Src and PI3 K inhibitors affect the virulence factors of Entamoeba histolytica L. LÓPEZ-CONTRERAS, V. I. HERNÁNDEZ-RAMÍREZ, Y. FLORES-GARCÍA, B. CHÁVEZ-MUNGUÍA and P. TALAMÁS-ROHANA* Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies, Av. IPN 2508, San Pedro Zacatenco, 07360, Mexico City, Mexico (Received 16 March 2012; revised 20 June 2012; accepted 19 August 2012; first published online 12 October 2012) SUMMARY Protein kinases (PKs) of parasitic protozoa are being evaluated as drug targets. A large number of protein kinases within the protein kinome of Entamoeba histolytica strongly suggest that protein phosphorylation is a key component of pathogenesis regulation by this parasite. PI3 K and Src are kinases previously described in this parasite, but their role is poorly understood. Here, the effect of Src-1-inhibitor and PI3 K inhibitor (Wortmannin) on the virulence factors of E. histolytica was evaluated. Results show that both inhibitors affect the actin cytoskeleton and the amoebic movement. Also, the proteolytic activity is diminished by Wortmannin, but not by Src-inhibitor-1; however, the phagocytic capacity is diminished by Wortmannin and Src-1-inhibitor. Finally, we found that the virulence in vivo of E. histolytica is affected by Wortmannin but not by Src-1-inhibitor. This study opens the way for the design of anti-amoebic drugs based on kinase inhibition. Key words: Entamoeba histolytica, PI3 K, Src, virulence factors. INTRODUCTION Entamoeba histolytica, the protozoan parasite capable of invading the intestinal mucosa and spreading to other organs, mainly the liver, is a significant source of morbidity and mortality in developing countries (Haque et al. 2003; World Health Organization, 2011). In humans, invasion starts when trophozoites residing in the colon deplete the mucus, interact with vulnerable enterocytes, dismantle cell junctions, and lyse host cells. Once the epithelial layer is disrupted, amoebae cross the basal lamina and the extracellular matrix (ECM); this is a process invol- ving parasite motility and cytotoxicity towards host cells by proteolytic activity and phagocytic capacity (Meza, 2000). Some trophozoites, which reside in the colon, penetrate the portal system and follow the bloodstream to the hepatic portal venule and sinu- soids (Tsutsumi et al. 1984). The latter are the main structures where amoebae cross the endothelium to reach the parenchyma, with a concomitant initiation of inflammatory foci and abscesses. The classical amoebic liver abscess (ALA) is due to necrotic lysis of the liver tissue, which varies in size from a few centimetres to a large lesion (Que and Reed, 2000; Labruyère and Guillén, 2006; Campos-Rodríguez et al. 2009; Santi-Roca et al. 2009). It is not yet clear what factors or signals, in virulent trophozoites, are particularly involved in tissue invasion; therefore, studies of signalling proteins and pathways may help us to understand the processes that may have an impact on the invasive disease. Recently, the protein kinome of E. histolytica has been described; however, the role of the identified kinases is unknown on this eukaryotic parasite (Loftus et al. 2005; Anamika et al. 2008). Src and PI3 K kinases are two proteins implicated in cell adhesion, motility and invasion (Heinz et al. 1999; Thamilselvan et al. 2007; Sanchez et al. 2010). These processes are essential for E. histolytica invasiveness and, interestingly, these two kinases have also been described in this parasite; however, their function is poorly characterized. There is evidence that PI3 K signalling pathways regulate phagocytosis and migration in E. histolytica tropho- zoites (Batista and De Souza, 2004; Blazquez et al. 2008). In the case of Src, only its presence has been reported on the protein kinome of E. histolytica by in silico analysis (Anamika et al. 2008). Previous work in our laboratory described the involvement of Src during trophozoite adherence to fibronectin, suggesting a possible role for this protein in the parasitic adhesion process (Flores-Robles et al. 2003). In this work, we decided to study the effect of Wortmannin and Src-1-inhibitor on relevant amoe- bic virulence factors. Results show that both inhibi- tors affect amoebic motion by a direct effect on the structure of the actin cytoskeleton. The proteolytic activity was diminished by Wortmannin, but not by Src-inhibitor; however, the phagocytic capacity of the parasite was significantly diminished by * Corresponding author: E-mail: ptr@cinvestav.mx 202 Parasitology (2013), 140, 202–209. © Cambridge University Press 2012 doi:10.1017/S0031182012001540