A Library of Functional Recombinant Cell-surface and Secreted P. falciparum Merozoite Proteins

Malaria, an infectious disease caused by parasites of the Plasmodium genus, is one of the world''s major public health concerns causing up to a million deaths annually, mostly because of P. falciparum infections. All of the clinical symptoms are associated with the blood stage of the disease, an obligate part of the parasite life cycle, when a form of the parasite called the merozoite recognizes and invades host erythrocytes. During erythrocyte invasion, merozoites are directly exposed to the host humoral immune system making the blood stage of the parasite a conceptually attractive therapeutic target. Progress in the functional and molecular characterization of P. falciparum merozoite proteins, however, has been hampered by the technical challenges associated with expressing these proteins in a biochemically active recombinant form. This challenge is particularly acute for extracellular proteins, which are the likely targets of host antibody responses, because they contain structurally critical post-translational modifications that are not added by some recombinant expression systems. Here, we report the development of a method that uses a mammalian expression system to compile a protein resource containing the entire ectodomains of 42 P. falciparum merozoite secreted and cell surface proteins, many of which have not previously been characterized. Importantly, we are able to recapitulate known biochemical activities by showing that recombinant MSP1-MSP7 and P12-P41 directly interact, and that both recombinant EBA175 and EBA140 can bind human erythrocytes in a sialic acid-dependent manner. Finally, we use sera from malaria-exposed immune adults to profile the relative immunoreactivity of the proteins and show that the majority of the antigens contain conformational (heat-labile) epitopes. We envisage that this resource of recombinant proteins will make a valuable contribution toward a molecular understanding of the blood stage of P. falciparum infections and facilitate the comparative screening of antigens as blood-stage vaccine candidates.Parasites of the Plasmodium genus are the etiological agents responsible for malaria, an infectious disease mostly occurring in developing countries with up to 40% of the world''s population described as being at risk of the disease. Among the Plasmodium species that can affect humans, Plasmodium falciparum is responsible for the highest mortality, causing around one million deaths annually, mostly in children under the age of five (1). The clinical symptoms of malaria occur during the cyclic asexual blood stage of the parasite lifecycle when merozoites, that have invaded and replicated within host erythrocytes, are released into the bloodstream before invading new red blood cells (2). Despite intensive efforts from the research community there is currently no licensed vaccine for malaria. The leading candidate RTS,S/AS01, which targets the pre-erythrocytic stage of the disease and was tested in phase III trials, conferred 30 to 50% protection from clinical malaria, depending on the age group studied (3, 4). This limited efficacy has led to calls for a more effective vaccine and many have suggested that a combinatorial vaccine that additionally targets the blood stage may increase efficacy.A vaccine targeting the proteins expressed on the surface of the blood stage of the parasite is conceptually attractive because merozoites are repeatedly and directly exposed to the human humoral immune system and naturally acquired antibodies against these proteins have been shown to confer at least partial immunity (5–8). Despite this, only a few antigens discovered before the completion of the parasite genome sequence have been assessed in detail (9) and clinical vaccine trials using antigens that target the blood stage have so far shown limited efficacy, mostly caused by antigenic diversity (10). The sequencing of the parasite genome (11) has identified all possible targets but the systematic screening of these new candidates to assess their potential as a vaccine is hampered by the inability to systematically express recombinant Plasmodium proteins in their native conformation (12–15). Likely explanations might be the high (∼80%) A:T content of the P. falciparum genome resulting in low codon usage compatibility in heterologous expression systems, the large size (> 50 kDa) of many proteins, the presence of long stretches of highly repetitive amino acids, and the difficulty in identifying clear structural domains within these proteins using standard prediction computer programs (11). Extracellular proteins, in particular, present an additional challenge because they often have signal peptides and transmembrane regions that can negatively impact expression (16–18) and contain structurally important disulfide bonds. However, unlike most other eukaryotic extracellular proteins, Plasmodium cell surface and secreted proteins are not modified by N-linked glycans because of the absence of the necessary enzymes (19).To express Plasmodium proteins for basic research and vaccine development, a diverse range of expression systems have been tried (12) ranging from bacteria (17, 18), yeast (13), Dictyostelium (20), and plants (21) to mammalian cells (22) and cell-free systems (23–25). To circumvent the problem of codon usage, bacterial (26) and yeast (27) strains with modified tRNA pools have been developed, or sequences of the gene of interest synthesized and codon-optimized to match that of the expression host (28, 29). Although Escherichia coli has been the most popular expression system because of its relative simplicity and cost effectiveness, large-scale production of soluble functional Plasmodium falciparum recombinant proteins remains challenging with success rates ranging from just 6 to 21% (17, 18) and is often hindered by the need for complex refolding procedures. Similarly, attempts have been made to compile large panels of parasite proteins using in vitro translation systems (23, 25, 30, 31). These systems, however, require reducing conditions and are therefore not generally suitable for the systematic expression of extracellular proteins that occupy an oxidizing environment and critically require the formation of disulfide bonds for proper function. As a result, functional analyses of extracellular parasite proteins have often been restricted to smaller subfragments of the proteins that can be expressed in a soluble form rather than the entire extracellular region. Although eukaryotic expression systems are able to add disulfide bonds, they also often inappropriately glycosylate parasite proteins, adding further complication (32). A generic method that would overcome these technical challenges to express, in a systematic way, panels of recombinant Plasmodium proteins that have retained their native function and conformation would therefore be a valuable resource for the molecular investigations of erythrocyte invasion and the development of a blood stage vaccine.To generate a resource of correctly folded recombinant merozoite proteins, we used a mammalian expression system and established the parameters necessary for high-level expression. Using this method, we compiled a panel of 42 proteins that corresponds to the repertoire of abundant cell surface and secreted merozoite proteins of the 3D7 strain of Plasmodium falciparum. Biochemical activity of these proteins was demonstrated by recapitulating known protein interactions and by showing conformation-sensitive immunoreactivity of the recombinant proteins using immune sera.     

Vinyl Sulfones as Antiparasitic Agents and a Structural Basis for Drug Design

Cysteine proteases of the papain superfamily are implicated in a number of cellular processes and are important virulence factors in the pathogenesis of parasitic disease. These enzymes have therefore emerged as promising targets for antiparasitic drugs. We report the crystal structures of three major parasite cysteine proteases, cruzain, falcipain-3, and the first reported structure of rhodesain, in complex with a class of potent, small molecule, cysteine protease inhibitors, the vinyl sulfones. These data, in conjunction with comparative inhibition kinetics, provide insight into the molecular mechanisms that drive cysteine protease inhibition by vinyl sulfones, the binding specificity of these important proteases and the potential of vinyl sulfones as antiparasitic drugs.Sleeping sickness (African trypanosomiasis), caused by Trypanosoma brucei, and malaria, caused by Plasmodium falciparum, are significant, parasitic diseases of sub-Saharan Africa (1). Chagas'' disease (South American trypanosomiasis), caused by Trypanosoma cruzi, affects approximately, 16–18 million people in South and Central America. For all three of these protozoan diseases, resistance and toxicity to current therapies makes treatment increasingly problematic, and thus the development of new drugs is an important priority (2–4).T. cruzi, T. brucei, and P. falciparum produce an array of potential target enzymes implicated in pathogenesis and host cell invasion, including a number of essential and closely related papain-family cysteine proteases (5, 6). Inhibitors of cruzain and rhodesain, major cathepsin L-like papain-family cysteine proteases of T. cruzi and T. brucei rhodesiense (7–10) display considerable antitrypanosomal activity (11, 12), and some classes have been shown to cure T. cruzi infection in mouse models (11, 13, 14).In P. falciparum, the papain-family cysteine proteases falcipain-2 (FP-2)⁶ and falcipain-3 (FP-3) are known to catalyze the proteolysis of host hemoglobin, a process that is essential for the development of erythrocytic parasites (15–17). Specific inhibitors, targeted to both enzymes, display antiplasmodial activity (18). However, although the abnormal phenotype of FP-2 knock-outs is “rescued” during later stages of trophozoite development (17), FP-3 has proved recalcitrant to gene knock-out (16) suggesting a critical function for this enzyme and underscoring its potential as a drug target.Sequence analyses and substrate profiling identify cruzain, rhodesain, and FP-3 as cathepsin L-like, and several studies describe classes of small molecule inhibitors that target multiple cathepsin L-like cysteine proteases, some with overlapping antiparasitic activity (19–22). Among these small molecules, vinyl sulfones have been shown to be effective inhibitors of a number of papain family-like cysteine proteases (19, 23–27). Vinyl sulfones have many desirable attributes, including selectivity for cysteine proteases over serine proteases, stable inactivation of the target enzyme, and relative inertness in the absence of the protease target active site (25). This class has also been shown to have desirable pharmacokinetic and safety profiles in rodents, dogs, and primates (28, 29). We have determined the crystal structures of cruzain, rhodesain, and FP-3 bound to vinyl sulfone inhibitors and performed inhibition kinetics for each enzyme. Our results highlight key areas of interaction between proteases and inhibitors. These results help validate the vinyl sulfones as a class of antiparasitic drugs and provide structural insights to facilitate the design or modification of other small molecule inhibitor scaffolds.     

Evidence for Catalytic Roles for Plasmodium falciparum Aminopeptidase P in the Food Vacuole and Cytosol

The metalloenzyme aminopeptidase P catalyzes the hydrolysis of amino acids from the amino termini of peptides with a prolyl residue in the second position. The human malaria parasite Plasmodium falciparum expresses a homolog of aminopeptidase P during its asexual intraerythrocytic cycle. P. falciparum aminopeptidase P (PfAPP) shares with mammalian cytosolic aminopeptidase P a three-domain, homodimeric organization and is most active with Mn(II) as the cofactor. A distinguishing feature of PfAPP is a 120-amino acid amino-terminal extension that appears to be removed from the mature protein. PfAPP is present in the food vacuole and cytosol of the parasite, a distribution that suggests roles in vacuolar hemoglobin catabolism and cytosolic peptide turnover. To evaluate the plausibility of these putative functions, the stability and kinetic properties of recombinant PfAPP were evaluated at the acidic pH of the food vacuole and at the near-neutral pH of the cytosol. PfAPP exhibited high stability at 37 °C in the pH range 5.0–7.5. In contrast, recombinant human cytosolic APP1 was unstable and formed a high molecular weight aggregate at acidic pH. At both acidic and slightly basic pH values, PfAPP efficiently hydrolyzed the amino-terminal X-Pro bond of the nonapeptide bradykinin and of two globin pentapeptides that are potential in vivo substrates. These results provide support for roles for PfAPP in peptide catabolism in both the food vacuole and the cytosol and suggest that PfAPP has evolved a dual distribution in response to the metabolic needs of the intraerythrocytic parasite.Malaria remains one of the most deadly global infectious diseases with an estimated 500 million clinical cases and 2 million deaths annually (1, 2). Clinical manifestations of the disease arise as the protozoan malaria parasite replicates asexually within human erythrocytes. Five species of the genus Plasmodium infect humans. The cytoadherent properties of red blood cells infected with Plasmodium falciparum, coupled with the ability of the parasite to reach high parasitemia, make it the most virulent species. The emergence of strains of P. falciparum that are resistant to affordable anti-malarial drugs such as chloroquine has complicated efforts to manage malaria, and new drugs are urgently needed.Aminopeptidases catalyze the hydrolysis of amino acids from the amino termini of proteins and peptides. They participate in a wide range of biological processes, including peptide catabolism, protein maturation, antigen presentation on immune cells, and regulation of hormone activity. During the asexual erythrocytic replication cycle of the malaria parasite, aminopeptidases contribute to the catabolism of peptides generated by two major proteolytic pathways. One of these is initiated at the proteasome, a multifunctional protease that plays an important role in the turnover of ubiquitinated cellular proteins in the cytosol (3–5). In addition, the parasite transports host red blood cell cytosol (consisting primarily of hemoglobin) to an acidic degradative organelle, the food vacuole, where it is degraded in a proteasome-independent pathway (6, 7). As up to 75% of the host cell hemoglobin is catabolized during the intraerythrocytic cycle (8, 9), flux through the vacuolar pathway is substantial. Three aminopeptidases have been identified as key players in recycling amino acids from peptides generated by the proteasomal and vacuolar catabolic pathways: leucine aminopeptidase, aminopeptidase N (PfA-M1), and aminopeptidase P (10–14). The latter two enzymes have been found in the food vacuole and therefore may play a direct role in hemoglobin catabolism (11). An aspartyl aminopeptidase is also expressed in asexual stage parasites and hydrolyzes amino-terminal aspartyl and glutamyl substrates (15); however, disruption of its gene does not prevent efficient intraerythrocytic replication (11).Aminopeptidase P (APP)² homologs exhibit high specificity for proline in the second position of the substrate (the P1′ position in the nomenclature of Schechter and Berger (16)) and catalyze the hydrolysis of the X-Pro amide bond, where X is any aminoacyl residue (17). Because of the cyclic nature of the proline side chain, X-Pro-containing peptides are not easily accommodated in the active sites of broad specificity aminopeptidases (17). In mammals, three APP isozymes have been identified. APP1 is found in the cytosolic fraction of cell lysates and has been characterized from a variety of tissues (18–20). Although this enzyme has not, to our knowledge, been localized in intact cells, the apparent lack of specific targeting information is consistent with a role in cytosolic peptide turnover. Active cytosolic forms of APP have been reported in plants (21), fruit flies (22), the microsporidian parasite Encephalitozoon cuniculi (23), and in intestinal cells in Caenorhabditis elegans (24), where it is believed to play a role in the catabolism of peptides produced from ingested bacteria. Mammalian APP2 is a glycosylated ectoenzyme anchored into the membrane of endothelial and epithelial cells with a glycosylphosphatidylinositol attachment (25). The best characterized role of APP2 is the inactivation of the plasma hormone bradykinin, a nonapeptide, through cleavage of the Arg-Pro amino-terminal peptide bond (26, 27). Inhibition of APP2 potentiates the vasodilatory and cardioprotective properties of bradykinin, and APP2 has been considered a target for the development of cardiovascular drugs (28–30). A third isoform, APP3, has been identified in the human genome and may be a mitochondrial enzyme but has not yet been characterized (31). Prokaryotic APP homologs contribute to intracellular peptide turnover (32).P. falciparum aminopeptidase P (PfAPP) appears to be important for intraerythrocytic growth, as parasites with a disrupted PfAPP gene could not be isolated (11). We have previously localized a PfAPP-yellow fluorescent protein fusion to the food vacuole and the cytosol of the parasite (11). The cytosolic pool of PfAPP probably fulfills a role in peptide turnover and amino acid recycling that is orthologous to those of the cytosolic enzymes described above. In contrast, there is no report to our knowledge of an aminopeptidase P homolog functioning in an acidic environment akin to the malarial food vacuole. Moreover, characterization of mammalian aminopeptidase P homologs typically reveals a pH optimum of 7–8 with relatively little, if any, activity in the pH range 5.0–5.5 (18–20). Although we have previously detected PfAPP activity at acidic pH (11), the catalytic efficiency of the enzyme has not been characterized. Thus, at the outset of this study it was not clear whether PfAPP has a significant catalytic role in the food vacuole.Here we have localized untagged, native PfAPP in the parasite and have confirmed the dual cytosolic/vacuolar distribution of the enzyme. The domain organization, quaternary structure, and metal requirement of PfAPP were characterized. To evaluate the plausibility of a catalytic role for PfAPP at acidic and near-neutral pH, its stability in the pH range 5.0–7.5 was assessed and compared with that of human cytosolic APP1, an enzyme that does not, to our knowledge, have a physiological role in an acidic environment. The catalytic efficiency of PfAPP at a range of pH values was characterized with three X-Pro-containing peptides, two of which are found in the sequences of human α- and β-globin and therefore represent potentially physiological substrates.     

Repeated Domains of Leptospira Immunoglobulin-like Proteins Interact with Elastin and Tropoelastin

Leptospira spp., the causative agents of leptospirosis, adhere to components of the extracellular matrix, a pivotal role for colonization of host tissues during infection. Previously, we and others have shown that Leptospira immunoglobulin-like proteins (Lig) of Leptospira spp. bind to fibronectin, laminin, collagen, and fibrinogen. In this study, we report that Leptospira can be immobilized by human tropoelastin (HTE) or elastin from different tissues, including lung, skin, and blood vessels, and that Lig proteins can bind to HTE or elastin. Moreover, both elastin and HTE bind to the same LigB immunoglobulin-like domains, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12 as demonstrated by enzyme-linked immunosorbent assay (ELISA) and competition ELISAs. The LigB immunoglobulin-like domain binds to the 17th to 27th exons of HTE (17–27HTE) as determined by ELISA (LigBCon4, K_D = 0.50 μm; LigBCen7′–8, K_D = 0.82 μm; LigBCen9, K_D = 1.54 μm; and LigBCen12, K_D = 0.73 μm). The interaction of LigBCon4 and 17–27HTE was further confirmed by steady state fluorescence spectroscopy (K_D = 0.49 μm) and ITC (K_D = 0.54 μm). Furthermore, the binding was enthalpy-driven and affected by environmental pH, indicating it is a charge-charge interaction. The binding affinity of LigBCon4D341N to 17–27HTE was 4.6-fold less than that of wild type LigBCon4. In summary, we show that Lig proteins of Leptospira spp. interact with elastin and HTE, and we conclude this interaction may contribute to Leptospira adhesion to host tissues during infection.Pathogenic Leptospira spp. are spirochetes that cause leptospirosis, a serious infectious disease of people and animals (1, 2). Weil syndrome, the severe form of leptospiral infection, leads to multiorgan damage, including liver failure (jaundice), renal failure (nephritis), pulmonary hemorrhage, meningitis, abortion, and uveitis (3, 4). Furthermore, this disease is not only prevalent in many developing countries, it is reemerging in the United States (3). Although leptospirosis is a serious worldwide zoonotic disease, the pathogenic mechanisms of Leptospira infection remain enigmatic. Recent breakthroughs in applying genetic tools to Leptospira may facilitate studies on the molecular pathogenesis of leptospirosis (5–8).The attachment of pathogenic Leptospira spp. to host tissues is critical in the early phase of Leptospira infection. Leptospira spp. adhere to host tissues to overcome mechanical defense systems at tissue surfaces and to initiate colonization of specific tissues, such as the lung, kidney, and liver. Leptospira invade hosts tissues through mucous membranes or injured epidermis, coming in contact with subepithelial tissues. Here, certain bacterial outer surface proteins serve as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)² to mediate the binding of bacteria to different extracellular matrices (ECMs) of host cells (9). Several leptospiral MSCRAMMs have been identified (10–18), and we speculate that more will be identified in the near future.Lig proteins are distributed on the outer surface of pathogenic Leptospira, and the expression of Lig protein is only found in low passage strains (14, 16, 17), probably induced by environmental cues such as osmotic or temperature changes (19). Lig proteins can bind to fibrinogen and a variety of ECMs, including fibronectin (Fn), laminin, and collagen, thereby mediating adhesion to host cells (20–23). Lig proteins also constitute good vaccine candidates (24–26).Elastin is a component of ECM critical to tissue elasticity and resilience and is abundant in skin, lung, blood vessels, placenta, uterus, and other tissues (27–29). Tropoelastin is the soluble precursor of elastin (28). During the major phase of elastogenesis, multiple tropoelastin molecules associate through coacervation (30–32). Because of the abundance of elastin or tropoelastin on the surface of host cells, several bacterial MSCRAMMs use elastin and/or tropoelastin to mediate adhesion during the infection process (33–35).Because leptospiral infection is known to cause severe pulmonary hemorrhage (36, 37) and abortion (38), we hypothesize that some leptospiral MSCRAMMs may interact with elastin and/or tropoelastin in these elastin-rich tissues. This is the first report that Lig proteins of Leptospira interact with elastin and tropoelastin, and the interactions are mediated by several specific immunoglobulin-like domains of Lig proteins, including LigBCon4, LigBCen7′–8, LigBCen9, and LigBCen12, which bind to the 17th to 27th exons of human tropoelastin (HTE).     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

Sex-partitioning of the Plasmodium falciparum Stage V Gametocyte Proteome Provides Insight into falciparum-specific Cell Biology

One of the critical gaps in malaria transmission biology and surveillance is our lack of knowledge about Plasmodium falciparum gametocyte biology, especially sexual dimorphic development and how sex ratios that may influence transmission from the human to the mosquito. Dissecting this process has been hampered by the lack of sex-specific protein markers for the circulating, mature stage V gametocytes. The current evidence suggests a high degree of conservation in gametocyte gene complement across Plasmodium, and therefore presumably for sex-specific genes as well. To better our understanding of gametocyte development and subsequent infectiousness to mosquitoes, we undertook a Systematic Subtractive Bioinformatic analysis (filtering) approach to identify sex-specific P. falciparum NF54 protein markers based on a comparison with the Dd2 strain, which is defective in producing males, and with syntenic male and female proteins from the reanalyzed and updated P. berghei (related rodent malaria parasite) gametocyte proteomes. This produced a short list of 174 male- and 258 female-enriched P. falciparum stage V proteins, some of which appear to be under strong diversifying selection, suggesting ongoing adaptation to mosquito vector species. We generated antibodies against three putative female-specific gametocyte stage V proteins in P. falciparum and confirmed either conserved sex-specificity or the lack of cross-species sex-partitioning. Finally, our study provides not only an additional resource for mass spectrometry-derived evidence for gametocyte proteins but also lays down the foundation for rational screening and development of novel sex-partitioned protein biomarkers and transmission-blocking vaccine candidates.Sexual stages represent only a small fraction of Plasmodium falciparum parasites that are present during human malaria infection, yet they alone are responsible for disease transmission (1). As such, the Malaria Eradication Research Agenda (malERA) has prioritized the need for studies that specifically address these transmission stages, with the hope of developing new transmission-blocking vaccines and drugs, as well as diagnostics that are specific for these sexual stages (2–4). In fact, one of the critical gaps in malaria transmission biology and surveillance centers on the lack of knowledge about the infectivity of symptomatic and asymptomatic gametocytemic individuals for mosquitoes. Many infected individuals harboring the Plasmodium falciparum sexual stage, or gametocyte, are asymptomatic carriers and they represent the primary reservoir for malaria transmission (5). Missing the opportunity to treat these carriers will increase the risk for epidemic malaria in regions that have approached the elimination phase. Thus, proper surveillance of gametocyte carriers is critical for evaluating ongoing malaria control and elimination programs. Surveillance is difficult, however, because gametocytes comprise only 0.1–2% of the total body parasite load during active infection (5), and are only observed in the bloodstream in their mature (Stage V) form, with the first four developing stages sequestered in tissues. Microscopy-based analysis for sex ratio determination and infectivity studies remains limited because of cost, training, and suitability for population-wide studies. Although light microscopy remains the gold standard for malaria diagnosis, the relatively low prevalence of circulating gametocytes makes it difficult to accurately detect much less quantify these stages. Moreover, because of variations in skill level of microscopists and inconsistency in method, exclusive use of light microscopy estimates of gametocyte carriage carries a high risk of error. Importantly, the presence of stage V gametocytes in the bloodstream alone, as determined by thick smear microscopy does not imply infectivity to mosquitoes. Ratios of male and female gametocytes in the blood circulation are skewed toward the female, but they can vary significantly based on co-infection, parasite and gametocyte density, and host environmental factors (6), and it is therefore hypothesized that this variation in sex ratios will influence mosquito infectivity. For example, mature gametocyte sex ratios can change during the course of infection in response to host cues or especially following antimalarial treatment resulting in an increase in the number of males (6, 7). However, it remains unknown whether the transmission potential to mosquitoes of the individuals in these studies fluctuated because of the changes in sex ratio.There are currently no uncomplicated tools to distinguish male and female mature P. falciparum gametocytes (of which at least one of each is required for fertilization and ookinete development in the mosquito) at the molecular level. Although the proteome of Plasmodium gametocytes has been described (8–11), these previous analyses fell just short of providing the partitioned male and female proteomes for P. falciparum. Moreover, the availability of the genomes of human, primate, and rodent malaria parasites and the acquisition of sequence information for recent field isolates of P. falciparum have created the opportunity to understand gene diversity and conservation in sexual stage development across Plasmodia. Identifying markers that differ between male and female P. falciparum stage V gametocytes is critical in informing transgenic approaches aimed at separating the two. It has been argued that the inherent evolutionary differences between rodent and human malaria parasites, especially for the sexual stages, limit the utility of the P. berghei gametocyte proteome (11) in providing a priori knowledge of these markers. Several iterations and improvements to the P. berghei genome have been made available since 2005, whereas MS search engines have improved commensurately, further compounding the issue. However, we would also argue that the current evidence suggests a high degree of conservation in gametocyte gene complement across Plasmodium (12, 13), and therefore presumably in sex-specific genes - despite key differences such as gametocyte sequestration and morphology. Here, we report on our effort to address these scientific gaps to a certain extent and to test our gametocyte gene conservation hypothesis through the use of comparative protein bioinformatics analyses of the mature stage V gametocyte proteomes of two distinct P. falciparum strains with our update of the bioinformatic analysis of the P. berghei male and female gametocyte proteomes.