Mosquito salivary apyrase regulates blood meal hemostasis and facilitates malaria parasite transmission.

The evolution of hematophagy involves a series of adaptations that allow blood-feeding insects to access and consume blood efficiently while managing and circumventing the host's hemostatic and immune responses. Mosquito, and other insects, utilize salivary proteins to regulate these responses at the bite site during and after blood feeding. We investigated the function of Anopheles gambiae salivary apyrase (AgApyrase) in regulating hemostasis in the mosquito blood meal and in Plasmodium transmission. Our results demonstrate that salivary apyrase, a known inhibitor of platelet aggregation, interacts with and activates tissue plasminogen activator, facilitating the conversion of plasminogen to plasmin, a human protease that degrades fibrin and facilitates Plasmodium transmission. We show that mosquitoes ingest a substantial amount of apyrase during blood feeding, which reduces coagulation in the blood meal by enhancing fibrin degradation and inhibiting platelet aggregation. AgApyrase significantly enhanced Plasmodium infection in the mosquito midgut, whereas AgApyrase immunization inhibited Plasmodium mosquito infection and sporozoite transmission. This study highlights a pivotal role for mosquito salivary apyrase for regulation of hemostasis in the mosquito blood meal and for Plasmodium transmission to mosquitoes and to the mammalian host, underscoring the potential for strategies to prevent malaria transmission.

Single-cell analyses of polyclonal Plasmodium vivax infections and their consequences on parasite transmission.

Most Plasmodium vivax infections contain genetically distinct parasites, but the consequences of this polyclonality on the development of asexual parasites, their sexual differentiation, and their transmission remain unknown. We describe infections of Saimiri monkeys with two strains of P. vivax and the analyses of 80,024 parasites characterized by single cell RNA sequencing and individually genotyped. In our model, consecutive inoculations fail to establish polyclonal infections. By contrast, simultaneous inoculations of two strains lead to sustained polyclonal infections, although without detectable differences in parasite regulation or sexual commitment. Analyses of sporozoites dissected from mosquitoes fed on coinfected monkeys show that all genotypes are successfully transmitted to mosquitoes. However, after sporozoite inoculation, not all genotypes contribute to the subsequent blood infections, highlighting an important bottleneck during pre-erythrocytic development. Overall, these studies provide new insights on the mechanisms regulating the establishment of polyclonal P. vivax infections and their consequences for disease transmission.

Variant surface protein GP60 contributes to host infectivity of Cryptosporidium parvum.

Biological studies of the determinants of Cryptosporidium infectivity are lacking despite the fact that cryptosporidiosis is a major public health problem. Recently, the 60-kDa glycoprotein (GP60) has received attention because of its high sequence polymorphism and association with host infectivity of isolates and protection against reinfection. However, studies of GP60 function have been hampered by its heavy O-linked glycosylation. Here, we used advanced genetic tools to investigate the processing, fate, and function of GP60. Endogenous gene tagging showed that the GP60 cleavage products, GP40 and GP15, are both highly expressed on the surface of sporozoites, merozoites and male gametes. During invasion, GP40 translocates to the apical end of the zoites and remains detectable at the parasite-host interface. Deletion of the signal peptide, GPI anchor, and GP15 sequences affects the membrane localization of GP40. Deletion of the GP60 gene significantly reduces parasite growth and severity of infection, and replacement of the GP60 gene with sequence from an avirulent isolate reduces the pathogenicity of a highly infective isolate. These results have revealed dynamic changes in GP60 expression during parasite development. They further suggest that GP60 is a key protein mediating host infectivity and pathogenicity.

Experimental vaccination by single dose sporozoite injection of blood-stage attenuated malaria parasites.

Malaria vaccination approaches using live Plasmodium parasites are currently explored, with either attenuated mosquito-derived sporozoites or attenuated blood-stage parasites. Both approaches would profit from the availability of attenuated and avirulent parasites with a reduced blood-stage multiplication rate. Here we screened gene-deletion mutants of the rodent parasite P. berghei and the human parasite P. falciparum for slow growth. Furthermore, we tested the P. berghei mutants for avirulence and resolving blood-stage infections, while preserving sporozoite formation and liver infection. Targeting 51 genes yielded 18 P. berghei gene-deletion mutants with several mutants causing mild infections. Infections with the two most attenuated mutants either by blood stages or by sporozoites were cleared by the immune response. Immunization of mice led to protection from disease after challenge with wild-type sporozoites. Two of six generated P. falciparum gene-deletion mutants showed a slow growth rate. Slow-growing, avirulent P. falciparum mutants will constitute valuable tools to inform on the induction of immune responses and will aid in developing new as well as safeguarding existing attenuated parasite vaccines.

Late sporogonic stages of Plasmodium parasites are susceptible to the melanization response in Anopheles gambiae mosquitoes.

The malaria-causing parasites have to complete a complex infection cycle in the mosquito vector that also involves attack by the insect's innate immune system, especially at the early stages of midgut infection. However, Anopheles immunity to the late Plasmodium sporogonic stages, such as oocysts, has received little attention as they are considered to be concealed from immune factors due to their location under the midgut basal lamina and for harboring an elaborate cell wall comprising an external layer derived from the basal lamina that confers self-properties to an otherwise foreign structure. Here, we investigated whether Plasmodium berghei oocysts and sporozoites are susceptible to melanization-based immunity in Anopheles gambiae. Silencing of the negative regulator of melanization response, CLIPA14, increased melanization prevalence without significantly increasing the numbers of melanized oocysts, while co-silencing CLIPA14 with CLIPA2, a second negative regulator of melanization, resulted in a significant increase in melanized oocysts and melanization prevalence. Only late-stage oocysts were found to be melanized, suggesting that oocyst rupture was a prerequisite for melanization-based immune attack, presumably due to the loss of the immune-evasive features of their wall. We also found melanized sporozoites inside oocysts and in the hemocoel, suggesting that sporozoites at different maturation stages are susceptible to melanization. Silencing the melanization promoting factors TEP1 and CLIPA28 rescued oocyst melanization in CLIPA2/CLIPA14 co-silenced mosquitoes. Interestingly, silencing of CTL4, that protects early stage ookinetes from melanization, had no effect on oocysts and sporozoites, indicating differential regulation of immunity to early and late sporogonic stages. Similar to previous studies addressing ookinete stage melanization, the melanization of Plasmodium falciparum oocysts was significantly lower than that observed for P. berghei. In summary, our results provide conclusive evidence that late sporogonic malaria parasite stages are susceptible to melanization, and we reveal distinct regulatory mechanisms for ookinete and oocyst melanization.

Monoclonal antibodies to the circumsporozoite proteins as an emerging tool for malaria prevention.

Despite various public health strategies, malaria caused by Plasmodium falciparum parasites remains a major global health challenge that requires development of new interventions. Extended half-life human monoclonal antibodies targeting the P. falciparum circumsporozoite protein on sporozoites, the infective form of malaria parasites, prevent malaria in rodents and humans and have been advanced into clinical development. The protective epitopes on the circumsporozoite protein targeted by monoclonal antibodies have been defined. Cryogenic electron and multiphoton microscopy have enabled mechanistic structural and functional investigations of how antibodies bind to the circumsporozoite protein and neutralize sporozoites. Moreover, innovations in bioinformatics and antibody engineering have facilitated enhancement of antibody potency and durability. Here, we summarize the latest scientific advances in understanding how monoclonal antibodies to the circumsporozoite protein prevent malaria and highlight existing clinical data and future plans for how this emerging intervention can be used alone or alongside existing antimalarial interventions to control malaria across at-risk populations.

A single-pass type I membrane protein, mannose-specific L-type lectin, potentially involved in the adhesion and invasion of Cryptosporidium parvum.

Cryptosporidium is a globally distributed zoonotic protozoan parasite that can cause severe diarrhea in humans and animals. L-type lectins are carbohydrate-binding proteins involved in multiple pathways in animals and plants, including protein transportation, secretion, innate immunity, and the unfolded protein response signaling pathway. However, the biological function of the L-type lectins remains unknown in Cryptosporidium parvum. Here, we preliminarily characterized an L-type lectin in C. parvum (CpLTL) that contains a lectin-leg-like domain. Immunofluorescence assay confirmed that CpLTL is located on the wall of oocysts, the surface of the mid-anterior region of the sporozoite and the cytoplasm of merozoites. The involvement of CpLTL in parasite invasion is partly supported by experiments showing that an anti-CpLTL antibody could partially block the invasion of C. parvum sporozoites into host cells. Moreover, the recombinant CpLTL showed binding ability with mannose and the surface of host cells, and competitively inhibited the invasion of C. parvum. Two host cell proteins were identified by proteomics which should be prioritized for future validation of CpLTL-binding. Our data indicated that CpLTL is potentially involved in the adhesion and invasion of C. parvum.

Title: Une protéine mono-transmembranaire, lectine de type L spécifique du mannose, potentiellement impliquée dans l'adhésion et l'invasion de Cryptosporidium parvum. Abstract: Cryptosporidium est un parasite protozoaire zoonotique répandu dans le monde entier qui peut provoquer de graves diarrhées chez les humains et les animaux. Les lectines de type L sont des protéines liant les glucides impliquées dans de multiples voies chez les animaux et les plantes, notamment le transport des protéines, la sécrétion, l'immunité innée et la voie de signalisation de la réponse protéique dépliée. Cependant, la fonction biologique des lectines de type L reste inconnue chez Cryptosporidium parvum. Ici, nous avons caractérisé de manière préliminaire une lectine de type L chez C. parvum (CpLTL) qui contient un domaine de type jambe de lectine. Le test d'immunofluorescence a confirmé que CpLTL est localisée sur la paroi des oocystes, la surface de la région médio-antérieure du sporozoïte et le cytoplasme des mérozoïtes. L'implication de CpLTL dans l'invasion parasitaire est en partie étayée par des expériences montrant qu'un anticorps anti-CpLTL peut bloquer partiellement l'invasion des sporozoïtes de C. parvum dans les cellules hôtes. De plus, la CpLTL recombinante a montré une capacité de liaison avec le mannose et la surface des cellules hôtes et a inhibé de manière compétitive l'invasion de C. parvum. Deux protéines de cellules hôtes ont été identifiées par protéomique et devraient être prioritaires pour la validation future de la liaison avec CpLTL. Nos données indiquent que CpLTL est potentiellement impliquée dans l'adhésion et l'invasion de C. parvum.

Comparative study of Eimeria tenella development in different cell culture systems.

Cell culture systems have long been recognised as great resources to mitigate the use of animals in research, offering effective solutions for replacement or reduction with benefits commonly including lower costs, shorter duration and improved reproducibility. The use of in vitro culture methods has been extensively explored for many apicomplexan parasites, supporting significant research advances, but studies with Eimeria are often limited since they still depend on the animal host. In this study we have used 2.5D and 3D culture systems for the first time to evaluate the growth of Eimeria tenella parasites using a panel of cell lines (MDBK, HD11, COLO-680N and HCC4006). Results were compared to growth in 2D monolayers following established protocols. Observations using the fluorescent transgenic strain Et-dYFP showed invasion and development of parasites inside cells suspended in a collagen matrix (2.5D or 3D), supporting the development of asexual stages with the release of first-generation merozoites. Similar findings were observed when Scaffold-free 3D cell spheroids of HD11 cells were infected with sporozoites. No subsequent developmental stages were identified while evaluating these cell lines and further work will be required to improve in vitro culture systems to a point where reduction and replacement of animal use becomes routine.

Plasmodium gametocyte carriage in humans and sporozoite rate in anopheline mosquitoes in Gondar zuria district, Northwest Ethiopia.

Although the overall burden of malaria is decreasing in Ethiopia, a recent report of an unpredictable increased incidence may be related to the presence of community-wide gametocyte-carrier individuals and a high proportion of infected vectors. This study aimed to reveal the current prevalence of gametocyte-carriage and the sporozoite infectivity rate of Anopheles vectors for Plasmodium parasites. A community-based cross-sectional study was conducted from May 01 to June 30/2019. A total of 53 households were selected using systematic random sampling and a 242 study participants were recruited. Additionally,515 adult female Anopheles mosquitoes were collected using Center for Diseases Control and Prevention (CDC) light traps and mouth aspirators. Parasite gametocytemia was determined using giemsa stain microscopy, while sporozoite infection was determined by giemsa staining microscopy and enzyme linked immunosorbent assay (ELISA). Among the total 242 study participants, 5.4% (95%, CI = 2.9-8.3) of them were positive for any of the Plasmodium species gametocyte. Furthermore, being female [AOR = 15.5(95%, CI = 1.71-140.39)], age group between 15-29 years old [AOR = 16.914 (95%, CI = 1.781-160.63)], no ITNs utilization [AOR = 16.7(95%, CI = 1.902 -146.727)], and high asexual parasite density [(95%, CI = 0.057-0.176, P = 0.001, F = 18.402)] were identified as statistically significant factors for gametocyte carriage. Whereas sporozoite infection rate was 11.6% (95%, CI = 8.2-15.5) and 12.7% (95%, CI = 9.6-16.3) by microscopy and ELISA, respectively. Overall, this study indicated that malaria remains to be an important public health problem in Gondar Zuria district where high gametocyte carriage rate and sporozoite infection rate could sustain its transmission and burden. Therefore, in Ethiopia, where malaria elimination program is underway, frequent, and active community-based surveillance of gametocytemia and sporozoite infection rate is important.

Flp/FRT-mediated disruption of ptex150 and exp2 in Plasmodium falciparum sporozoites inhibits liver-stage development.

Plasmodium falciparum causes severe malaria and assembles a protein translocon (PTEX) complex at the parasitophorous vacuole membrane (PVM) of infected erythrocytes, through which several hundred proteins are exported to facilitate growth. The preceding liver stage of infection involves growth in a hepatocyte-derived PVM; however, the importance of protein export during P. falciparum liver infection remains unexplored. Here, we use the FlpL/FRT system to conditionally excise genes in P. falciparum sporozoites for functional liver-stage studies. Disruption of PTEX members ptex150 and exp2 did not affect sporozoite development in mosquitoes or infectivity for hepatocytes but attenuated liver-stage growth in humanized mice. While PTEX150 deficiency reduced fitness on day 6 postinfection by 40%, EXP2 deficiency caused 100% loss of liver parasites, demonstrating that PTEX components are required for growth in hepatocytes to differing degrees. To characterize PTEX loss-of-function mutations, we localized four liver-stage Plasmodium export element (PEXEL) proteins. P. falciparum liver specific protein 2 (LISP2), liver-stage antigen 3 (LSA3), circumsporozoite protein (CSP), and a Plasmodium berghei LISP2 reporter all localized to the periphery of P. falciparum liver stages but were not exported beyond the PVM. Expression of LISP2 and CSP but not LSA3 was reduced in ptex150-FRT and exp2-FRT liver stages, suggesting that expression of some PEXEL proteins is affected directly or indirectly by PTEX disruption. These results show that PTEX150 and EXP2 are important for P. falciparum development in hepatocytes and emphasize the emerging complexity of PEXEL protein trafficking.

Transmission-blocking activities of artesunate, chloroquine, and methylene blue on Plasmodium vivax gametocytes.

Plasmodium vivax is now the main cause of malaria outside Africa. The gametocytocidal effects of antimalarial drugs are important to reduce malaria transmissibility, particularly in low-transmission settings, but they are not well characterized for P. vivax. The transmission-blocking effects of chloroquine, artesunate, and methylene blue on P. vivax gametocytes were assessed. Blood specimens were collected from patients presenting with vivax malaria, incubated with or without the tested drugs, and then fed to mosquitos from a laboratory-adapted colony of Anopheles dirus (a major malaria vector in Southeast Asia). The effects on oocyst and sporozoite development were analyzed under a multi-level Bayesian model accounting for assay variability and the heterogeneity of mosquito Plasmodium infection. Artesunate and methylene blue, but not chloroquine, exhibited potent transmission-blocking effects. Gametocyte exposures to artesunate and methylene blue reduced the mean oocyst count 469-fold (95% CI: 345 to 650) and 1,438-fold (95% CI: 970 to 2,064), respectively. The corresponding estimates for the sporozoite stage were a 148-fold reduction (95% CI: 61 to 470) and a 536-fold reduction (95% CI: 246 to 1,311) in the mean counts, respectively. In contrast, high chloroquine exposures reduced the mean oocyst count only 1.40-fold (95% CI: 1.20 to 1.64) and the mean sporozoite count 1.34-fold (95% CI: 1.12 to 1.66). This suggests that patients with vivax malaria often remain infectious to anopheline mosquitos after treatment with chloroquine. Use of artemisinin combination therapies or immediate initiation of primaquine radical cure should reduce the transmissibility of P. vivax infections.

A Micronemal Protein, Scot1, Is Essential for Apicoplast Biogenesis and Liver Stage Development in Plasmodium berghei.

Plasmodium sporozoites invade hepatocytes, transform into liver stages, and replicate into thousands of merozoites that infect erythrocytes and cause malaria. Proteins secreted from micronemes play an essential role in hepatocyte invasion, and unneeded micronemes are subsequently discarded for replication. The liver-stage parasites are potent immunogens that prevent malarial infection. Late liver stage-arresting genetically attenuated parasites (GAPs) exhibit greater protective efficacy than early GAP. However, the number of late liver-stage GAPs for generating GAPs with multiple gene deletions is limited. Here, we identified Scot1 (Sporozoite Conserved Orthologous Transcript 1), which was previously shown to be upregulated in sporozoites, and by endogenous tagging with mCherry, we demonstrated that it is expressed in the sporozoite and liver stages in micronemes. Using targeted gene deletion in Plasmodium berghei, we showed that Scot1 is essential for late liver-stage development. Scot1 KO sporozoites grew normally into liver stages but failed to initiate blood-stage infection in mice due to impaired apicoplast biogenesis and merozoite formation. Bioinformatic studies suggested that Scot1 is a metal-small-molecule carrier protein. Remarkably, supplementation with metals in the culture of infected Scot1 KO cells did not rescue their phenotype. Immunization with Scot1 KO sporozoites in C57BL/6 mice confers protection against malaria via infection. These proof-of-concept studies will enable the generation of P. falciparum Scot1 mutants that could be exploited to generate GAP malaria vaccines.

The major surface protein of malaria sporozoites is GPI-anchored to the plasma membrane.

Glycosylphosphatidylinositol (GPI) anchor protein modification in Plasmodium species is well known and represents the principal form of glycosylation in these organisms. The structure and biosynthesis of GPI anchors of Plasmodium spp. has been primarily studied in the asexual blood stage of Plasmodium falciparum and is known to contain the typical conserved GPI structure of EtN-P-Man3GlcN-PI. Here, we have investigated the circumsporozoite protein (CSP) for the presence of a GPI anchor. CSP is the major surface protein of Plasmodium sporozoites, the infective stage of the malaria parasite. While it is widely assumed that CSP is a GPI-anchored cell surface protein, compelling biochemical evidence for this supposition is absent. Here, we employed metabolic labeling and mass-spectrometry-based approaches to confirm the presence of a GPI anchor in CSP. Biosynthetic radiolabeling of CSP with [3H]-palmitic acid and [3H]-ethanolamine, with the former being base-labile and therefore ester-linked, provided strong evidence for the presence of a GPI anchor on CSP, but these data alone were not definitive. To provide further evidence, immunoprecipitated CSP was analyzed for the presence of myo-inositol (a characteristic component of GPI anchor) using strong acid hydrolysis and GC-MS for highly sensitive and quantitative detection. The single ion monitoring (SIM) method for GC-MS analysis confirmed the presence of the myo-inositol component in CSP. Taken together, these data provide confidence that the long-assumed presence of a GPI anchor on this important parasite protein is correct.

Additional blood meals increase sporozoite infection in Anopheles mosquitoes but not Plasmodium falciparum genetic diversity.

The availability of nutrients from mosquito blood meals accelerates the development of Plasmodium falciparum laboratory strains in artificially infected Anopheles gambiae mosquitoes. The impact of multiple blood meals on the number of P. falciparum genotypes developing from polyclonal natural human malaria infections (field-isolates) remains unexplored. Here, we experimentally infect An. gambiae with P. falciparum field-isolates and measure the impact of an additional non-infectious blood meal on parasite development. We also assess parasite genetic diversity at the blood stage level of the parasite in the human host and of the sporozoites in the mosquito. Additional blood meals increase the sporozoite infection prevalence and intensity, but do not substantially affect the genetic diversity of sporozoites in the mosquito. The most abundant parasite genotypes in the human blood were transmitted to mosquitoes, suggesting that there was no preferential selection of specific genotypes. This study underlines the importance of additional mosquito blood meals for the development of parasite field-isolates in the mosquito host.

Geospatial joint modeling of vector and parasite serology to microstratify malaria transmission.

The World Health Organization identifies a strong surveillance system for malaria and its mosquito vector as an essential pillar of the malaria elimination agenda. Anopheles salivary antibodies are emerging biomarkers of exposure to mosquito bites that potentially overcome sensitivity and logistical constraints of traditional entomological surveys. Using samples collected by a village health volunteer network in 104 villages in Southeast Myanmar during routine surveillance, the present study employs a Bayesian geostatistical modeling framework, incorporating climatic and environmental variables together with Anopheles salivary antigen serology, to generate spatially continuous predictive maps of Anopheles biting exposure. Our maps quantify fine-scale spatial and temporal heterogeneity in Anopheles salivary antibody seroprevalence (ranging from 9 to 99%) that serves as a proxy of exposure to Anopheles bites and advances current static maps of only Anopheles occurrence. We also developed an innovative framework to perform surveillance of malaria transmission. By incorporating antibodies against the vector and the transmissible form of malaria (sporozoite) in a joint Bayesian geostatistical model, we predict several foci of ongoing transmission. In our study, we demonstrate that antibodies specific for Anopheles salivary and sporozoite antigens are a logistically feasible metric with which to quantify and characterize heterogeneity in exposure to vector bites and malaria transmission. These approaches could readily be scaled up into existing village health volunteer surveillance networks to identify foci of residual malaria transmission, which could be targeted with supplementary interventions to accelerate progress toward elimination.

Mendelian segregation and high recombination rates facilitate genetic analyses in Cryptosporidium parvum.

Very little is known about the process of meiosis in the apicomplexan parasite Cryptosporidium despite the essentiality of sex in its life cycle. Most cell lines only support asexual growth of Cryptosporidium parvum (C. parvum), but stem cell derived intestinal epithelial cells grown under air-liquid interface (ALI) conditions support the sexual cycle. To examine chromosomal dynamics during meiosis in C. parvum, we generated two transgenic lines of parasites that were fluorescently tagged with mCherry or GFP on chromosomes 1 or 5, respectively. Infection of ALI cultures or Ifngr1-/- mice with mCherry and GFP parasites resulted in cross-fertilization and the formation of "yellow" oocysts, which contain 4 haploid sporozoites that are the product of meiosis. Recombinant oocysts from the F1 generation were purified and used to infect HCT-8 cultures, and phenotypes of the progeny were observed by microscopy. All possible phenotypes predicted by independent segregation were represented equally (~25%) in the population, indicating that C. parvum chromosomes exhibit a Mendelian inheritance pattern. The most common pattern observed from the outgrowth of single oocysts included all possible parental and recombinant phenotypes derived from a single meiotic event, suggesting a high rate of crossover. To estimate the frequency of crossover, additional loci on chromosomes 1 and 5 were tagged and used to monitor intrachromosomal crosses in Ifngr1-/- mice. Both chromosomes showed a high frequency of crossover compared to other apicomplexans with map distances (i.e., 1% recombination) of 3-12 kb. Overall, a high recombination rate may explain many unique characteristics observed in Cryptosporidium spp. such as high rates of speciation, wide variation in host range, and rapid evolution of host-specific virulence factors.

Vectorial drivers of malaria transmission in Jabi Tehnan district, Amhara Regional State, Ethiopia.

Among the factors affecting the effectiveness of malaria control is poor knowledge of the entomologic drivers of the disease. We investigated anopheline populations as part of a baseline study to implement house screening of windows and doors as a supplementary malaria control tool towards elimination in Jabi Tehnan district, Amhara Regional State of Ethiopia. The samples were surveyed monthly using CDC light traps between June 2020 and May 2021. Mosquito trap density (< 3 mosquitoes/trap) was low, however, with a high overall Plasmodium sporozoite rate (9%; indoor = 4.3%, outdoor = 13.1%) comprising P. falciparum (88.9%) and P. vivax (11.1%). Anopheles gambiae s.l., mostly An. arabiensis, comprised > 80% of total anopheline captures and contributed ~ 42% of Plasmodium-infected mosquitoes. On the other hand, morphologically scored Anopheles funestus s.l., constituting about 6% of anopheline collections, accounted for 50% of sporozoite-infected mosquitoes. Most of the infected An. funestus s.l. specimens (86.7%) were grouped with previously unknown or undescribed Anopheles species previously implicated as a cryptic malaria vector in the western Kenyan highlands, confirming its wider geographic distribution in eastern Africa. Other species with Plasmodium infection included An. longipalpis C, An. theileri, An. demillioni, and An. nili. Cumulatively, 77.8% of the infected mosquitoes occurred outdoors. These results suggest efficient malaria parasite transmission despite the low vector densities, which has implications for effective endpoint indicators to monitor malaria control progress. Additionally, the largely outdoor infection and discovery of previously unknown and cryptic vectors suggest an increased risk of residual malaria transmission and, thus, a constraint on effective malaria prevention and control.

The novel Plasmodium berghei protein S14 is essential for sporozoite gliding motility and infectivity.

Plasmodium sporozoites are the infective forms of the malaria parasite in the mosquito and vertebrate host. Gliding motility allows sporozoites to migrate and invade mosquito salivary glands and mammalian hosts. Motility and invasion are powered by an actin-myosin motor complex linked to the glideosome, which contains glideosome-associated proteins (GAPs), MyoA and the myosin A tail-interacting protein (MTIP). However, the role of several proteins involved in gliding motility remains unknown. We identified that the S14 gene is upregulated in sporozoite from transcriptome data of Plasmodium yoelii and further confirmed its transcription in P. berghei sporozoites using real-time PCR. C-terminal 3×HA-mCherry tagging revealed that S14 is expressed and localized on the inner membrane complex of the sporozoites. We disrupted S14 in P. berghei and demonstrated that it is essential for sporozoite gliding motility, and salivary gland and hepatocyte invasion. The gliding and invasion-deficient S14 knockout sporozoites showed normal expression and organization of inner membrane complex and surface proteins. Taken together, our data show that S14 plays a role in the function of the glideosome and is essential for malaria transmission.

Immune responses associated with protection induced by chemoattenuated PfSPZ vaccine in malaria-naive Europeans.

Vaccination of malaria-naive volunteers with a high dose of Plasmodium falciparum sporozoites chemoattenuated by chloroquine (CQ) (PfSPZ-CVac [CQ]) has previously demonstrated full protection against controlled human malaria infection (CHMI). However, lower doses of PfSPZ-CVac [CQ] resulted in incomplete protection. This provides the opportunity to understand the immune mechanisms needed for better vaccine-induced protection by comparing individuals who were protected with those not protected. Using mass cytometry, we characterized immune cell composition and responses of malaria-naive European volunteers who received either lower doses of PfSPZ-CVac [CQ], resulting in 50% protection irrespective of the dose, or a placebo vaccination, with everyone becoming infected following CHMI. Clusters of CD4+ and γδ T cells associated with protection were identified, consistent with their known role in malaria immunity. Additionally, EMRA CD8+ T cells and CD56+CD8+ T cell clusters were associated with protection. In a cohort from a malaria-endemic area in Gabon, these CD8+ T cell clusters were also associated with parasitemia control in individuals with lifelong exposure to malaria. Upon stimulation with P. falciparum-infected erythrocytes, CD4+, γδ, and EMRA CD8+ T cells produced IFN-γ and/or TNF, indicating their ability to mediate responses that eliminate malaria parasites.

The influence of oviposition status on measures of transmission potential in malaria-infected mosquitoes depends on sugar availability.

BACKGROUND: Like other oviparous organisms, the gonotrophic cycle of mosquitoes is not complete until they have selected a suitable habitat to oviposit. In addition to the evolutionary constraints associated with selective oviposition behavior, the physiological demands relative to an organism's oviposition status also influence their nutrient requirement from the environment. Yet, studies that measure transmission potential (vectorial capacity or competence) of mosquito-borne parasites rarely consider whether the rates of parasite replication and development could be influenced by these constraints resulting from whether mosquitoes have completed their gonotrophic cycle. METHODS: Anopheles stephensi mosquitoes were infected with Plasmodium berghei, the rodent analog of human malaria, and maintained on 1% or 10% dextrose and either provided oviposition sites ('oviposited' herein) to complete their gonotrophic cycle or forced to retain eggs ('non-oviposited'). Transmission potential in the four groups was measured up to 27 days post-infection as the rates of (i) sporozoite appearance in the salivary glands ('extrinsic incubation period' or EIP), (ii) vector survival and (iii) sporozoite densities. RESULTS: In the two groups of oviposited mosquitoes, rates of sporozoite appearance and densities in the salivary glands were clearly dependent on sugar availability, with shorter EIP and higher sporozoite densities in mosquitoes fed 10% dextrose. In contrast, rates of appearance and densities in the salivary glands were independent of sugar concentrations in non-oviposited mosquitoes, although both measures were slightly lower than in oviposited mosquitoes fed 10% dextrose. Vector survival was higher in non-oviposited mosquitoes. CONCLUSIONS: Costs to parasite fitness and vector survival were buffered against changes in nutritional availability from the environment in non-oviposited but not oviposited mosquitoes. Taken together, these results suggest vectorial capacity for malaria parasites may be dependent on nutrient availability and oviposition/gonotrophic status and, as such, argue for more careful consideration of this interaction when estimating transmission potential. More broadly, the complex patterns resulting from physiological (nutrition) and evolutionary (egg-retention) trade-offs described here, combined with the ubiquity of selective oviposition behavior, implies the fitness of vector-borne pathogens could be shaped by selection for these traits, with implications for disease transmission and management. For instance, while reducing availability of oviposition sites and environmental sources of nutrition are key components of integrated vector management strategies, their abundance and distribution are under strong selection pressure from the patterns associated with climate change.