Journal of Proteome Research protein protein interactions, ranging from attachment to the midgut surface to penetration and crossing of the midgut apical plasma membrane.2 Experimental evidence from previous studies indicated that AgAPN1, ANXB9, ANXB10B, ANXB10C, CpbAg1, and SCRBQ2 mediate the ookinete invasion process (Table 1). Given our hypothesized role of DRMs in partitioning important midgut ligands at the site of ookinete invasion, a reasonable expectation would be the conﬁrmatory presence of these six proteins in our DRM fraction, since partitioning requires association of these proteins to mobile rafts. Our anal- yses of the DRM fractions revealed that ﬁve out of six of these proteins, AgAPN1, ANXB9, ANXB10B, CpbAg1, and SCRBQ2, are indeed DRM-associated (Table 1), providing inferred but plausible support for the role for DRM partitioning of proteins utilized by ookinetes to transverse the midgut epithelium. ANXB10C was the only known ookinete interacting protein not detected in any of our DRM preparations. Two of the known ookinete-interacting proteins, AgAPN1 and CpbAg1, are pepti- dases. Peptidases comprised 27.7% and 52.9% of the total extracellular DRM proteins and of the extracellular subset of identiﬁed GPI-anchored proteins, respectively, indicating that peptidases are highly abundant proteins on the midgut sur- face. From a pathogen’s perspective, selecting highly abun- dant proteins on the host surface would facilitate host cell recognition, attachment, and invasion. Fifty-two other DRM- associated peptidases were predicted to be extracellular and further characterization of these peptidases may lead to the characterization of other midgut proteins that mediate ookinete invasion. It can be argued that since our DRM preparations used sugar- fed midguts, it may not completely reﬂect the DRM proteome in which the ookinete interacts with in vivo. Upon ingestion of a blood meal, the PM separates the blood meal bolus from the apical microvillar surface. Both the formation of the PM, the distension of the midgut after a blood meal, as well as the presence of blood in the lumen of the midgut may inﬂuence protein expression and protein residency in DRMs. We used sugar-fed midguts in this study for a number of reasons. First, doing so facilitated the identiﬁcation by mass spectrometry of low abundance Anopheles midgut DRM-associated proteins that would have otherwise been diﬃcult to achieve due to the gross quantities of blood and serum proteins contaminating our DRM preparations. Second, although several microarray studies have shown diﬀerential expression of the Anopheles midgut transcrip- tome in response to Plasmodium invasion,29,30 these genes appear to be primarily involved in immunity and are upregulated after the ookinete has invaded the midgut epithelium. Thus, these changes in gene expression may have little eﬀect on the initial ookinete interactions with the midgut surface. Most importantly, to our knowledge, experimental validation by Western blotting of the assumed 1:1 concordance of transcript abundance with protein translation/presence for these immune-responsive genes is largely missing. In fact, this is the ﬁrst report detecting the presence of proteins from the immunoglobulin-like domain family of proteins in sugar-fed midguts, which further supports this assertion. Third, many apical membrane proteins present on the surface of blood-fed midguts are already surface-associated in sugar-fed midguts. For example, AgAPN111 and AgMUC-1,12 a midgut surface-associated mucin-like protein, as well as critical O-linked glycans on midgut surface glycoproteins2,57,59 have been shown to be surface associated in sugar-fed and blood-fed midguts by immunoﬂuorescence microscopy. Additionally, in ARTICLE our proteomic analysis of DRMs from sugar-fed midguts, we were able to detect the presence of ﬁve known ookinete-inter- acting proteins. While it can be argued that DRM-residency of proteins can change across time and space, especially postblood feeding, our data suggest that at least prior to acquiring an infectious bloodmeal, many ookinete-interacting proteins are already present in DRMs before the ingestion of an infected blood meal. Therefore, we believe that the sugar-fed midgut DRM proteome has physiological signiﬁcance in terms of ookinete midgut interactions but still recognize that our approach may not necessarily capture the complete proteomic landscape 18 24 h postblood feeding. A Model of Ookinete Interaction with Mosquito Midgut Host DRM (Lipid Raft) Proteins Throughout nature, DRMs are exploited by pathogens as entry points into host cells. In this study, we propose that Plasmodium ookinetes interact with midgut DRMs to cross the midgut epithelium. Also, we report the ﬁrst description of the DRM proteome of An. gambiae midguts in which we identiﬁed 1452 proteins. Furthermore, examination of the GPI proteome and immunoblots probed with speciﬁc antibodies demonstrated that our DRM fractions contain GPI-anchored proteins, such as AgAPN1. Analysis of the proteins identiﬁed in the DRM frac- tions revealed that ﬁve known ookinete-interacting proteins were present. These results provided the basis for a proposed model in which ookinetes interact with a subset of midgut DRM-asso- ciated proteins to transverse the midgut epithelium. Given our mass spectrometry evidence suggesting the pre- sence of known parasite “interacting” proteins in midgut DRMs, we propose a working model in which interactions between a subset of midgut DRM-associated proteins and the ookinete facilitate the invasion process (Figure 4). Lipid rafts allow for the partitioning of multiple ookinete-interacting proteins into dis- crete locations on the midgut surface, enhancing multivalent interactions between the ookinete and the midgut. Multivalent interactions have been suggested to be a conserved process for strengthening single, protein protein, or protein glycan inter- actions for several vector-borne pathogens, including Plasmodium.2 In one example, a complex set of events that require lipid raft integrity, lateral mobility, and partitioning on midgut surfaces has been shown to be essential for Cry1a toxin insertion and pore formation in Heliothis virescens and Manducta sexta.63 From our proteomic analysis, we know that, long before the ookinete contacts the midgut surface, many of the DRM- associated proteins utilized by the ookinete already exist in rafts (Figure 4a). This is evident by the fact that the ookinete- interacting proteins (AgAPN1, ANXB9, ANXB10B, CpbAg1, and SCRBQ2) were detected in our DRM preparations, which used sugar-fed midguts from mosquitoes that had no previous blood meal or exposure to Plasmodium parasites. Previous studies have shown that ookinetes glide along the midgut surface toward the junctions between cells before inva- sion occurs.64 After initial contact is made with the midgut microvillar surface, we propose that the ookinete engages a subset of speciﬁc rafts following one of two (or a combination of) diﬀerent mechanisms. The ﬁrst possibility (1) is that the ookinete interacts with single or multiple rafts already at the site of invasion (depicted by the red arrowhead, Figure 4b). Here, the fusion of rafts may not be necessary for ookinete invasion as the partitioning of host receptors in individual rafts may already be of suﬃcient density to promote multivalent interactions. 5159 dx.doi.org/10.1021/pr2006268 |J. Proteome Res. 2011, 10, 5150–5162