The DEAD-box RNA helicase PfDOZI imposes opposing actions on RNA metabolism in Plasmodium falciparum.

In malaria parasites, the regulation of mRNA translation, storage and degradation during development and life-stage transitions remains largely unknown. Here, we functionally characterized the DEAD-box RNA helicase PfDOZI in P. falciparum. Disruption of pfdozi enhanced asexual proliferation but reduced sexual commitment and impaired gametocyte development. By quantitative transcriptomics, we show that PfDOZI is involved in the regulation of invasion-related genes and sexual stage-specific genes during different developmental stages. PfDOZI predominantly participates in processing body-like mRNPs in schizonts but germ cell granule-like mRNPs in gametocytes to impose opposing actions of degradation and protection on different mRNA targets. We further show the formation of stress granule-like mRNPs during nutritional deprivation, highlighting an essential role of PfDOZI-associated mRNPs in stress response. We demonstrate that PfDOZI participates in distinct mRNPs to maintain mRNA homeostasis in response to life-stage transition and environmental changes by differentially executing post-transcriptional regulation on the target mRNAs.

Oral administration of IPI549 protects mice from neuropathology and an overwhelming inflammatory response during experimental cerebral malaria.

Infection with Plasmodium falciparum is often deadly when it results in cerebral malaria, which is associated with neuropathology described as an overwhelming inflammatory response and mechanical obstruction of cerebral microvascular. PI3Kγ is a critical component of intracellular signal transduction and plays a central role in regulating cell chemotaxis, migration, and activation. The purpose of this study was to examine the relationship between inhibiting the PI3Kγ pathway and the outcome of experimental cerebral malaria (ECM) in C57BL/6J mice infected with the mouse malaria parasite, Plasmodium berghei ANKA. We observed that oral administration of the PI3Kγ inhibitor IPI549 after infection completely protected mice from ECM. IPI549 treatment significantly dampened the magnitude of inflammatory responses, with reduced production of pro-inflammatory factors, decreased T cell activation, and altered differentiation of antigen-presenting cells. IPI549 treatment protected the infected mice from neuropathology, as assessed by an observed reduction of pathogenic T cells in the brain. Treating the infected mice with IPI549 three days after parasite inoculation improved the murine blood brain barrier (BBB) integrity and helped the mice pass the onset of ECM. Together, these data indicate that oral administration of the PI3Kγ inhibitor IPI549 has a suppressive role in host inflammation and alleviates cerebral pathology, which supports IPI549 as a new malaria treatment option with potential therapeutic implications for cerebral malaria.

Evaluation of different types of adjuvants in a malaria transmission-blocking vaccine.

Adjuvants are critical components for vaccines, which enhance the strength and longevity of the antibody response and influence the types of immune response. Limited research has been conducted on the immunogenicity and protective efficacy of various adjuvants in malaria transmission-blocking vaccines (TBVs). In this study, we formulated a promising TBV candidate antigen, the P. berghei ookinete surface antigen PSOP25, with different types of adjuvants, including the TLR4 agonist monophosphoryl lipid A (MPLA), the TLR9 agonist cytosine phosphoguanosine oligodeoxynucleotides (CpG ODN 1826) (CpG), a saponin adjuvant QS-21, aluminum hydroxide (Alum), and two combination adjuvants MPLA + QS-21 and QS-21 + CpG. We demonstrated that adjuvanted vaccines results in elevated elicited antibody levels, increased proliferation of plasma cells, and efficient formation of germinal centers (GCs), leading to enhanced long-term protective immune responses. Furthermore, CpG group exhibited the most potent inhibition of ookinete formation and transmission-blocking activity. We found that the rPSOP25 with CpG adjuvant was more effective than MPLA, QS-21, MPLA + QS-21, QS-21 + CpG adjuvants in dendritic cells (DCs) activation and differentiation. Additionally, the CpG adjuvant elicited more rubust immune memory response than Alum adjuvant. CpG and QS-21 adjuvants could activate the Th1 response and promote the secretion of IFN-γ and TNF-α. PSOP25 induced a higher number of Tfh cells in splenocytes when combined with MPLA, CpG, and QS-21 + CpG; and there was no increase in these cell populations when PSOP25 was administered with Alum. In conclusion, CpG may confer enhanced efficacy for the rPSOP25 vaccine, as evidenced by the ability of the elicited antisera to induce protective immune responses and improved transmission-blocking activity.

Functional characterization of a conserved membrane protein, Pbs54, involved in gamete fertilization in Plasmodium berghei.

The successful completion of gamete fertilization is essential for malaria parasite transmission, and this process can be targeted by intervention strategies. In this study, we identified a conserved gene (PBANKA_0813300) in the rodent malaria parasite Plasmodium berghei, which encodes a protein of 54 kDa (designated as Pbs54). Localization studies indicated that Pbs54 is associated with the plasma membranes of gametes and ookinetes. Functional studies by gene disruption showed that the Δpbs54 parasites had no defect in asexual proliferation, gametocyte development, or gametogenesis. However, the interactions between male and female gametes were significantly decreased compared with wild-type parasites. The Δpbs54 lines did not show a further reduction in zygote and ookinete numbers during in vitro culture, indicating that the defects were probably restricted to gamete fertilization. Consistent with this finding, mosquitoes fed on Δpbs54-infected mice showed a 30.1% reduction in infection prevalence and a 74.7% reduction in oocyst intensity. Cross-fertilization assay indicated that both male and female gametes were impaired in the Δpbs54 parasites. To evaluate its transmission-blocking potential, we obtained polyclonal antibodies from mice immunized with the recombinant Pbs54 (rPbs54) protein. In vitro assays showed that anti-rPbs54 sera inhibited ookinete formation by 42.7%. Our experiments identified Pbs54 as a fertility factor required for mosquito transmission and a novel candidate for a malaria transmission-blocking vaccine.

A dual-antigen malaria vaccine targeting Pb22 and Pbg37 was able to induce robust transmission-blocking activity.

BACKGROUND: Despite years of effort to develop an effective vaccine against malaria infection, a vaccine that provides individuals with sufficient protection against malaria illness and death in endemic areas is not yet available. The development of transmission-blocking vaccines (TBVs) is a promising strategy for malaria control. A dual-antigen malaria vaccine targeting both pre- and post-fertilization antigens could effectively improve the transmission-blocking activity of vaccines against the sexual stages of the parasite. METHODS: A chimeric recombinant protein Pb22-Pbg37 (Plasmodium berghei 22-P. berghei G37) composed of 19-218 amino acids (aa) of Pb22 and the N-terminal 26-88 aa of Pbg37 was designed and expressed in the Escherichia coli expression system. The antibody titers of the fusion (Pb22-Pbg37) and mixed (Pb22+Pbg37) antigens, as well as those of Pb22 and Pbg37 single antigens were evaluated by enzyme-linked immunosorbent assay. Immunofluorescence and western blot assays were performed to test the reactivity of the antisera with the native proteins in the parasite. The induction of transmission-blocking activity (TBA) by Pb22-Pbg37 and Pb22+Pbg37 were evaluated by in vitro gametocyte activation, gamete and exflagellation center formation, ookinete conversion, and in the direct mosquito feeding assay. RESULTS: The Pb22-Pbg37 fusion protein was successfully expressed in vitro. Co-administration of Pb22 and Pbg37 as a fusion or mixed protein elicited comparable antibody responses in mice and resulted in responses to both antigens. Most importantly, both the mixed and fusion antigens induced antibodies with significantly higher levels of TBA than did each of the individual antigens when administered alone. In addition, the efficacy of vaccination with the Pb22-Pbg37 fusion protein was equivalent to that of vaccination with the mixed single antigens. CONCLUSIONS: Dual-antigen vaccines, which expand/lengthen the period during which the transmission-blocking antibodies can act during sexual-stage development, can provide a promising higher transmission-reducing activity compared to single antigens.

RBMS3-induced circHECTD1 encoded a novel protein to suppress the vasculogenic mimicry formation in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a highly vascularized malignant cancer of the central nervous system, and the presence of vasculogenic mimicry (VM) severely limits the effectiveness of anti-vascular therapy. In this study, we identified downregulated circHECTD1, which acted as a key VM-suppressed factor in GBM. circHECTD1 elevation significantly inhibited cell proliferation, migration, invasion and tube-like structure formation in GBM. RIP assay was used to demonstrate that the flanking intron sequence of circHECTD1 can be specifically bound by RBMS3, thereby inducing circHECTD1 formation to regulate VM formation in GBM. circHECTD1 was confirmed to possess a strong protein-encoding capacity and the encoded functional peptide 463aa was identified by LC-MS/MS. Both circHECTD1 and 463aa significantly inhibited GBM VM formation in vivo and in vitro. Analysis of the 463aa protein sequence revealed that it contained a ubiquitination-related domain and promoted NR2F1 degradation by regulating the ubiquitination of the NR2F1 at K396. ChIP assay verified that NR2F1 could directly bind to the promoter region of MMP2, MMP9 and VE-cadherin, transcriptionally promoting the expression of VM-related proteins, which in turn enhanced VM formation in GBM. In summary, we clarified a novel pathway for RBMS3-induced circHECTD1 encoding functional peptide 463aa to mediate the ubiquitination of NR2F1, which inhibited VM formation in GBM. This study aimed to reveal new mechanisms of GBM progression in order to provide novel approaches and strategies for the anti-vascular therapy of GBM. The schematic illustration showed the inhibitory effect of circHECTD1-463aa in the VM formation in GBM.

A nanoparticle vaccine displaying the ookinete PSOP25 antigen elicits transmission-blocking antibody response against Plasmodium berghei.

BACKGROUND: Safe and effective vaccines are crucial for the control and eventual elimination of malaria. Novel approaches to optimize and improve vaccine efficacy are urgently required. Nanoparticle-based delivery platforms are considered potent and powerful tools for vaccine development. METHODS: In this study, we developed a transmission-blocking vaccine against malaria by conjugating the ookinete surface antigen PSOP25 to the Acinetobacter phage coat protein AP205, forming virus-like particles (VLPs) using the SpyTag/SpyCatcher adaptor system. The combination of AP205-2*SpyTag with PSOP25-SpyCatcher resulted in the formation of AP205-PSOP25 complexes (VLP-PSOP25). The antibody titers and avidity of serum from each immunization group were assessed by ELISA. Western blot and IFA were performed to confirm the specific reactivity of the elicit antisera to the native PSOP25 in Plasmodium berghei ookinetes. Both in vitro and in vivo assays were conducted to evaluate the transmission-blocking activity of VLP-PSOP25 vaccine. RESULTS: Immunization of mice with VLP-PSOP25 could induced higher levels of high-affinity antibodies than the recombinant PSOP25 (rPSOP25) alone or mixtures of untagged AP205 and rPSOP25 but was comparable to rPSOP25 formulated with alum. Additionally, the VLP-PSOP25 vaccine enhanced Th1-type immune response with remarkably increased levels of IgG2a subclass. The antiserum generated by VLP-PSOP25 specifically recognizes the native PSOP25 antigen in P. berghei ookinetes. Importantly, antisera generated by inoculation with the VLP-PSOP25 could inhibit ookinete development in vitro and reduce the prevalence of infected mosquitoes or oocyst intensity in direct mosquito feeding assays. CONCLUSIONS: Antisera elicited by immunization with the VLP-PSOP25 vaccine confer moderate transmission-reducing activity and transmission-blocking activity. Our results support the utilization of the AP205-SpyTag/SpyCatcher platform for next-generation TBVs development.

Plasmodium falciparum GAP40 Plays an Essential Role in Merozoite Invasion and Gametocytogenesis.

Cyclic invasion of red blood cells (RBCs) by Plasmodium merozoites is associated with the symptoms and pathology of malaria. Merozoite invasion is powered actively and rapidly by a parasite actomyosin motor called the glideosome. The ability of the glideosome to generate force to support merozoite entry into the host RBCs is thought to rely on its stable anchoring within the inner membrane complex (IMC) through membrane-resident proteins, such as GAP50 and GAP40. Using a conditional knockdown (KD) approach, we determined that PfGAP40 was required for asexual blood-stage replication. PfGAP40 is not needed for merozoite egress from host RBCs or for the attachment of merozoites to new RBCs. PfGAP40 coprecipitates with PfGAP45 and PfGAP50. During merozoite invasion, PfGAP40 is associated strongly with stabilizing the expression levels of PfGAP45 and PfGAP50 in the schizont stage. Although PfGAP40 KD did not influence IMC integrity, it impaired the maturation of gametocytes. In addition, PfGAP40 is phosphorylated, and mutations that block phosphorylation of PfGAP40 at the C-terminal serine residues S370, S372, S376, S405, S409, S420, and S445 reduced merozoite invasion efficiency. Overall, our findings implicate PfGAP40 as an important regulator for the gliding activity of merozoites and suggest that phosphorylation is required for PfGAP40 function. IMPORTANCE Red blood cell invasion is central to the pathogenesis of the malaria parasite, and the parasite proteins involved in this process are potential therapeutic targets. Gliding motility powers merozoite invasion and is driven by a unique molecular motor termed the glideosome. The glideosome is stably anchored to the parasite inner membrane complex (IMC) through membrane-resident proteins. In the present study, we demonstrate the importance of an IMC-resident glideosome component, PfGAP40, that plays a critical role in stabilizing the expression levels of glideosome components in the schizont stage. We determined that phosphorylation of PfGAP40 at C-terminal residues is required for efficient merozoite invasion.

Spatio-temporal trends of malaria incidence from 2011 to 2017 and environmental predictors of malaria transmission in Myanmar.

BACKGROUND: Myanmar bears the heaviest malaria burden in the Greater Mekong Subregion (GMS). This study assessed the spatio-temporal dynamics and environmental predictors of Plasmodium falciparum and Plasmodium vivax malaria in Myanmar. METHODS: Monthly reports of malaria cases at primary health centers during 2011-2017 were analyzed to describe malaria distribution across Myanmar at the township and state/region levels by spatial autocorrelation (Moran index) and spatio-temporal clustering. Negative binomial generalized additive models identified environmental predictors for falciparum and vivax malaria, respectively. RESULTS: From 2011 to 2017, there was an apparent reduction in malaria incidence in Myanmar. Malaria incidence peaked in June each year. There were significant spatial autocorrelation and clustering with extreme spatial heterogeneity in malaria cases and test positivity across the nation (P < 0.05). Areas with higher malaria incidence were concentrated along international borders. Primary clusters of P. falciparum persisted in western townships, while clusters of P. vivax shifted geographically over the study period. The primary cluster was detected from January 2011 to December 2013 and covered two states (Sagaing and Kachin). Annual malaria incidence was highest in townships with a mean elevation of 500‒600 m and a high variance in elevation (states with both high and low elevation). There was an apparent linear relationship between the mean normalized difference vegetative index and annual P. falciparum incidence (P < 0.05). CONCLUSION: The decreasing trends reflect the significant achievement of malaria control efforts in Myanmar. Prioritizing the allocation of resources to high-risk areas identified in this study can achieve effective disease control.

Evaluation of transmission-blocking potential of Pv22 using clinical Plasmodium vivax infections and transgenic Plasmodium berghei.

Antigens expressed during the sexual development of malaria parasites are transmission-blocking vaccine (TBV) targets. Pb22, a protein expressed and localized to the plasma membrane of gametes and ookinetes in Plasmodium berghei, is an excellent TBV candidate. Here, we evaluated the TB potential of the Plasmodium vivax ortholog Pv22 using a transgenic P. berghei parasite line and P. vivax clinical isolates. The full-length recombinant Pv22 (rPv22) protein was produced and used to immunize mice and rabbits to obtain antibodies. We generated a transgenic P. berghei line (TrPv22Pb) by inserting the pv22 gene into the pb22 locus and showed that Pv22 expression completely rescued the defects in male gametogenesis of the pb22 deletion parasite. Since Pv22 in the transgenic parasite showed similar expression and localization patterns to Pb22, we used the TrPv22Pb parasite as a surrogate to evaluate the TB potential of Pv22. In mosquito feeding assays, mosquitoes feeding on rPv22-immunized mice infected with TrPv22Pb parasites showed a 49.3-53.3 % reduction in the oocyst density compared to the control group. In vitro assays showed that the rPv22 immune sera significantly inhibited exflagellation and ookinete formation of the TrPv22Pb parasites. In a direct membrane feeding assay using three clinical P. vivax isolates, the rabbit anti-rPv22 antibodies also significantly decreased the oocyst density by 53.7, 30.2, and 26.2 %, respectively. This study demonstrated the feasibility of using transgenic P. berghei parasites expressing P. vivax antigens as a potential tool to evaluate TBV candidates. However, the much weaker TB activity of Pv22 obtained from two complementary assays suggest that Pv22 may not be a promising TBV candidate for P. vivax.

Chloroquine treatment influences immunologicalmemory through the PD-1/PD-L1 pathway during the initiation of Plasmodium chabaudi infection.

Growing evidence describes the host immune response mechanism involved in malaria. Despite the spread of drug resistance, chloroquine (CQ) remains the main antimalarial drug in most countries in Latin America and Asia. Studies have indicated an immunomodulatory activity of CQ, however, the potential implications for CQ on immunological memory recognizing the malaria parasite are still being elucidated. Our study suggests that CQ treatment significantly delayed the initiation of parasitemia during infection of mice with the rodent malaria parasite, Plasmodium chabaudi (P.c.). Additionally, there was a decrease in T follicular helper cells (Tfh), CD4+ effector memory T cells, memory B cells (MBC), IgG2a memoryB cells, along with IgG2a plasma cells; while antibody production was not affected atthe observation time points. After PD-1 blockade and CQ treatment, no reductions in the numbers of CD4+ effector memory T cells, MBC, and IgG2a memoryB cells were observed compared with the P.c. group. Therefore, CQ might regulate immunological memory via the PD-1/PD-L1 signaling pathway. Compared with antibody secretion, the inhibition of CQ on immune memory cells was a more sensitive indicator.

Multidisciplinary Investigations of Sustained Malaria Transmission in the Greater Mekong Subregion.

In the course of malaria elimination in the Greater Mekong Subregion (GMS), malaria epidemiology has experienced drastic spatiotemporal changes with residual transmission concentrated along international borders and the rising predominance of Plasmodium vivax. The emergence of Plasmodium falciparum parasites resistant to artemisinin and partner drugs renders artemisinin-based combination therapies less effective while the potential spread of multidrug-resistant parasites elicits concern. Vector behavioral changes and insecticide resistance have reduced the effectiveness of core vector control measures. In recognition of these problems, the Southeast Asian International Center of Excellence for Malaria Research (ICEMR) has been conducting multidisciplinary research to determine how human migration, antimalarial drug resistance, vector behavior, and insecticide resistance sustain malaria transmission at international borders. These efforts allow us to comprehensively understand the ecology of border malaria transmission and develop population genomics tools to identify and track parasite introduction. In addition to employing in vivo, in vitro, and molecular approaches to monitor the emergence and spread of drug-resistant parasites, we also use genomic and genetic methods to reveal novel mechanisms of antimalarial drug resistance of parasites. We also use omics and population genetics approaches to study insecticide resistance in malaria vectors and identify changes in mosquito community structure, vectorial potential, and seasonal dynamics. Collectively, the scientific findings from the ICEMR research activities offer a systematic view of the factors sustaining residual malaria transmission and identify potential solutions to these problems to accelerate malaria elimination in the GMS.

Genetic diversity in the transmission-blocking vaccine candidate Plasmodium vivax gametocyte protein Pvs230 from the China-Myanmar border area and central Myanmar.

BACKGROUND: Sexual stage surface antigens are potential targets of transmission-blocking vaccines (TBVs). The gametocyte and gamete surface antigen P230, a leading TBV candidate, is critical for red blood cell binding during exflagellation and subsequent oocyst development. Here, the genetic diversity of Pvs230 was studied in Plasmodium vivax parasite isolates from the China-Myanmar border (CMB) and central Myanmar. METHODS: Plasmodium vivax isolates were collected in clinics from malaria-endemic areas of the CMB (143 samples) and Myanmar (23 samples). The interspecies variable part (IVP, nucleotides 1-807) and interspecies conserved part (ICP, 808-2862) of Pvs230 were amplified by PCR and sequenced. Molecular evolution studies were conducted to evaluate the genetic diversity, signature of selection, population differentiation, haplotype network, and population structure of the study parasite populations and publicly available Pvs230 sequences from six global P. vivax populations. RESULTS: Limited genetic diversity was observed for the CMB (π = 0.002) and Myanmar (π = 0.001) isolates. Most amino acid substitutions were located in the IVP and cysteine-rich domain of Pvs230. Evidence of positive selection was observed for IVP and purifying selection for ICP. Codon-based tests identified specific codons under natural selection in both IVP and ICP. The fixation index (FST) showed low genetic differentiation between East and Southeast Asian populations, with FST ranging from 0.018 to 0.119. The highest FST value (FST = 0.503) was detected between the Turkey and Papua New Guinea populations. A total of 92 haplotypes were identified in global isolates, with the major haplotypes 2 and 9 being the most abundant and circulating in East and Southeast Asia populations. Several detected non-synonymous substitutions were mapped in the predicted structure and B-cell epitopes of Pvs230. CONCLUSIONS: We detected low levels of genetic diversity of Pvs230 in global P. vivax populations. Geographically specific haplotypes were identified for Pvs230. Some mutations are located within a potential B-cell epitope region and need to be considered in future TBV designs.

The Plasmodium falciparum Nuclear Protein Phosphatase NIF4 Is Required for Efficient Merozoite Invasion and Regulates Artemisinin Sensitivity.

Artemisinin resistance in Plasmodium falciparum has been associated with a mutation in the NLI-interacting factor-like phosphatase PfNIF4, in addition to the mutations in the Kelch13 protein as the major determinant. We found that PfNIF4 was predominantly expressed at the schizont stage and localized in the nuclei of the parasite. To elucidate the functions of PfNIF4 in P. falciparum, we performed PfNIF4 knockdown (KD) using the inducible ribozyme system. PfNIF4 KD attenuated merozoite invasion and affected gametocytogenesis. PfNIF4 KD parasites also showed significantly increased in vitro susceptibility to artemisinins. Transcriptomic and proteomic analysis revealed that PfNIF4 KD led to the downregulation of gene categories involved in invasion and artemisinin resistance (e.g., mitochondrial function, membrane, and Kelch13 interactome) at the trophozoite and/or schizont stage. Consistent with PfNIF4 being a protein phosphatase, PfNIF4 KD resulted in an overall upregulation of the phosphoproteome of infected erythrocytes. Quantitative phosphoproteomic profiling identified a set of PfNIF4-regulated phosphoproteins with functional similarity to FCP1 substrates, particularly proteins involved in chromatin organization and transcriptional regulation. Specifically, we observed increased phosphorylation of Ser2/5 of the tandem repeats in the C-terminal domain (CTD) of RNA polymerase II (RNAPII) upon PfNIF4 KD. Furthermore, using the TurboID-based proteomic approach, we identified that PfNIF4 interacted with the RNAPII components, AP2-domain transcription factors, and chromatin-modifiers and binders. These findings suggest that PfNIF4 may act as the RNAPII CTD phosphatase, regulating the expression of general and parasite-specific cellular pathways during the blood-stage development. IMPORTANCE Protein phosphorylation regulates a multitude of cellular processes. The eukaryotic FCP1 phosphatase acts as a CTD-phosphatase to critically balance the phosphorylation status of the CTD of the RNAPII, controlling the accurate execution of the transcription process. Here, we identified PfNIF4 as the FCP1-like phosphatase in P. falciparum. PfNIF4 KD specifically downregulated genes involved in merozoite invasion, resulting in the attenuation of this process. Consistent with the earlier finding of the association of PfNIF4 mutations with artemisinin resistance in Southeast Asian parasite populations, PfNIF4 KD significantly increased in vitro susceptibility to artemisinins. The regulation of these cellular processes in P. falciparum by PfNIF4 is likely realized through RNAPII-mediated transcription, because PfNIF4 was found to interact with RNAPII subunits and KD of PfNIF4 caused CTD hyperphosphorylation. Our results reveal the functions of the PfNIF4 phosphatase in controlling the transcription of invasion- and resistance-related genes in the malaria parasite.

Phosphatase inhibitors BVT-948 and alexidine dihydrochloride inhibit sexual development of the malaria parasite Plasmodium berghei.

BACKGROUND: With the emergence of resistance to front-line antimalarials, there is an urgent need to develop new medicines, including those targeting sexual development. This study aimed to assess the activity of a panel of phosphatase inhibitors against the sexual development of Plasmodium berghei and evaluate their potential as transmission-blocking agents. METHODS: Twenty-five compounds were screened for transmission-blocking activity in vitro using the P. berghei ookinete culture assay. The inhibitory effects on male gametogenesis, gamete-ookinete, and zygote-ookinete formation were evaluated. The transmission-blocking activity of two compounds was evaluated using an in vivo mosquito feeding assay. Their cytotoxic effects were assessed on the human cell line HepG2. RESULTS: Twelve compounds inhibited P. berghei ookinete formation with an IC50 < 10 µM. Two compounds, BVT-948 and alexidine dihydrochloride, significantly inhibited different developmental stages from gametogenesis through ookinete maturation. They also showed a substantial in vivo transmission-blocking activity by the mosquito feeding assay. CONCLUSIONS: Some phosphatase inhibitors effectively inhibited Plasmodium sexual development and exhibited evident transmission-blocking activity, suggesting that phosphatases are valid targets for antimalarial development.

Genetic diversity of Plasmodium vivax reticulocyte binding protein 2b in global parasite populations.

BACKGROUND: Plasmodium vivax reticulocyte binding protein 2b (PvRBP2b) plays a critical role in parasite invasion of reticulocytes by binding the transferrin receptor 1. PvRBP2b is a vaccine candidate based on the negative correlation between antibody titers against PvRBP2b recombinant proteins and parasitemia and risk of vivax malaria. The aim of this study was to analyze the genetic diversity of the PvRBP2b gene in the global P. vivax populations. METHODS: Near full-length PvRBP2b nucleotide sequences (190-8349 bp) were obtained from 88 P. vivax isolates collected from the China-Myanmar border (n = 44) and Thailand (n = 44). An additional 224 PvRBP2b sequences were retrieved from genome sequences from parasite populations worldwide. The genetic diversity, neutral selection, haplotype distribution and genetic differentiation of PvRBP2b were examined. RESULTS: The genetic diversity of PvRBP2b was distributed unevenly, with peak diversity found in the reticulocyte binding region in the N-terminus. Neutrality analysis suggested that this region is subjected to balancing selection or population bottlenecks. Several amino acid variants were found in all or nearly all P. vivax endemic regions. However, the critical residues responsible for reticulocyte binding were highly conserved. There was substantial population differentiation according to the geographical separation. The distribution of haplotypes in the reticulocyte binding region varied among regions; even the two major haplotypes Hap_6 and Hap_8 were found in only five populations. CONCLUSIONS: Our data show considerable genetic variations of PvRBPb in global parasite populations. The geographic divergence may pose a challenge to PvRBP2b-based vaccine development.

Retraction Notice to: miR-181a Induces Macrophage Polarized to M2 Phenotype and Promotes M2 Macrophage-mediated Tumor Cell Metastasis by Targeting KLF6 and C/EBPα.

[This retracts the article DOI: 10.1038/mtna.2016.71.].

Characterization of PSOP26 as an ookinete surface antigen with improved transmission-blocking activity when fused with PSOP25.

BACKGROUND: The Plasmodium zygote-to-ookinete developmental transition is an essential step for establishing an infection in the mosquito vector, and antigens expressed during this stage are potential targets for transmission-blocking vaccines (TBVs). The secreted ookinete protein 26 (PSOP26) is a newly identified ookinete surface protein. The anti-PSOP26 serum has moderate transmission-blocking activity, indicating the benefit of further investigating this protein as a target for TBVs. METHODS: The function of psop26 was analyzed by targeted gene disruption. A chimeric PSOP25-PSOP26 protein was expressed in the Escherichia coli system. The PSOP25-PSOP26 fusion protein, along with mixed (PSOP25 + PSOP26) or single proteins (PSOP26 or PSOP25), were used for the immunization of mice. The antibody titers and immunogenicity of individual sera were analyzed by enzyme-linked immunoassay (ELISA), indirect immunofluorescence assay (IFA), and Western blot. The transmission-blocking activity of sera from different immunization schemes was assessed using in vitro and in vivo assays. RESULTS: PSOP26 is a surface protein expressed in Plasmodium gametes and ookinetes. The protein is dispensable for asexual blood-stage development, gametogenesis, and zygote formation, but is essential for the zygote-to-ookinete developmental transition. Specifically, both the prevalence of infections and oocyst densities were decreased in mosquitoes fed on psop26-null mutants. Mixtures of individual PSOP25 and PSOP26 fragments (PSOP25 + PSOP26), as well as chimeras (PSOP25-PSOP26), elicited high antibody levels in mice, with no immunological interference. Antisera against the mixed and fusion proteins elicited higher transmission-reducing activity (TRA) than antisera against the single PSOP26 antigen, but comparable to antisera against PSOP25 antigen alone. CONCLUSIONS: PSOP26 plays a critical role in the zygote-to-ookinete developmental transition. PSOP25 is a promising TBV candidate that could be used alone to target the ookinete stage.

A G-Protein-Coupled Receptor Modulates Gametogenesis via PKG-Mediated Signaling Cascade in Plasmodium berghei.

Gametogenesis is essential for malaria parasite transmission, but the molecular mechanism of this process remains to be refined. Here, we identified a G-protein-coupled receptor 180 (GPR180) that plays a critical role in signal transduction during gametogenesis in Plasmodium. The P. berghei GPR180 was predominantly expressed in gametocytes and ookinetes and associated with the plasma membrane in female gametes and ookinetes. Knockout of pbgpr180 (Δpbgpr180) had no noticeable effect on blood-stage development but impaired gamete formation and reduced transmission of the parasites to mosquitoes. Transcriptome analysis revealed that a large proportion of the dysregulated genes in the Δpbgpr180 gametocytes had assigned functions in cyclic nucleotide signal transduction. In the Δpbgpr180 gametocytes, the intracellular cGMP level was significantly reduced, and the cytosolic Ca2+ mobilization showed a delay and a reduction in the magnitude during gametocyte activation. These results suggest that PbGPR180 functions upstream of the cGMP-protein kinase G-Ca2+ signaling pathway. In line with this functional prediction, the PbGPR180 protein was found to interact with several transmembrane transporter proteins and the small GTPase Rab6 in activated gametocytes. Allele replacement of pbgpr180 with the P. vivax ortholog pvgpr180 showed equal competence of the transgenic parasite in sexual development, suggesting functional conservation of this gene in Plasmodium spp. Furthermore, an anti-PbGPR180 monoclonal antibody and the anti-PvGPR180 serum possessed robust transmission-blocking activities. These results indicate that GPR180 is involved in signal transduction during gametogenesis in malaria parasites and is a promising target for blocking parasite transmission. IMPORTANCE Environmental changes from humans to mosquitoes activate gametogenesis of the malaria parasite, an obligative process for parasite transmission, but how the signals are relayed remains poorly understood. Here, we show the identification of a Plasmodium G-protein-coupled receptor, GPR180, and the characterization of its function in gametogenesis. In P. berghei, GPR180 is dispensable for asexual development and gametocytogenesis, but its deletion impairs gametogenesis and reduces transmission to mosquitoes. GPR180 appears to function upstream of the cGMP-protein kinase G-Ca2+ signaling pathway and is required for the maximum activity of this pathway. Genetic complementation shows that the GPR180 ortholog from the human malaria parasite P. vivax was fully functional in P. berghei, indicating functional conservation of GPR180 in Plasmodium spp. With predominant expression and membrane association of GPR180 in sexual stages, GPR180 is a promising target for blocking transmission, and antibodies against GPR180 possess robust transmission-blocking activities.

G6pd-Deficient Mice Are Protected From Experimental Cerebral Malaria and Liver Injury by Suppressing Proinflammatory Response in the Early Stage of Plasmodium berghei Infection.

Epidemiological studies provide compelling evidence that glucose-6-phosphate dehydrogenase (G6PD) deficiency individuals are relatively protected against Plasmodium parasite infection. However, the animal model studies on this subject are lacking. Plus, the underlying mechanism in vivo is poorly known. In this study, we used a G6pd-deficient mice infected with the rodent parasite Plasmodium berghei (P.berghei) to set up a malaria model in mice. We analyzed the pathological progression of experimental cerebral malaria (ECM) and acute liver injury in mice with different G6pd activity infected with P.berghei. We performed dual RNA-seq for host-parasite transcriptomics and validated the changes of proinflammatory response in the murine model. G6pd-deficient mice exhibited a survival advantage, less severe ECM and mild liver injury compared to the wild type mice. Analysis based on dual RNA-seq suggests that G6pd-deficient mice are protected from ECM and acute liver injury were related to proinflammatory responses. Th1 differentiation and dendritic cell maturation in the liver and spleen were inhibited in G6pd-deficient mice. The levels of proinflammatory cytokines were reduced, chemokines and vascular adhesion molecules in the brain were significantly down-regulated, these led to decreased cerebral microvascular obstruction in G6pd-deficient mice. We generated the result that G6pd-deficiency mediated protection against ECM and acute liver injury were driven by the regulatory proinflammatory responses. Furthermore, bioinformatics analyses showed that P.berghei might occur ribosome loss in G6pd-deficient mice. Our findings provide a novel perspective of the underlying mechanism of G6PD deficiency mediated protection against malaria in vivo.