A. Gomes a, * a Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazil b Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, MG, Brazil article info Article history: Received 13 August 2008 Received in revised form 27 March 2009 Accepted 13 April 2009 Available online 21 April 2009 Keywords: Entamoeba histolytica Cysteine proteinase 5 Pathogenesis of amoebiasis abstract Cysteine proteinase (CP) activity and CP5 mRNA levels were analyzed in eleven samples of Entamoeba histolytica isolated from patients presenting different clinical profiles. The virulence degree of the isolates, determined in hamster liver, correlated well with the clinical form of the patient and culture conditions. CP5 mRNA levels were also determined in sample freshly picked up directly from liver amoebic abscess. Differ- ences were not observed in the levels of CP5 mRNA and CP specific activity among the cultured samples. However, different levels of CP5 mRNA were observed in trophozoite freshly isolated from hepatic amoebic lesions. These results reinforce the importance of CP5 for the virulence of amoebae and the need for studies with the parasite present in lesions to validate mechanisms involved in pathogenesis of amoebiasis. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction The great variability in the pathogenic potential of Entamoeba histolytica reflects in differences on its virulence to the host. An in- fected person may be asymptomatic or present invasive form, responsible for intestinal and extra-intestinal infections. Data in the literature have pointed many molecular factors as important for amoeba virulence, a necessary condition to the parasite reach tissues and to produce lesion (Olivos-Garcia et al., 2004; Stanley et al., 1995). Among factors that influence the invasive process, the cysteine proteinases (CPs) are particularly worthy of note. It seems there is a correlation between the pathogenicity of the iso- lates and the cysteine proteinases expression levels in E. histolytica (Bruchhaus et al., 2003; Garcia-Rivera et al., 1999). The activity lev- els of these enzymes appear to determine the gravity of the clinical profile in amoebiasis (Que and Reed, 1997). CPs degrade human co- lonic mucus allowing subsequent cytolysis dependent on contact with the intestinal epithelium (Moncada et al., 2003), degrade extracellular matrix proteins (Keene et al., 1990; Li et al., 1995) and provoke microvillar effacement (Lauwaet et al., 2003). They also interact with the host immune system activating the comple- ment system and interleukin (Zhang et al., 2000), as well as degrading IgG, IgA, C3a and C5a (Reed et al., 1995). With the presentation of E. histolytica genome by Loftus et al. (Loftus et al., 2005), 10 new CP genes (ehcp) have been identified in addition to that already described. Among these genes, one cod- ing for CP1 appears to have been lost in Entamoeba dispar (Bruch- haus et al., 1996) that does not invade tissues. Another gene, ehcp5, which codes for CP5, a membrane-bound proteinase, is highly degenerated in E. dispar (Willhoeft et al., 1999). The presence of CP5 on the surface of the amoeba, associated with its absence in E. dispar makes it an excellent candidate for a key molecule in the invasive capacity of E. histolytica (Moncada et al., 2006). To as- sess the importance of CPs for the growth and pathogenicity of E. histolytica, Nowak et al. (2004) cultivated amoebae in the pres- ence of various cysteine proteinase inhibitors. E64, one of these inhibitors was deleterious to amoebae, which rapidly presented a significant reduction in its virulence due to the inhibition of CP5 expression. This amoeba showed to be ineffective to degrade colo- nic mucin, a prerequisite for invasion of the colonic mucosa. Prob- ably such reduction of virulence is related to the inability of trophozoites to invade mucosa. In this study, we analyzed the CP5 mRNA and CP activity levels in E. histolytica strains isolated from patients presenting different clinical forms of the disease in order to clarify the contribution of this enzyme for the virulence of the parasite. To access the CP5 mRNA levels on the locus of abscess, total RNA was extracted from pieces of liver presenting amoebic abscess and used it in RT-PCR. 2. Materials and methods 2.1. Entamoeba histolytica strains Nine strains of E. histolytica obtained from patients presenting different clinical profiles were studied. These strains have been 0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2009.04.005 * Corresponding author. Fax: +55 31 34092970. E-mail address: magomes@icb.ufmg.br (M.A. Gomes). Experimental Parasitology 122 (2009) 306–309 Contents lists available at ScienceDirect Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr