Cellular Microbiology (2005) 7(2), 181–190 doi:10.1111/j.1462-5822.2004.00445.x © 2004 Blackwell Publishing Ltd Blackwell Science, LtdOxford, UKCMICellular Microbiology 1462-5814Blackwell Publishing Ltd, 20047 2181190Original ArticleSerpin overexpressionin Plasmodium-infected midgut cells A. Danielli et al. Received 6 April, 2004; revised 7 July, 2004; accepted 8 July, 2004. *For correspondence with Professor Kafatos. E-mail dg-office@embl.de; Tel. (+49) 6221 387 200; Fax 49 6221 387 211 and for corrrespondence with Dr Loukeris. E-mail loukeris@mailhost.imbb.forth.gr; Tel. (+30) 2810 391178; Fax (+30) 2810 391101. Overexpression and altered nucleocytoplasmic distribution of Anopheles ovalbumin-like SRPN10 serpins in Plasmodium-infected midgut cells Alberto Danielli, 1,2 Carolina Barillas-Mury, 3,4 Sanjeev Kumar, 4 Fotis C. Kafatos 1 * and Thanasis G. Loukeris 1,5 * 1 European Molecular Biology Laboratory (EMBL), Meyerhoftrasse 1, 69117 Heidelberg, Germany. 2 Dipartimento di Biologia Evoluzionistica Sperimentale, Università di Bologna, Via Selmi 3, 40126 Bologna, Italy. 3 Colorado State University, Department of Microbiology, Immunology and Pathology (MIP), 1619 Campus Delivery, Fort Collins, CO, USA. 4 Laboratory of Malaria and Vector Research, National Institutes of Health, 4 Center Drive, Bethesda, MD 20892, USA. 5 Institute of Molecular Biology and Biotechnology (IMBB/ FORTH), Vassilika Vouton, GR71110, Heraklion, Greece. Summary The design of effective, vector-based malaria trans- mission blocking strategies relies on a thorough understanding of the molecular and cellular interac- tions that occur during the parasite sporogonic cycle in the mosquito. During Plasmodium berghei invasion, transcription from the SRPN10 locus, encoding four serine protease inhibitors of the ovalbumin family, is strongly induced in the mosquito midgut. Herein we demonstrate that intense induction as well as redistri- bution of SRPN10 occurs specifically in the parasite- invaded midgut epithelial cells. Quantitative analysis establishes that in response to epithelial invasion, SRPN10 translocates from the nucleus to the cyto- plasm and this is followed by strong SRPN10 overex- pression. The invaded cells exhibit signs of apoptosis, suggesting a link between this type of intracellular serpin and epithelial damage. The SRPN10 gene prod- ucts constitute a novel, robust and cell-autonomous marker of midgut invasion by ookinetes. The SRPN10 dynamics at the subcellular level confirm and further elaborate the ‘time bomb’ model of P. berghei invasion in both Anopheles stephensi and Anopheles gambiae. In contrast, this syndrome of responses is not elicited by mutant P. berghei ookinetes lacking the major ook- inete surface proteins, P28 and P25. Molecular mark- ers with defined expression patterns, in combination with mutant parasite strains, will facilitate dissection of the molecular mechanisms underlying vector com- petence and development of effective transmission blocking strategies. Introduction In arthropods, including insects, serpins have to date been identified principally as secreted haemolymph pro- teins (Kanost, 1999). Genetic and biochemical studies in Drosophila have identified extracellular serpins, together with their target proteases, as principal regulators of anti- microbial responses, the melanization cascade and establishment of the dorsoventral axis (Ligoxygakis et al., 2002a; Ligoxygakis et al., 2002b; Ligoxygakis et al., 2003). Lately, genome sequencing projects have revealed a large number of annotated serpins in both the fruit fly (Adams et al., 2000; Kruger et al., 2002) and Anopheles gambiae (Christophides et al., 2002; Holt et al., 2002), many of which lack an obvious hydrophobic signal sequence and therefore are predicted to be intracellular. Functional characterization of these serpins and their tar- gets is expected to uncover additional biological roles of this important class of molecules. Recently, we have cloned the A. gambiae SRPN10 locus and determined that it encodes four alternatively spliced serpins with similarity to intracellular, nucleocyto- plasmic serpins of the ovalbumin clade (Danielli et al., 2003), some of which are involved in modulation of apo- ptotic processes in mammals (Bird, 1998; Silverman et al., 2001). We have shown that at least three SPRN10 isoforms are functional inhibitors of different types of pro- teases in vitro, including bacterial subtilisins, and that the SRPN10 gene is strongly upregulated in the midgut after infection by the model rodent parasite Plasmodium berghei. This finding is particularly interesting as the mid- gut appears to be an especially difficult barrier for the parasite in order to complete the sporogonic cycle in the