Role of Pfs47 in the dispersal of ancestral Plasmodium falciparum malaria through adaptation to different anopheline vectors
Contributed by Carolina Barillas-Mury; received August 9, 2022; accepted December 30, 2022; reviewed by Daniel E. Neafsey and Douglas E. Norris
Significance
Plasmodium falciparum malaria remains a devastating parasitic disease with high morbidity and mortality. This disease originated in sylvatic regions of Africa from the transfer P. praefalciparum, a malaria parasite that infects gorillas, to humans. Human infections were likely initiated by bites from infected sylvan mosquitoes, but once the parasite adapted to humans, global dispersal of ancestral P. falciparum also required adaptation to different anopheline species present in new geographic regions. Here, we explore the extent to which parasite selection by the mosquito immune system was an important barrier for early dispersal of ancestral P. falciparum from sylvatic regions in Central Africa to other Sub-Saharan regions and to the Asian continent.
Abstract
Plasmodium falciparum malaria originated when Plasmodium praefalciparum, a gorilla malaria parasite transmitted by African sylvan anopheline mosquitoes, adapted to humans. Pfs47, a protein on the parasite surface mediates P. falciparum evasion of the mosquito immune system by interacting with a midgut receptor and is critical for Plasmodium adaptation to different anopheline species. Genetic analysis of 4,971 Pfs47 gene sequences from different continents revealed that Asia and Papua New Guinea harbor Pfs47 haplotypes more similar to its ortholog in P. praefalciparum at sites that determine vector compatibility, suggesting that ancestral P. falciparum readily adapted to Asian vectors. Consistent with this observation, Pfs47-receptor gene sequences from African sylvan malaria vectors, such as Anopheles moucheti and An. marshallii, were found to share greater similarity with those of Asian vectors than those of vectors of the African An. gambiae complex. Furthermore, experimental infections provide direct evidence that transformed P. falciparum parasites carrying Pfs47 orthologs of P. praefalciparum or P. reichenowi were more effective at evading the immune system of the Asian malaria vector An. dirus than An. gambiae. We propose that high compatibility of ancestral P. falciparum Pfs47 with the receptors of Asian vectors facilitated the early dispersal of human malaria to the Asian continent, without having to first adapt to sub-Saharan vectors of the An. gambiae complex.
Sign up for PNAS alerts.
Get alerts for new articles, or get an alert when an article is cited.
Despite recent progress, Plasmodium falciparum malaria remains a devastating parasitic disease with high morbidity and mortality. In 2020, an estimated 241 million cases occurred, resulting in 627,000 deaths, mostly of children from Sub-Saharan Africa (1). Previous studies indicate that P. falciparum originated from P. praefalciparum, a malaria parasite that infects gorillas in Central Africa (2). Transmission to humans was likely initiated by infectious bites from sylvan mosquitoes, such as Anopheles vinckei, An. moucheti, and An. marshallii, which transmit nonhuman ape malarias (3). Once the parasite adapted to humans, global dispersal of ancestral P. falciparum also required adaptation to different anopheline species (4, 5). However, some of the major malaria vectors are evolutionarily distant from sylvan vectors of ape malarias (6). Additionally, it is not clear to what extent vector adaptation was an important barrier for early dispersal of ancestral P. falciparum from sylvatic regions in central Africa to other regions in sub-Saharan Africa and to the Asian continent.
The ability of P. falciparum to evade the mosquito immune system is critical for parasite survival in the mosquito vector and for disease transmission to humans (7). The parasite surface protein Pfs47 has three domains, and polymorphisms in the central domain are major determinants of compatibility between P. falciparum strains from different continents and their mosquito vector species (8, 9). Pfs47 is expressed in female gametocytes, and on the surface of female gametes and motile ookinetes, the stage that traverses the mosquito midgut (10, 11). Pfs47 is required for immune evasion through interaction with compatible Pfs47 midgut receptors (12). Genetic diversity in Pfs47 shows exceptionally high geographic structure at the continent level (7, 13–16), indicative of natural selection by local mosquito vectors (7). Functional studies revealed that P. falciparum parasites are more efficient evading the immune system of sympatric vectors (7). Furthermore, elimination of incompatible parasites can be prevented by disrupting the mosquito complement-like system, and genetic exchange of the Pfs47 haplotype in transformed parasites is sufficient to change vector/parasite compatibility (7). A “lock and key” working model has been established, proposing that only those parasites with a Pfs47 variant compatible with the Pfs47 receptor of a given anopheline vector can evade the mosquito immune system and establish a local malaria transmission cycle (7). Here, we explored the genetic diversity of Plasmodium Pfs47 and of the mosquito Pfs47-receptor of anopheline vectors of malaria from different geographic regions. Experimental infections, with transformed P. falciparum parasites expressing orthologs of Pfs47 from Plasmodium species that infect other African apes, were also used to establish whether the mosquito immune system was a barrier for adaptation of the inferred ancestral P. falciparum Pfs47 genotype to major mosquito vectors of human malaria. This in-depth genetic analysis of Pfs47 and its receptor revealed unexpected aspects of the evolutionary history of ancestral P. falciparum that appear to be determined by parasite compatibility with different mosquito vectors.
Results
Most South Asian and Papua New Guinean Plasmodium falciparum Isolates Harbor the Same Amino Acid Sequence as P. praefalciparum P47 in a Region Critical for Vector Compatibility.
The genetic diversity of Pfs47 was explored by analyzing 4,971 genomic DNA sequences from different geographic regions (SI Appendix, Table S1) identifying 209 distinct haplotypes (SI Appendix and File S2). Network analysis of Pfs47 haplotypes (Fig. 1A) confirmed marked genetic differentiation of Pfs47 haplotypes circulating in different continents, as previously reported (7, 14–16). Furthermore, differences in Pfs47 haplotype frequency distribution at a sub-continent level were detected between West and East Africa, South Asia and Southeast Asia, and between Coastal South America and Amazonia (Fig. 1A). The Pfs47 haplotype network makes evident the genetic diversity present in Papua New Guinea (PNG), which includes haplotypes characteristic of Africa, South and Southeast Asia, and South America (Fig. 1A).
Fig. 1.
To gain new insights into the origin of Pfs47, five full-length P. praefalciparum P47 (Pp47) sequences were also included in the network analysis. Remarkably, these Pp47 sequences showed very little diversity from each other and were also most similar to four Pfs47 haplotypes found in South and Southeast Asia and PNG (Hap 172, 161, 167, and 182) and one haplotype found in Africa (Hap 40) (Fig. 1A). Moreover, the Pp47 sequences, which are presumably similar to ancestral Pfs47, were found at the center of the network, connecting to two distinct haplotype clusters, one African and another Asian. South American haplotypes are most similar to a central haplotype (Hap 161) in the network that is currently present in South Asia, Southeast Asia and PNG (Fig. 1A). Furthermore, four polymorphic amino acids between residues C230 and C260 in domain 2 of Pfs47 (I236T, S242L, V247A, and L248I), which have been shown to determine vector compatibility (8, 9), are identical between P. praefalciparum P47 and the most common P. falciparum Pfs47 haplotypes in South Asia (66%) and PNG (55%) (Fig. 1B and SI Appendix, Table S2), leading to the most parsimonious inference that this sequence is representative of the ancestral allelic state. Three of these (I236, S242, and L248) are also conserved among all Laverania P47 sequences, except for 248R in P. lomamiensis (SI Appendix, Table S2 and Figs. S1 and S2), reinforcing their likely ancestral allelic state. While Pfs47 haplotypes with the putative ancestral allelic state for polymorphisms I236T, S242L, V247A, and L248I are also represented in Southeast Asia (3%), the L240I polymorphism is highly prevalent (96.5%) in that continent. Interestingly, the I236T polymorphism is almost fixed (99.8%) in Africa for the putative derived allelic state, which is not present in any other Laverania and is rare in P. falciparum from other continents except PNG (Fig. 1B and SI Appendix, Table S2). In most cases (74%), a second polymorphism (L248I) in the putative derived allelic state is also present in Africa. The sample set from the Americas is almost fixed (99.2%) for the putative derived allelic state of two other polymorphisms (S242L and V247A) that are present at very low frequency in PNG (2%) and Africa (0.03%), and are absent in South and Southeast Asia (Fig. 1B and SI Appendix, Table S2).
Taken together, these observations suggest that some Pfs47 haplotypes currently present in Asia and PNG are most similar, in key polymorphisms that determine vector compatibility, to the inferred ancestral allelic states present at the time when the gorilla P. praefalciparum parasites in sylvatic regions of Africa gained the ability to infect humans. Previous studies have shown that P. falciparum parasites with a Pfs47 haplotype compatible with a given vector are able to evade the mosquito immune system and be effectively transmitted (7, 12). This raises the possibility that the ancestral Pfs47 haplotypes, presumably adapted to sylvan anophelines, may have been more compatible with anophelines from Asia and PNG than to vectors of the An. gambiae complex in Africa.
Pfs47-Receptor Sequences of Ape Malaria Vectors Are More Similar to Those of Major Asian Vectors of Human Malaria than to African Vectors of the An. gambiae Complex.
The compatibility of P. falciparum parasites with different anopheline vectors is determined by molecular interactions between Pfs47 on the parasite’s surface and a mosquito Pfs47-receptor in midgut epithelial cells (12). The hypothesis that higher similarity between the Pfs47-receptors of sylvatic vectors of P. praefaciparum and the receptors from Asian anophelines may have facilitated dispersal of ancestral P. falciparum to Asia was explored. Specimens of ape malaria vectors An. moucheti and An. marshallii were collected in Cameroon, identified by morphology and molecular taxonomy (SI Appendix, Fig. S3 and File S2), and the gene encoding their Pfs47-receptor ortholog was sequenced (File S3). Distance analysis of Pfs47-receptor sequences indicates that the orthologs from An. moucheti and An. marshallii are more similar to those of An. funestus and of some major Asian vectors such as An. dirus and An. culicifacies, as well as An. farauti from PNG, than to the receptors of African vectors of the An. gambiae complex (Fig. 2). This suggests that current Pfs47 haplotypes that are more similar to those of P. praefalciparum parasites, presumably with polymorphisms in the ancestral allelic state that were adapted to sylvatic vectors, could have been more compatible with some Asian and PNG anophelines vectors, than with African vectors of the An. gambiae complex.
Fig. 2.
P47 from Ape Malaria Parasites Are More Compatible with An. dirus Mosquitoes from Asia than with An. gambiae from Africa.
We hypothesized that Pfs47 orthologs adapted to malaria vectors of great apes are more compatible with Asian vectors than with vectors of the An. gambiae complex. This was experimentally tested by infecting different anophelines with transgenic P. falciparum parasites expressing P47 from ape malaria parasites that infect either gorillas (P. praefalciparum, Pp47) or chimpanzees (P. reichenowi, Pr47). Pp47 and Pr47 were cloned and used to genetically complement a P. falciparum Pfs47-KO line (SI Appendix, Fig. S4). The An. stephensi Nijmegen strain, genetically selected to be highly susceptible to P. falciparum infection (17), was used to ensure the quality of the gametocyte cultures, as this line is readily infected by parasites with all different Pfs47 haplotypes and even by those lacking Pfs47 (Pfs47-KO) (9, 18). The Pfs47-KO line was unable to infect An. dirus and had low infectivity in An. gambiae (Fig. 3A), as previously reported (7). Genetic complementation of the KO line with Pp47 (gorilla malaria) or Pr47 (chimpanzee malaria) dramatically increased the infectivity in An. dirus (P < 0.0001, Mann–Whitney test), reaching an infection prevalence of 74% and 75%, respectively (Fig. 3 B and C). In contrast, genetic complementation did not rescue the infectivity in An. albimanus mosquitoes from Central and South America (Fig. 3 B and C). Although both Pp47 and Pr47 complemented lines were able to infect An. gambiae, the infectivity with Pp47 was similar to that of the An. stephensi control (Fig. 3B), while the infectivity of An. gambiae with Pr47 was significantly lower (P < 0.0001, Mann–Whitney test) (Fig. 3C).
Fig. 3.
Incompatible mosquito/parasite combinations activate the mosquito complement system and this immune response limits infection (7). As a result, a significant enhancement in the infection level is observed when the complement system is disrupted by silencing key effectors, such as the thioester-containing protein 1 (TEP1) or the leucin-rich repeat protein LRIM1, a protein that stabilizes TEP1 (19). In contrast, highly compatible mosquito/parasite combinations avoid activating the complement system, therefore silencing either TEP1 or LRIM1 has no significant effect on the level of infection (7, 9, 20). The parasite’s ability to evade the mosquito immune system was determined by evaluating the effect of disrupting the mosquito complement system on the infectivity of the transgenic lines complemented with P47 from ape malarias (Pp47 and Pr47).
Disruption of the complement-like system by silencing TEP1 in An. gambiae significantly increased infection with the control Pfs47-KO P. falciparum line and with the Pp47 and Pr47 complemented Pfs47-KO lines (Fig. 4A), indicating that both Pfs47 ape orthologs are not fully compatible with An. gambiae. In contrast, LRIM1 silencing in An. dirus significantly increased infection with the control Pfs47-KO P. falciparum line (P < 0.01, Mann–Whitney test) (Fig. 4B), but had no significant effect on the infectivity with the Pp47- and Pr47-complemented Pfs47-KO lines (Fig. 4B), showing that both ape Pfs47 orthologs are compatible with An. dirus and can evade the complement system of this anopheline. This functional analysis of Pfs47 orthologs is consistent with the hypothesis that the ancestral P. falciparum Pfs47 haplotype was highly compatible with Asian vectors, and this may have facilitated the transmission and dispersal of ancestral P. falciparum from Africa to the Asian continent.
Fig. 4.
Discussion
P. falciparum malaria originated from P. praefalciparum in sylvatic regions of Central Africa. The parasite had to adapt to different anopheline mosquitoes, most of them with a restricted geographic distribution within a continent, to spread to other regions of Africa and other continents. To understand the evolutionary history and epidemiology of malaria, it is therefore critical to identify factors that allowed P. falciparum to adapt to different mosquito vectors.
Several lines of evidence presented here suggest that the Pfs47 haplotype of ancestral P. falciparum, presumably adapted to sylvan mosquitoes in tropical Africa, had greater compatibility with anophelines in Asia/PNG than with the An. gambiae complex. The evidence includes i) the identity of Pfs47 amino acid residues which determine vector compatibility among Pfs47 haplotypes from Asia/PNG and ape malaria Pfs47 orthologs, (Fig. 1B and SI Appendix, Figs. S1 and S2), ii) the greater Pfs47-receptor sequence similarity of P. falciparum vectors in Asian/PNG to ape malaria vectors (Fig. 2), and iii) the higher compatibility of transgenic P. falciparum parasites expressing the Pfs47 orthologs of P. praefalciparum and P. reichenowi with An. dirus, a major Asian vector, than with An. gambiae (Fig. 4).
The Pfs47 receptor is an ancient gene with clear orthologs in all anophelines and culicine species, and it is highly conserved within a species (21). The receptor appears to have diverged during the speciation of the anopheline subgenus (Nyssorhynchus, Anopheles, and Cellia) (Fig. 2). The receptor is highly conserved in mosquito species from the An. gambiae complex, which form a monophyletic clade (Fig. 2), separate from those of other species of the Cellia subgenus (22). A fortuitous interaction of the Plasmodium surface protein P47 with an ancestral mosquito protein allowed the parasite to evade the mosquito immune system, survive, and be transmitted very effectively.
The current absence in Africa of Pfs47 haplotypes most similar to the putative ancestral allelic states in key amino acids that determine vector compatibility suggests that the dispersal of ancestral P. falciparum to Asia/PNG did not require prior adaptation of P. falciparum to mosquitoes of the An. gambiae complex. Nevertheless, the less parsimonious possibility that some P. falciparum parasites first adapted to the An. gambiae complex and later mutated back to adapt to Asian vectors cannot be ruled out. Initial dispersal of ancestral P. falciparum out of tropical Africa would have required adaptation to nonsylvan mosquito species. Interestingly, the Pfs47-receptor gene sequence of An. funestus —a vector of human malaria in Africa—is more similar to those of An. moucheti and An. marshallii and to Asian vectors compared with those of the An. gambiae complex (Fig. 2) (12). This suggests that ancestral P. falciparum could have been compatible with An. funestus, which may have been important during the initial expansion of ancestral P. falciparum within Africa, a critical step for the parasite to reach Asia.
The high prevalence of Pfs47 haplotypes in South Asia and PNG that most closely resemble the orthologs from P. praefalciparum and P. reichenowi, especially in polymorphic amino acid residues that determine vector compatibility (Fig. 1B and SI Appendix, Table S2), indicates that the main malaria vectors in those regions were particularly receptive to ancestral P. falciparum coming out of Africa. The major vectors in South Asia include An. stephensi and An. culicifacies and in PNG the An. punctulatus complex, which includes An. farauti. It is worth noting that the recent establishment of An. stephensi in Africa (23) is of particular concern, as it could accelerate the dispersal of Asian P. falciparum parasites back to Africa, including artemisinin-resistant strains.
The fact that a Pfs47 polymorphic site which determines vector compatibility (I236T) is nearly fixed (99.8%) for the derived allelic state in Africa, suggests that adaptation of ancestral P. falciparum to mosquitoes of the An. gambiae complex—the dominant vectors in Africa—resulted in a stringent selection of Pfs47. Such adaptation of P. falciparum to the An. gambiae complex presumably took place some 4,000 to 6,000 y ago with the beginning of agriculture in Africa and likely coincides with the domestication of An. gambiae. The adaptation of the parasite to the An. gambiae complex was followed by a vast expansion of the P. falciparum population due to the fast growth in the human population with the advent of agriculture.
We hypothesize that Asian P. falciparum may harbor unique variants in other genes besides Pfs47—especially genes required for development in the mosquito vector—and, counterintuitively, that their genomes have retained ancestral character states, unlike most parasites currently circulating in Africa, the continent where P. falciparum originated. PfAP2-G, an essential regulator of gametocytogenesis, is an example of such a gene, as it has an insertion of ~200 bp in the ape malaria parasites P. praefalciparum and P. reichenowi, and in P. falciparum isolates from Asia, relative to the reference P. falciparum 3D7 strain. That longer allele is not present in African parasites, suggesting that a deletion took place when the P. falciparum parasite population expanded in Africa, but not in those ancestral parasites that dispersed to Asia (24). Genetic analysis and functional studies of Pfs47 presented here reveal that the evolutionary history of P. falciparum has been sculpted, at least in part, by the constant selection for parasites that evade the immune system of compatible mosquito vector species.
Materials and Methods
Pfs47 Gene Sequences.
P. falciparum Pfs47 gene sequences were retrieved from the literature (7, 24) and from the Malaria Genomic Epidemiology Network (Malaria-GEN) (25) as previously described (16). A total of 4,971 Pfs47 sequences were used, originating from Africa (3,126), New World (121), Asia (1,675), and PNG (49). The Pfs47 sequences from Pf3K polygenomic samples were obtained after deconvolution using DEploid as described (26). In the case of the Pfs47 sequences in samples from other sources, only the major allele was analyzed as described (24).
This publication uses data from the MalariaGEN P. falciparum Community Project, PfCP (www.malariagen.net/projects/p-falciparum-communityproject) and the Pf3K project (2016) pilot data release 5 (www.malariagen.net/data/pf3k-5). Genome sequencing was performed by the Wellcome Trust Sanger Institute and the Community Projects, coordinated by the MalariaGEN Resource Centre with funding from the Wellcome Trust (098051, 090770).
Ape P47 Sequences.
P47 sequences from P. praefalciparum were obtained from PlasmoDB (https://plasmodb.org/) (PPRFGO1_1348600, Gabon) or amplified by limiting dilution PCR (also termed single genome amplification or SGA) using DNA extracted from gorilla fecal (NDggg3203_FL_V2.7) or blood (SAggg3157_FL_SGA30.7, SAggg3157_FL_SGA30.12, SAggg3157_FL_SGA60) samples, all of which were obtained with permission from the respective authorities and shipped in compliance with the regulations of the Convention on International Trade in Endangered Species of Wild Fauna and Flora as previously described (27). The Pp47 NDggg3203_FL_V2.7 sequence used for complementation was amplified as two nonoverlapping fragments from a gorilla fecal sample collected in Cameroon and assembled by filling a 23 bp gap with a sequence obtained from a second sample (GTggg118_SGA10.3) collected in the Republic of Congo (SI Appendix, Fig. S5). Three additional full-length Pp47 sequences (SAggg3157_FL_SGA30.7, SAggg3157_FL_SGA30.12, and SAggg3157_FL_SGA60.1) were SGA amplified from a gorilla bushmeat sample (28). Full-length and partial P47 sequences from P. reichenowi, P. adleri, P. gaboni, P. billcollinsi, P. blacklocki, and P. lomamiensis were retrieved from PlasmoDB (PRCDC_1345800, PADL01_13455900, PGABG01_1344800-t36_1-p1, PGSY75_1346800-t31_1-p1, PBILCG01_1347000-t36_1-p1, and PBLACG01_1344400-t36_1-p1) and from the literature (27, 29).
Genetic Diversity and Network Analysis of P47 Gene Sequences.
DnaSP6 (30) was used to identify distinct Pfs47 haplotypes from full-length sequences, 1,320 bp. A Nexus file generated in DnaSP6 was used in a DNA network analysis (TCS) of the 209 Pfs47 haplotypes identified and five Pp47 sequences in PopArt (31). The “trait” for the Pfs47 haplotypes was the geographic origin region (West Africa Central Africa, East Africa, South America, Amazonia, South America Coastal, Bangladesh, Southeast Asia, and PNG).
Pfs47-Receptor Sequences in Anopheline Mosquitoes.
Anopheles moucheti and An. marshallii female mosquitoes were collected in Cameroon, morphologically identified and dried with silica gel. DNA was extracted from individual dried mosquitoes as previously described (32). Morphological species identification was confirmed by molecular taxonomy of the mitochondrial cytochrome c oxidase subunit II (COII) gene. A 567 bp fragment of the COII was PCR amplified as previously described (33) using primers: forward: 5′-TCTAATATGGCAGATTAGTGC A-3′, and reverse: 5′-ACTTGCTTTCAGTCATCTAATG-3′. NCBI Blast analysis of 4 An. marshallii and 8 An. moucheti COII gene sequences (File S2) presented the highest homology with the corresponding species.
The Pfs47-receptor ortholog in An. moucheti and An. marshallii field collected samples was PCR amplified and later sequenced in two overlapping fragments using primers BF 5′-GATTTAACTGGATTCCGTGGAC-3′ with Reverse_579 5′-GTCCGAATCGTGTCCCGC-3′; and CF 5′-GGTGCGGAACAAAGCTTC-3′ with CR 5′-TCAGTGTTCGATCAGCACCTC-3′, which were designed based on An. funestus Pfs47-receptor gene sequence. The Pfs47-receptor gene consensus sequence was aligned with Pfs47-receptor orthologs from different mosquito species retrieved from VectorBase (https://vectorbase.org) and the introns removed manually, generating a 873-bp coding sequence. Gene sequence clustering was done using the neighbor-joining method and genetic distance of Pfs47-receptor-coding gene sequences was estimated using the Nei-Goyobori method (Jukes-Cantor correction) with MEGA X (34).
Anopheles Mosquitoes and Plasmodium Parasites.
The mosquito strains used were An. gambiae G3, An. stephensi Nijmegen, An. dirus X s.s. (An. dirus A), and An. albimanus. Mosquito rearing was done at 27 °C and 80% humidity on a 12-h light–dark cycle under standard laboratory conditions as previously shown (20). The P. falciparum strains —NF54, NF54-Pfs47KO and Pfs47KO complemented lines with P. praefalciparum Pp47, and P. reichenowi Pr47—were cultured in O+ human erythrocytes at 5% hematocrit in RPMI 1640 medium supplemented with 25 mM NaHCO3, 10 mg/L hypoxanthine, 25 mM Hepes, and 10% (vol/vol) heat-inactivated O+ human serum (Interstate Blood Bank, Memphis, TE) at 37 °C and with a gas mixture of 5% CO2, 5% O2, and balance N2 (35).
Experimental Infection of Mosquitoes with P. falciparum.
Mosquito females were infected artificially by membrane feeding with P. falciparum gametocyte cultures prepared as previously described (36). Briefly, the RBCs of a 14 to 17-d old gametocyte culture (stages IV and V) were separated by centrifugation at 37 °C (3 min, 2,500 g). The infected RBCs were resuspended in one volume of human serum at 37 °C and then diluted to 0.15 to 0.2% stage V gametocytemia with O+ human RBC at 40% in human serum at 37 °C. All manipulations were done maintaining the tubes in water at 37 °C. A 400-uL sample of diluted gametocytes was delivered to a prewarmed (37 °C) water jacketed glass feeder with parafilm as membrane for 30 min. Mosquito midguts were dissected 8 to 10 d after feeding and were stained with 0.1% (wt/vol) mercurochrome in water to count oocysts by light microscopy. The distribution of parasite numbers in individual mosquitoes from control and experimental groups was compared using the nonparametric Mann–Whitney test (GraphPad, Prism). All standard membrane feeding assays were confirmed in two-to-three independent experiments.
dsRNA-Mediated Gene Knockdown.
Female An. gambiae or An. dirus X s.s. mosquitoes were injected 1 to 2 d after emergence as shown previously (7). Individual mosquitoes were injected 69 nL dsRNA solution (3 μg/μL) in water, 3 to 4 d before membrane feeding with a Plasmodium-infected blood meal. The control dsRNA (LacZ), and An. dirus LRIM1 and An. gambiae TEP1 dsRNA were produced as previously described (7) with the MEGAscript RNAi Kit (Ambion, Austin, TX) using DNA templates generated by nested PCR on cDNA from whole female mosquitoes. Gene silencing for An. gambiae TEP1 and An. dirus LRIM1 was estimated in whole sugar-fed mosquitoes by quantitative real-time PCR (qPCR) using the S7 ribosomal protein gene as internal reference, as shown previously (7). The silencing efficiency in dsRNA-injected mosquitoes was 81 to 84% for TEP1 in An. gambiae, and 60 to 80% for LRIM1 in An. dirus, relative to dsLacZ-injected controls.
Episomal Genetic Complementation of P. falciparum Pfs47KO Line with Pp47 and Pr47.
The P. falciparum Pfs47KO line (9) was complemented with different orthologs of P47. The 8.3-kb pPfs47attP plasmids were assembled following several modifications to the previously developed pCBM-BSD plasmid, which contains a full-length Pfs47 from P. falciparum surrounded by upstream (1 kb) and downstream (0.16 kb) noncoding regions of the Pfs47 ORF, flanked by two Afl II restriction enzyme sites (7). To introduce different orthologs of P47 into the pPfs47attP plasmid, full-length synthetized Pp47 (NDggg3203_FL_V2.7, SI Appendix) and Pr47 (PRCDC_1345800.1, SI Appendix) were PCR-amplified with In-Fusion–designed oligonucleotides and subcloned into an Afl II digested- pPfs47attP plasmid, obtaining pPp47 and pPr47 plasmids. Cloning procedures were carried out in Stellar bacteria (Takara, San Jose, CA), and every construct was fully sequenced.
Transfection was performed by electroporation of either pPp47 or pPr47 plasmids into uninfected erythrocytes. P. falciparum Pfs47KO trophozoite stage infected cells were enriched by the Percoll-sorbitol method and cultivated with these plasmid-loaded cells. The transfected culture was selected with blasticidin at 2.5 µg/mL. After 2 to 3 wk, parasite growth was detected by Giemsa-staining.
RNA and Protein Expression.
The expression of P47 mRNA in the transfected P. falciparum lines was confirmed by qPCR using cDNA from stage IV–V gametocytes, and with the P. falciparum gene Pf10_0203 (ADP ribosylation factor) as an internal reference gene (7). The ΔΔCt method was used to estimate gene dosage and mRNA expression level. P47 protein expression was confirmed by western blot of gametocyte protein from the different P. falciparum lines as previously shown (7). Briefly, P. falciparum gametocytes were isolated from cultures by saponin treatment and stored at −70 °C until used. The gametocyte frozen pellet was resuspended in 100 μL water, an aliquot of 5 μL was mixed with NuPage LDS sample buffer and heated at 70 °C for 10 min before electrophoresis in a 4 to 12% NuPage Bis Tris gradient gel. Gametocyte protein was transferred from the gel to a nitrocellulose membrane with the iBlot dry blotting system (Invitrogen, Thermo Fisher Scientific, Waltham, MA). Blocking of the blot was done with 5% (wt/vol) milk in Tris-buffered saline with Tween 20 (0.05 M Tris, 0.138 M NaCl, 0.0027 M KCl, pH 8; 0.05% Tween 20) (TBST) overnight at 4 °C, followed by incubation (2 h at RT) with anti Pfs47 mouse monoclonal antibody (1 mg/mL) diluted 1:500 in TBST-milk solution. Afterward, the blot was incubated (1 h at RT) with anti-mouse IgG alkaline phosphatase conjugate (1 mg/mL; Promega, Madison, WI) diluted 1:10,000 in TBST-milk solution. Bound conjugated antibody was detected with Western Blue stabilized substrate (Promega, Madison, WI) as previously shown (7).
Data, Materials, and Software Availability
GenBank accession numbers for new derived Pp47 and An. moucheti and An. marshallii COII and Pfs47 receptor sequences are shown in SI Appendix, Table S3. All study data are included in the article and/or SI Appendix.
Acknowledgments
This work was supported by the Intramural Research Program of the Division of Intramural Research Z01AI000947, NIAID, NIH as well as extramural funding to B.H.H. (R01 AI 091595). We would like to thank Jacob Almagro for his help with Pfs47 sequence data gathering and deconvolution. We thank Kevin Lee, Yonas Gebremicale, and André Laughinghouse for insectary support, and Asher Kantor and Micah Young for editorial assistance. We sincerely appreciate the valuable comments and suggestions from the two reviewers, which helped us in improving the quality of the manuscript.
Author contributions
A.M.-C., G.E.C., A.D., C.A.-N, B.H.H., J.C.S., and C.B.-M. designed research; A.M.-C., G.E.C., A.D., W.L., N.R., and C.A.-N. performed research; W.L. and B.H.H. contributed new reagents/analytic tools; A.M.-C., G.E.C., A.D., W.L., N.R., J.C.C., and C.B.-M. analyzed data; and A.M.-C., G.E.C., and C.B.-M. wrote the paper.
Competing interest
The authors declare no competing interest.
Supporting Information
Appendix 01 (PDF)
- Download
- 482.02 KB
Dataset S01 (TXT)
- Download
- 286.38 KB
Dataset S02 (XLSX)
- Download
- 37.62 KB
Dataset S03 (TXT)
- Download
- 41.36 KB
Dataset S04 (TXT)
- Download
- 16.51 KB
References
1
WHO, “World Malaria Report 2021” (World Health Organization, Geneva, 2021), p. 322.
2
W. Liu et al., Origin of the human malaria parasite Plasmodium falciparum in gorillas. Nature 467, 420–425 (2010).
3
B. Makanga et al., Ape malaria transmission and potential for ape-to-human transfers in Africa. Proc. Natl. Acad. Sci. U.S.A. 113, 5329–5334 (2016).
4
A. Molina-Cruz, C. Barillas-Mury, Mosquito vectors of ape malarias: Another piece of the puzzle. Proc. Natl. Acad. Sci. U.S.A. 113, 5153–5154 (2016).
5
A. Molina-Cruz, M. M. Zilversmit, D. E. Neafsey, D. L. Hartl, C. Barillas-Mury, Mosquito vectors and the globalization of Plasmodium falciparum Malaria. Annu. Rev. Genetics 50, 447–465 (2016).
6
R. E. Harbach, “The phylogeny and classification of Anopheles” in Anopheles Mosquitoes - New Insights into Malaria Vectors, S. Manguin, Ed. (InTech, 2013).
7
A. Molina-Cruz et al., Plasmodium evasion of mosquito immunity and global malaria transmission: The lock-and-key theory. Proc. Natl. Acad. Sci. U.S.A. 112, 15178–15183 (2015).
8
G. E. Canepa, A. Molina-Cruz, C. Barillas-Mury, Molecular analysis of Pfs47-mediated Plasmodium evasion of mosquito immunity. PLoS One 11, e0168279 (2016).
9
A. Molina-Cruz et al., The human malaria parasite Pfs47 Gene mediates evasion of the mosquito immune system. Science 340, 984–987 (2013).
10
S. A. Arredondo, S. H. Kappe, The s48/45 six-cysteine proteins: Mediators of interaction throughout the Plasmodium life cycle. Int J. Parasitol. 47, 409–423 (2016), https://doi.org/10.1016/j.ijpara.2016.10.002.
11
A. Molina-Cruz, G. E. Canepa, C. Barillas-Mury, Plasmodium P47: A key gene for malaria transmission by mosquito vectors. Curr. Opin. Microbiol. 40, 168–174 (2017).
12
A. Molina-Cruz et al., Plasmodium falciparum evades immunity of anopheline mosquitoes by interacting with a Pfs47 midgut receptor. Proc. Natl. Acad. Sci. U.S.A. 117, 2597–2605 (2020).
13
A. Ahouidi et al., An open dataset of Plasmodium falciparum genome variation in 7,000 worldwide samples. Wellcome Open Res. 6, 42 (2021).
14
T. G. Anthony, S. D. Polley, A. P. Vogler, D. J. Conway, Evidence of non-neutral polymorphism in Plasmodium falciparum gamete surface protein genes Pfs47 and Pfs48/45. Mol. Biochem. Parasitol. 156, 117–123 (2007).
15
M. Manske et al., Analysis of Plasmodium falciparum diversity in natural infections by deep sequencing. Nature 487, 375–379 (2012).
16
A. Molina-Cruz et al., A genotyping assay to determine geographic origin and transmission potential of Plasmodium falciparum malaria cases. Commun. Biol. 4, 1145 (2021).
17
A. M. Feldmann, T. Ponnudurai, Selection of Anopheles stephensi for refractoriness and susceptibility to Plasmodium falciparum. Med. Veterinary Entomol. 3, 41–52 (1989).
18
B. C. van Schaijk et al., Pfs47, paralog of the male fertility factor Pfs48/45, is a female specific surface protein in Plasmodium falciparum. Mol. Biochem. Parasitol. 149, 216–222 (2006).
19
S. Blandin et al., Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae. Cell 116, 661–670 (2004).
20
A. Molina-Cruz et al., Some strains of Plasmodium falciparum, a human malaria parasite, evade the complement-like system of Anopheles gambiae mosquitoes. Proc. Natl. Acad. Sci. U.S.A. 109, E1957–E1962 (2012), https://doi.org/10.1073/pnas.1121183109.
21
A. Molina-Cruz, Plasmodium falciparum evades immunity of anopheline mosquitoes by interacting with a Pfs47 midgut receptor. Proc. Natl. Acad. Sci. U.S.A. 117, 2597–2605 (2020), https://doi.org/10.1073/pnas.1917042117, 201917042.
22
D. E. Neafsey et al., Mosquito genomics. Highly evolvable malaria vectors: The genomes of 16 Anopheles mosquitoes. Science 347, 1258522 (2015).
23
M. E. Sinka et al., A new malaria vector in Africa: Predicting the expansion range of Anopheles stephensi and identifying the urban populations at risk. Proc. Natl. Acad. Sci. U.S.A. 117, 24900–24908 (2020).
24
K. A. Moser et al., Strains used in whole organism Plasmodium falciparum vaccine trials differ in genome structure, sequence, and immunogenic potential. Genome Med. 12, 6 (2020).
25
T. M. E. Network, A global network for investigating the genomic epidemiology of malaria. Nature 456, 732–737 (2008).
26
S. J. Zhu, J. Almagro-Garcia, G. McVean, Deconvolution of multiple infections in Plasmodium falciparum from high throughput sequencing data. Bioinformatics 34, 9–15 (2017).
27
W. Liu et al., Multigenomic delineation of Plasmodium species of the Laverania subgenus infecting wild-living chimpanzees and gorillas. Genome Biol. Evol. 8, 1929–1939 (2016).
28
S. A. Sundararaman et al., Genomes of cryptic chimpanzee Plasmodium species reveal key evolutionary events leading to human malaria. Nat. Commun. 7, 11078 (2016).
29
W. Liu et al., Wild bonobos host geographically restricted malaria parasites including a putative new Laverania species. Nat. Commun. 8, 1635 (2017).
30
P. Librado, J. Rozas, DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451–1452 (2009).
31
J. W. Leigh, D. Bryant, popart: Full-feature software for haplotype network construction. Methods Ecol. Evol. 6, 1110–1116 (2015).
32
A. M. De Merida et al., Mitochondrial DNA variation among Anopheles albimanus populations. Am. J. Trop. Med. Hyg. 61, 230–239 (1999).
33
Y. Ma, S. Li, J. Xu, Molecular identification and phylogeny of the Maculatus group of Anopheles mosquitoes (Diptera: Culicidae) based on nuclear and mitochondrial DNA sequences. Acta Trop. 99, 272–280 (2006).
34
K. Tamura, G. Stecher, D. Peterson, A. Filipski, S. Kumar, MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729 (2013).
35
J. W. Zolg, A. J. MacLeod, I. H. Dickson, J. G. Scaife, Plasmodium falciparum: Modifications of the in vitro culture conditions improving parasitic yields. J. Parasitol. 68, 1072–1080 (1982).
36
T. Ifediba, J. P. Vanderberg, Complete in vitro maturation of Plasmodium falciparum gametocytes. Nature 294, 364–366 (1981).
Information & Authors
Information
Published in
Classifications
Copyright
Copyright © 2023 the Author(s). Published by PNAS. This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).
Data, Materials, and Software Availability
GenBank accession numbers for new derived Pp47 and An. moucheti and An. marshallii COII and Pfs47 receptor sequences are shown in SI Appendix, Table S3. All study data are included in the article and/or SI Appendix.
Submission history
Received: August 9, 2022
Accepted: December 30, 2022
Published online: January 23, 2023
Published in issue: January 31, 2023
Keywords
Acknowledgments
This work was supported by the Intramural Research Program of the Division of Intramural Research Z01AI000947, NIAID, NIH as well as extramural funding to B.H.H. (R01 AI 091595). We would like to thank Jacob Almagro for his help with Pfs47 sequence data gathering and deconvolution. We thank Kevin Lee, Yonas Gebremicale, and André Laughinghouse for insectary support, and Asher Kantor and Micah Young for editorial assistance. We sincerely appreciate the valuable comments and suggestions from the two reviewers, which helped us in improving the quality of the manuscript.
Author contributions
A.M.-C., G.E.C., A.D., C.A.-N, B.H.H., J.C.S., and C.B.-M. designed research; A.M.-C., G.E.C., A.D., W.L., N.R., and C.A.-N. performed research; W.L. and B.H.H. contributed new reagents/analytic tools; A.M.-C., G.E.C., A.D., W.L., N.R., J.C.C., and C.B.-M. analyzed data; and A.M.-C., G.E.C., and C.B.-M. wrote the paper.
Competing interest
The authors declare no competing interest.
Notes
Reviewers: D.E. Neafsey, Harvard University; and D.E. Norris, Johns Hopkins University Bloomberg School of Public Health.
Authors
Metrics & Citations
Metrics
Citation statements
Altmetrics
Citations
Cite this article
120 (5) e2213626120,
Export the article citation data by selecting a format from the list below and clicking Export.
Cited by
Loading...
View Options
View options
PDF format
Download this article as a PDF file
DOWNLOAD PDFLogin options
Check if you have access through your login credentials or your institution to get full access on this article.
Personal login Institutional LoginRecommend to a librarian
Recommend PNAS to a LibrarianPurchase options
Purchase this article to access the full text.