The effect of kinase, actin, myosin and dynamin inhibitors on host cell egress by Toxoplasma gondii Lucio Ayres Caldas a, b , Sergio Henrique Seabra c , Márcia Attias a, b, ⁎, Wanderley de Souza a, b, d a Laboratório de Ultraestrutura Celular Hertha Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil b Instituto Nacional de Ciência e Tecnologia em Biologia Estrutural e Bioimagens, Inbeb, Brazil c Universidade Estadual da Zona Oeste, Rio de Janeiro, Brazil d Instituto Nacional de Metrologia, Qualidade e Tecnologia, Inmetro, Rio de Janeiro, Brazil abstract article info Article history: Received 22 October 2012 Received in revised form 26 March 2013 Accepted 18 April 2013 Available online xxxx Keywords: Apicomplexa Calcium ionophore Toxoplasma egress Kinase Actin Myosin Dynamin Toxoplasma gondii is a protozoan parasite that can infect the nucleated cells of all warm-blooded animals. De- spite its medical and veterinary importance, the egress of T. gondii from host cells has not been fully elucidat- ed. This process is usually studied with calcium ionophores, which artificially trigger T. gondii egress. Among the diverse signaling events that take place during egress, kinases appear to play a crucial role. In this work we employed several kinase inhibitors to examine their role in egress: although parasite egress was only slightly impaired by treatment with the PI3K and PKC inhibitors wortmannin and staurosporine, the addition of the tyrosine kinase-specific inhibitor genistein efficiently blocked the exit of parasites by more than 50%. IPA-3, a non-ATP-competitive inhibitor of p21-activated kinases, which play a role in actin cytoskeleton re- modeling inhibited egress of T. gondii by only 15%. The myosin motor inhibitor blebbistatin and the actin po- lymerization inhibitor cytochalasin D also blocked the egress of T. gondii. Nevertheless, dynasore, which is known to block the GTPase activity of dynamin, had little or no effect on T. gondii egress. © 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction The protozoan parasite Toxoplasma gondii can infect virtually any warm-blooded animal, and it is responsible for toxoplasmosis, an impor- tant cause of abortion and malformation in unborn children, life- threatening encephalitis in immunocompromised individuals and other significant effects on the behavior of people and animals [1–3]. Livestock, such as pigs and cattle, play important roles in the transmission process, and they are associated with seasonal outbreaks in certain countries [4–6]. Acute toxoplasmosis results from the rapidly dividing tachyzoite form, which invades host cells and ultimately resides in a parasitophorous vacuole (PV) that increases in size as the parasite divides [7,8]. Upon suc- cessive rounds of replication, the parasites leave the PV, eventually carry- ing remnants of the PV membrane (PVM) and the endoplasmic reticulum membranes towards the extracellular medium [9]. Calcium concentrations within T. gondii have been shown to in- crease prior to both invasion and egress from the host cell [10,11]. It has also been reported that cytoplasmic K + and perforins play a role in egress [12,13]. Nevertheless, very little is known about this process, which has predominantly been studied in cells that have been treated with the calcium ionophore A23817 [14–17]. Some reports have pointed to similarities between the processes of host cell invasion and egress by T. gondii [10]. Recent studies have shown that some T. gondii calcium-dependent protein kinases, such as TgCDPK1 and TgCPDK3, are crucial for intracellular parasite microneme secretion [18], the latter being specially involved in parasite egress [19]. However, the intracellular cycle of T. gondii also depends on host cell factors as previously demonstrated and some kinase inhibitors – wortmannin, staurosporine and genistein, which are known to block PI3K, PKC and tyrosine kinase activities, respectively – have been shown to effectively block calcium ionophore A23817-induced invasion [20] and egress at early stages of infection [17]. The effects of the myosin II inhibitor blebbistatin [21] and the actin depolymerizing drug cytocha- lasin D on host cell egress were also investigated. Certain cytoskeletal proteins, such as actin and myosin, are known to play a role in host cell invasion by T. gondii [22]. These components are involved in pro- cesses such as plasma membrane rearrangement, which takes place during endocytosis, as well as vesicular and vacuolar trafficking [23,24]. While blebbistatin blocks the ATPase activity of myosin II, an actin-based molecular motor, cytochalasin D interferes both with para- site invasion and egress [25–27]. Actin filaments were labeled with col- loidal gold and fluorescent markers to examine their possible role as a substrate for T. gondii gliding during egress. The involvement of actin polymerization and cell motility makes use of IPA-3, a highly selective Parasitology International xxx (2013) xxx–xxx ⁎ Corresponding author at: Instituto de Biofísica Carlos Chagas Filho, Av. Carlos Chagas Filho, 373 Prédio do CCS, Bloco G- sala 19 Cidade Universitária. Ilha do Fundão, Cep 21941-902 Rio de Janeiro, RJ, Brazil. Tel./fax: +55 21 22602364. E-mail address: mattias@biof.ufrj.br (M. Attias). PARINT-01095; No of Pages 8 1383-5769/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.parint.2013.04.006 Contents lists available at SciVerse ScienceDirect Parasitology International journal homepage: www.elsevier.com/locate/parint Please cite this article as: Caldas LA, et al, The effect of kinase, actin, myosin and dynamin inhibitors on host cell egress by Toxoplasma gondii, Par- asitology International (2013), http://dx.doi.org/10.1016/j.parint.2013.04.006