UNCORRECTED PROOF Cellular Microbiology (2005) doi:10.1111/j.1462-5822.2004.00486.x © 2005 Blackwell Publishing Ltd cmi_486.fm Blackwell Science, LtdOxford, UKCMICellular Microbiology 1462-5814Blackwell Publishing Ltd, 2005 20057••••Original ArticleBarrier penetration by ToxoplasmaA. Barragan, F. Brossier and L. D. Sibley Received 19 July, 2004; revised 14 October, 2004; accepted 1 November, 2004. *For correspondence. E-mail Antonio. Barragan@medhs.ki.se; Tel. (+46) 84572524; Fax: (+46) 87467637. Transepithelial migration of Toxoplasma gondii involves an interaction of intercellular adhesion molecule 1 (ICAM-1) with the parasite adhesin MIC2 Antonio Barragan, 1,2 * Fabien Brossier 1 and L. David Sibley 1 1 Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110, USA. 2 Center for Infectious Medicine, Karolinska Institutet and the Swedish Institute for Infectious Disease Control, SE-171 82 Stockholm, Sweden. Summary Toxoplasma gondii crosses non-permissive biologi- cal barriers such as the intestine, the blood–brain barrier and the placenta thereby gaining access to tissues where it most commonly causes severe pathology. Herein we show that in the process of migration Toxoplasma initially concentrates around intercellular junctions and probably uses a paracellu- lar pathway to transmigrate across biological barri- ers. Parasite transmigration required viable and actively motile parasites. Interestingly, the integrity of host cell barriers was not altered during parasite transmigration. As intercellular adhesion molecule 1 (ICAM-1) is upregulated on cellular barriers during Toxoplasma infection, we investigated the role of this receptor in parasite transmigration. Soluble human ICAM-1 and ICAM-1 antibodies inhibited transmigra- tion of parasites across cellular barriers implicating this receptor in the process of transmigration. Fur- thermore, human ICAM-1 immunoprecipitated the mature form of the parasite adhesin MIC2 present on the parasite surface, indicating that this interaction may contribute to cellular migration. These findings reveal that Toxoplasma exploits the natural cell traf- ficking pathways in the host to cross cellular barriers and disseminate to deep tissues. Introduction Biological barriers play crucial roles in homeostasis and represent the first line of defence against microbial infec- 1 tions that would otherwise lead to disseminated disease. Whereas the mechanisms leading to bacterial and viral penetration of biological barriers have been partly eluci- dated (Kerr, 1999; Hornef et al., 2002), very little is known about mechanisms used by parasites. During natural infections, Toxoplasma initially crosses the intestinal epi- thelium, disseminates into the deep tissues and traverses biological barriers in the placenta, the blood–brain barrier and the blood–retina barrier (Barragan and Sibley, 2003). Within these immunologically privileged sites, it causes severe pathology in the developing fetus (Remington et al., 1995), immunocompromised individuals (Luft et al., 1993), and ocular pathology in immunocompetent individ- uals (Roberts and McLeod, 1999). We recently demon- strated that Toxoplasma actively crosses polarized cell monolayers in vitro and that this ability is linked to parasite motility and virulence in the mouse model (Barragan and Sibley, 2002). Toxoplasma lacks cilia or flagella and uses a unique mode of locomotion, termed gliding motility, to rapidly enter host cells by active penetration (Dobrowolski and Sibley, 1996). Gliding motility is also characteristic of other apicomplexan parasites that are important causative agents of human diseases including Plasmodium, the aetiological agent of malaria (Sibley, 2004). Toxoplasma tachyzoites invade a wide variety of cells from different vertebrates, suggesting the presence of ligands for wide- spread host cell surface molecules. Consistent with this model, interactions with sialic acid (Monteiro et al., 1998) and heparan sulphate-like proteoglycans (Ortega-Barria and Boothroyd, 1999; Carruthers et al., 2000a; Harper et al., 2004) are important in host cell recognition. Whether there are also specific protein–protein interac- tions that mediate polarized attachment, entry and tissue migration remained uncertain, as these specific interac- tions have not been extensively studied. Apicomplexan parasites share an evolutionary con- served family of transmembrane adhesins first described in malaria as TRAP (thrombospondin-related anonymous protein) (Robson et al., 1988), and called MIC2 in Toxo- plasma (Wan et al., 1997). The extracellular domains of MIC2 and TRAP contain two adhesive modules, an inte- grin I/A-domain and one or more thrombospondin type I repeats; these domains bear significant similarities to 2