ARTICLE pubs.acs.org/jpr Ookinete-Interacting Proteins on the Microvillar Surface are Partitioned into Detergent Resistant Membranes of Anopheles gambiae Midguts Lindsay A. Parish,† David R. Colquhoun,†,‡ Ceereena Ubaida Mohien,†,‡ Alexey E. Lyashkov,‡ David R. Graham,‡ and Rhoel R. Dinglasan*,† †W. Harry Feinstone Department of Molecular Microbiology & Immunology, Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, Maryland 21205, United States ‡Department of Molecular and Comparative Pathobiology, Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, Maryland 21205, United States bS Supporting Information ABSTRACT: Lipid raft microdomains, a component of detergent resistant membranes (DRMs), are routinely exploited by pathogens during host-cell entry. Multiple membrane-surface proteins mediate Plasmodium ookinete invasion of the Anopheles midgut, a critical step in the parasite life cycle that is successfully targeted by transmission- blocking vaccines (TBV). Given that lipid rafts are a common feature of host-pathogen interactions, we hypothesized that they promote the partitioning of midgut surface proteins and thus facilitate ookinete invasion. In support of this hypothesis, we found that ﬁve of the characterized Anopheles TBV candidates, including the leading Ano- pheles TBV candidate, AgAPN1, are present in Anopheles gambiae DRMs. Therefore, to extend the repertoire of putative midgut ligands that can be targeted by TBVs, we analyzed midgut DRMs by tandem mass spectro- metry. We identiﬁed 1452 proteins including several markers of DRMs. Since glycosylphosphotidyl inositol (GPI)-anchored proteins partition to DRMs, we characterized the GPI subproteome of An. gambiae midgut brush-border microvilli and found that 96.9% of the proteins identiﬁed in the GPI-anchored fractions were also present in DRMs. Our study vastly expands the number of candidate malarial TBV targets for subsequent analysis by the broader community and provides an inferred role for midgut plasmalemma microdomains in ookinete cell invasion. KEYWORDS: Plasmodium, ookinete, Anopheles gambiae, midgut, transmission blocking vaccine, detergent resistant membranes, lipid rafts, malaria ’ INTRODUCTION Malaria remains a signiﬁcant cause of morbidity and mortality across the globe despite eﬀorts to control the disease. The spread of drug resistant parasites and insecticide resistant mosquitoes highlight the need for innovative strategies that can eventually lead to the elimination of malaria. One approach that has recently regained momentum is the concept of transmission-blocking vaccines (TBV), which interrupt the development of Plasmodium parasites, the causative agents of malaria, within the Anopheles mosquito.1 In order to be transmitted to a human host, parasites must travel from the mosquito midgut lumen to the salivary glands. Inside the midgut lumen, Plasmodium gametocytes that are ingested with a blood meal transform into invasive ookinetes, which then interact with the midgut surface prior to active cell invasion. Following cell traversal to the basal side of the midgut cell, the ookinete develops into an oocyst, which ultimately releases thousands of sporozoites that invade the mosquito salivary glands. Once in the salivary glands, these sporozoites are now primed and ready to infect a vertebrate host once the mosquito takes its next blood meal. Ookinete invasion of the midgut represents the ﬁrst invasion bottleneck in the parasite’s complex life cycle within the mosquito, oﬀering a unique opportunity to interrupt malaria transmission.1 Therefore, deﬁn- ing the molecular interactions between the ookinete and the lumenal surface of the Anopheles midgut is crucial to under- standing the biology of transmission and for the development of novel transmission-blocking interventions. Previous studies have proposed that ookinetes interact with multiple glycans and glycoproteins on the apical (lumenal) surface of the midgut, (reviewed in refs 2 and 3), and these glycoconjugates represent a set of potential targets for mosquito- based malaria TBVs (Table 1). As multiple midgut surface macromolecules appear to be necessary for midgut invasion by Received: July 4, 2011 Published: September 09, 2011 r 2011 American Chemical Society 5150 dx.doi.org/10.1021/pr2006268 | J. Proteome Res. 2011, 10, 5150–5162