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.