Journal of Proteome Research Figure 4. Model illustrating how partitioned midgut DRM-associated proteins can be utilized by Plasmodium ookinetes to adhere to the midgut epithelium. (a) Ookinete-interacting proteins exist in DRMs (rafts) before the ookinete makes contact with the midgut surface. (b) Potential model of ookinetes utilizing individual DRMs (rafts) containing ookinete-interacting proteins at the site of invasion to invade the midgut epithelium. (c) Potential model of DRMs (rafts) fusing to form a large platform containing ookinete- interacting proteins at the site of invasion, which would facilitate ookinete invasion of the midgut epithelium. Red arrowhead, hypothetical site of ookinete interaction along the midgut plasmalemma. The second model (2) is that the ookinete induces multiple small rafts to mobilize and fuse forming a large platform of parti- tioned ookinete-interacting proteins at the site of invasion on the midgut surface (Figure 4c). Both hypotheses include the central theme of ookinetes utilizing rafts as a means of accessing partitioned host receptors in a speciﬁc region of the midgut apical membrane. Unfortunately, it is diﬃcult to test whether or not raft fusion is involved in the invasion process. Currently, a polarized Anopheles midgut epithelial cell line does not exist; thus, our experimental design is limited to studies involving whole organ- isms. Development of a midgut epithelial cell line would aid in elucidating the precise molecular interactions between midgut DRM-associated proteins and the ookinete and in determining if raft fusion occurs in the invasion process. Lastly, we cannot exclude the possibility that proteins that are not associated with DRMs may also be involved in the invasion process. ANXB10C along with several other non-DRM asso- ciated proteins may in fact be present at the site of invasion. It is possible that accumulation of the DRM-associated proteins in close proximity to, if not in complex with, these non-DRM ligands, facilitates the ookinete invasion process. Unfortunately, to date a comprehensive analysis of a whole host of apical microvilli glycoproteins has not yet been achieved. ARTICLE Plasmodium Subversion of Host Red Cell DRMs While this study is the ﬁrst to propose an interaction between Plasmodium ookinetes with midgut DRMs, previous studies have suggested that Plasmodium merozoites may engage host DRMs to enter erythrocytes.65,66 During inva- sion, the erythrocyte plasma membrane indents forming a vacuole around the invading merozoite known as the para- sitophorous vacuole membrane (PVM), where the parasite will reside for the duration of its intraerythrocytic cycle. Erythrocyte DRM-associated proteins CD55, CD59, Duﬀy, ﬂotillin-1, and ﬂotillin-2 are detected in the PVM but glyco- phorin A, glycophorin C, CD47, and ankyrin, which are not present in DRMs, are excluded from the PVM.65 Since the PVM originates from the point of merozoite entry, the presence of erythrocyte DRM-associated proteins in the PVM imply that DRMs are utilized by the merozoite at some point during the invasion process. Moreover, some erythrocyte DRM-associated proteins are excluded from the PVM suggesting that merozoites interact only with a particular subset of rafts on the surface of the erythrocyte.65 The biological relevance of lipid raft involvement in parasite invasion is further supported by the observation that treat- ment of uninfected erythrocytes with lidocaine, which disrupts lipid rafts, inhibits merozoite invasion of these erythrocytes.66 Although ookinetes do not form a PVM during invasion, both merozoite inva- sion of erythrocytes and ookinete invasion of the midgut epithe- lium appear to be mechanistically similar in that host DRM- associated proteins are involved in mediating the invasion process. In conclusion, our proteomic analysis of An. gambiae midgut epithelial surface DRMs suggests that Plasmodium ookinetes may interact with midgut proteins that are partitioned in membrane microdomains. These ﬁndings advance signiﬁcantly our current understanding of the mosquito midgut microvillar surface and open new avenues in the study of ookinete invasion of the midgut, an essential step for Plasmodium transmission through the mosquito. Uncharacterized DRM-associated proteins identi- ﬁed in this study may prove to be necessary for eﬃcient midgut invasion by ookinetes. A thorough interrogation of the results is needed to screen for important targets of Plasmodium interaction that, in turn, may lead to the development of novel mosquito- based transmission-blocking vaccines. ’ ASSOCIATED CONTENT bS Supporting Information Figure S1. Lipid Raft Fractions IV VI. SDS-PAGE SYPRO Ruby stained 4 12% Invitrogen gel of fractions 4, 5, 6, 7, 8, 9, 10,11, pellet, and a sample of the midgut lysate before separation on a sucrose gradient. Approximately 2 μg of protein was loaded per well. Lane a, Mark12 ladder (Invitrogen); Lane b, fraction 4; Lane c, fraction 5; Lane d, fraction 6; Lane e, fraction 7; Lane f, fraction 8; Lane g, fraction 9; Lane h, fraction 10; Lane i, fraction 11; Lane j, pellet; Lane k, total midgut lysate before separation on a sucrose gradient. The DRM fractions are highlighted by an asterisk. This representative gel from all three biological replicate fractionations was imaged using a 9210 Typhoon imager (PMT of 500 V and 25 μM resolution). This mate- rial is available free of charge via the Internet at http://pubs.acs.org. ’ AUTHOR INFORMATION Corresponding Author *Rhoel R. Dinglasan, rdinglas@jhsph.edu. Phone +1-410-614-4839. Fax +1-410-955-0105. 5160 dx.doi.org/10.1021/pr2006268 |J. Proteome Res. 2011, 10, 5150–5162