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Malaria remains a significant global public health concern, with a recent increase in the number of zoonotic malaria cases in Southeast Asian countries. However, limited reports on the vector for zoonotic malaria exist owing to difficulties in detecting parasite DNA in Anopheles mosquito vectors. Herein, we demonstrate for the first time that several Anopheles mosquitoes contain simian malaria parasite DNA using droplet digital PCR (ddPCR), a highly sensitive PCR method. An entomological survey was conducted to identify simian malaria vector species at Phra Phothisat Temple (PPT), central Thailand, recognized for a high prevalence of simian malaria in wild cynomolgus macaques. A total of 152 mosquitoes from six anopheline species were collected and first analyzed by a standard 18S rRNA nested-PCR analysis for malaria parasite which yielded negative results in all collected mosquitoes. Later, ddPCR was used and could detect simian malaria parasite DNA, i.e. Plasmodium cynomolgi, in 25 collected mosquitoes. And this is the first report of simian malaria parasite DNA detection in Anopheles sawadwongporni. This finding proves that ddPCR is a powerful tool for detecting simian malarial parasite DNA in Anopheles mosquitoes and can expand our understanding of the zoonotic potential of malaria transmission between monkeys and humans., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This review discusses the potential of liposomes as drug delivery systems for antimalarial therapies. Malaria continues to be a significant cause of mortality and morbidity, particularly among children and pregnant women. Drug resistance due to patient non-compliance and troublesome side effects remains a significant challenge in antimalarial treatment. Liposomes, as targeted and efficient drug carriers, have garnered attention owing to their ability to address these issues. Liposomes encapsulate hydrophilic and/or hydrophobic drugs, thus providing comprehensive and suitable therapeutic drug delivery. Moreover, the potential of passive and active drug delivery enables drug concentration in specific target tissues while reducing adverse effects. However, successful liposome formulation is influenced by various factors, including drug physicochemical characteristics and physiological barriers encountered during drug delivery. To overcome these challenges, researchers have explored modifications in liposome nanocarriers to achieve efficient drug loading, controlled release, and system stability. Computational approaches have also been adopted to predict liposome system stability, membrane integrity, and drug-liposome interactions, improving formulation development efficiency. By leveraging computational methods, optimizing liposomal drug delivery systems holds promise for enhancing treatment efficacy and minimizing side effects in malaria therapy. This review consolidates the current understanding and highlights the potential of liposome strategies against malaria., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

The Plasmodium life cycle involves differentiation into multiple morphologically distinct forms, a process regulated by developmental stage-specific gene expression. Histone proteins are involved in epigenetic regulation in eukaryotes, and the histone variant H3.3 plays a key role in the regulation of gene expression and maintenance of genomic integrity during embryonic development in mice. However, the function of H3.3 through multiple developmental stages in Plasmodium remains unknown. To examine the function of H3.3, h3.3-deficient mutants (Δh3.3) were generated in P. berghei. The deletion of h3.3 was not lethal in blood stage parasites, although it had a minor effect of the growth rate in blood stage; however, the in vitro ookinete conversion rate was significantly reduced, and the production of the degenerated form was increased. Regarding the mosquito stage development of Δh3.3, oocysts number was significantly reduced, and no sporozoite production was observed. The h3.3 gene complemented mutant have normal development in mosquito stage producing mature oocysts and salivary glands contained sporozoites, and interestingly, the majority of H3.3 protein was detected in female gametocytes. However, Δh3.3 male and female gametocyte production levels were comparable to the wild-type levels. Transcriptome analysis of Δh3.3 male and female gametocytes revealed the upregulation of several male-specific genes in female gametocytes, suggesting that H3.3 functions as a transcription repressor of male-specific genes to maintain sexual identity in female gametocytes. This study provides new insights into the molecular biology of histone variants H3.3 which plays a critical role on zygote-to-oocyst development in primitive unicellular eukaryotes., (Copyright © 2024. Published by Elsevier B.V.)

Recent studies have suggested that CD8 + liver-resident memory T (T RM ) cells are crucial in the protection against liver-stage malaria. We used liver-directed mRNA-containing lipid nanoparticles (mRNA-LNPs) to induce liver T RM cells in a murine model. Single-dose intravenous injections of ovalbumin mRNA-LNPs effectively induced antigen-specific cytotoxic T lymphocytes in a dose-dependent manner in the liver on day 7. T RM cells (CD8 + CD44 hi CD62L lo CD69 + KLRG1 - ) were induced 5 weeks after immunization. To examine the protective efficacy, mice were intramuscularly immunized with two doses of circumsporozoite protein mRNA-LNPs at 3-week intervals and challenged with sporozoites of Plasmodium berghei ANKA. Sterile immunity was observed in some of the mice, and the other mice showed a delay in blood-stage development when compared with the control mice. mRNA-LNPs therefore induce memory CD8 + T cells that can protect against sporozoites during liver-stage malaria and may provide a basis for vaccines against the disease., Competing Interests: SaM is an employee of SHIONOGI & CO., LTD. This work was supported by SHIONOGI & CO., LTD. Osaka, Japan. Nagasaki University and SHIONOGI & CO., LTD. launched the Shionogi Global Infectious Division SHINE at the Institute of Tropical Medicine NEKKEN, Nagasaki University. Based on the contract for this collaboration, SHIONOGI & CO., LTD. provided funding and contributed to improving the work through frequent discussions with the authors. However, SHIONOGI & CO., LTD. had no role in the experiments, data analysis, writing, or reviewing the paper., (Copyright © 2023 Nakamae, Miyagawa, Ogawa, Kamiya, Taniguchi, Ono, Kawaguchi, Teklemichael, Jian, Araki, Katagami, Mukai, Annoura, Yui, Hirayama, Kawakami and Mizukami.)

Malaria is a mosquito-borne fatal infectious disease that affects humans and is caused by Plasmodium parasites, primarily Plasmodium falciparum. Widespread drug resistance compels us to discover novel compounds and alternative drug discovery targets. The coenzyme A (CoA) biosynthesis pathway is essential for the malaria parasite P. falciparum. The last enzyme in CoA biosynthesis, dephospho-CoA kinase (DPCK), is essential to the major life cycle development stages but has not yet been exploited as a drug target in antimalarial drug discovery. We performed a high-throughput screen of a 210,000-compound library using recombinant P. falciparum DPCK ( Pf DPCK). A high-throughput enzymatic assay using a 1,536-well platform was developed to identify potential Pf DPCK inhibitors. Pf DPCK inhibitors also inhibited parasite growth in a P. falciparum whole-cell asexual blood-stage assay in both drug-sensitive and drug-resistant strains. Hit compounds were selected based on their potency in cell-free ( Pf DPCK) and whole-cell ( Pf 3D7 and Pf Dd2) assays, selectivity over the human orthologue ( Hs COASY) and no cytotoxicity (HepG2). The compounds were ranked using a multiparameter optimization (MPO) scoring model, and the specific binding and the mechanism of inhibition were investigated for the most promising compounds.

Glutaminyl cyclase (QC) modifies N-terminal glutamine or glutamic acid residues of target proteins into cyclic pyroglutamic acid (pGlu). Here, we report the biochemical and functional analysis of Plasmodium QC. We show that sporozoites of QC-null mutants of rodent and human malaria parasites are recognized by the mosquito immune system and melanized when they reach the hemocoel. Detailed analyses of rodent malaria QC-null mutants showed that sporozoite numbers in salivary glands are reduced in mosquitoes infected with QC-null or QC catalytically dead mutants. This phenotype can be rescued by genetic complementation or by disrupting mosquito melanization or phagocytosis by hemocytes. Mutation of a single QC-target glutamine of the major sporozoite surface protein (circumsporozoite protein; CSP) of the rodent parasite Plasmodium berghei also results in melanization of sporozoites. These findings indicate that QC-mediated posttranslational modification of surface proteins underlies evasion of killing of sporozoites by the mosquito immune system.

The infectious protozoan parasite Entamoeba histolytica is responsible for amebiasis causing colitis and liver abscesses, which is an epidemic in developing countries. To develop a drug discovery strategy targeting the iron source required for the proliferation of E. histolytica , an untapped chemical group consisting of low-molecular-weight compounds with metal-binding affinity was investigated. Electrochemically neutral polypyridine compounds, PHN-R 2 , that showed specific Fe(II)-binding affinity and growth inhibitory ability against E. histolytica were identified. Furthermore, the iron-dependent IC 50 values of PHN-R 2 and the spectrometric analytical data of their iron complexes clarified the relationship between the antiamebic activity and the iron-targeting specificity. Notably, when PHN-H 2 was administrated to E. histolytica -infected hamsters as an animal model of amebiasis, it exhibited a prominent therapeutic efficacy to completely cure liver abscesses without serious side effects. Deciphering the antiamebic activity of iron-targeting compounds in vitro and in vivo provides valuable insights into the development of a next-generation drug against amebiasis.

Background: Plasmodium falciparum has acquired resistance to artemisinin in Southeast Asia, with mutations in the P. falciparum Kelch-13 (Pfk13) gene associated with the resistance phenotype. The widespread use of Artemisinin-based combination therapy (ACT)s in Southeast Asia has led to the selection and spread of parasites carrying mutations in Pfk13. We characterised the allele diversity of Pfk13 and pfg377, an artemisinin-resistance neutral polymorphic gene, in parasite DNA extracted human blood from in southern Vietnam in 2003, 2012, 2015 and 2018., Method: This study was conducted in Bu Gia Map commune, Binh Phuoc province, Vietnam, from May 2018 to January 2019. Twenty-four samples from 2018 to 2019, 30 from 2003, 24 from 2012 and 32 from 2015 were analysed. Malaria-infected human blood was collected by finger-prick and used for molecular analysis. A nested-PCR targeting the small subunit ribosomal RNA gene was used for Plasmodium species identification, followed by amplification and nucleotide sequencing of Pfk13 and region 3 of pfg377. Archived blood samples collected in the same region in 2012 and 2015 were also analysed as above for comparison., Results: The genetic diversity of Pfk13 and pfg377 was lower in 2018-2019 compared to 2012 and 2015. The number of distinct Pfk13 mutants decreased from three in 2012 and 2015, P553L, V568G and C580Y, to one, C580Y in 2018-2019. In 2018-2019, the frequency of C580Y mutant strains was 71% (17/24 isolates). All samples were wild type in 2003. In 2012 and 2015, there were single-strain infections as well as co-infections with two mutant strains or with mutant and wild strains, whereas there were no co-infections in 2018. pfg377 allele diversity decreased from five alleles in 2012 to two alleles in 2018-2019., Conclusion: The genetic diversity of P. falciparum was reduced at the two genetic loci surveyed in this study, Pfk13 and pfg377. In the case of the former gene, we observed an increase in the prevalence of parasites carrying the C580Y gene, known to confer reduced susceptibility to ACTs. The reduction in the diversity of pfg377 may be linked to the clonal expansion of parasite strains carrying the C580Y mutation, leading to an overall reduction in parasite genetic diversity across the population., (© 2022. The Author(s).)

Coenzyme A (CoA) is a well-known cofactor that plays an essential role in many metabolic reactions in all organisms. In Plasmodium falciparum , the most deadly among Plasmodium species that cause malaria, CoA and its biosynthetic pathway have been proven to be indispensable. The first and rate-limiting reaction in the CoA biosynthetic pathway is catalyzed by two putative pantothenate kinases ( Pf PanK1 and 2) in this parasite. Here we produced, purified, and biochemically characterized recombinant Pf PanK1 for the first time. Pf PanK1 showed activity using pantetheine besides pantothenate, as the primary substrate, indicating that CoA biosynthesis in the blood stage of P. falciparum can bypass pantothenate. We further developed a robust and reliable screening system to identify inhibitors using recombinant Pf PanK1 and identified four Pf PanK inhibitors from natural compounds., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Nurkanto, Jeelani, Santos, Rahmawati, Mori, Nakamura, Goto, Saikawa, Annoura, Tozawa, Sakura, Inaoka, Shiomi and Nozaki.)

Plasmodium falciparum extensively remodels human erythrocytes by exporting hundreds of parasite proteins. This remodeling is closely linked to the Plasmodium virulence-related functions and immune evasion. The N-terminal export signal named PEXEL ( Plasmodium export element) was identified to be important for the export of proteins beyond the PVM, however, the issue of how these PEXEL-positive proteins are transported and regulated by Rab GTPases from the endoplasmic reticulum (ER) to the cell surface has remained poorly understood. Previously, we identified new aspects of the trafficking of N-myristoylated adenylate kinase 2 (PfAK2), which lacks the PEXEL motif and is regulated by the PfRab5b GTPase. Overexpression of PfRab5b suppressed the transport of PfAK2 to the parasitophorous vacuole membrane and PfAK2 was accumulated in the punctate compartment within the parasite. Here, we report the identification of PfRab5b associated proteins and dissect the pathway regulated by PfRab5b. We isolated two membrane trafficking GTPases PfArf1 and PfRab1b by coimmunoprecipitation with PfRab5b and via mass analysis. PfArf1 and PfRab1b are both colocalized with PfRab5b adjacent to the ER in the early erythrocytic stage. A super-resolution microgram of the indirect immunofluorescence assay using PfArf1 or PfRab1b- expressing parasites revealed that PfArf1 and PfRab1b are localized to different ER subdomains. We used a genetic approach to expresses an active or inactive mutant of PfArf1 that specifically inhibited the trafficking of PfAK2 to the parasitophorous vacuole membrane. While expression of PfRab1b mutants did not affect in the PfAK2 transport. In contrast, the export of the PEXEL-positive protein Rifin was decreased by the expression of the inactive mutant of PfRab1b or PfArf1. These data indicate that the transport of PfAK2 and Rifin were recognized at the different ER subdomain by the two independent GTPases: PfAK2 is sorted by PfArf1 into the pathway for the PV, and the export of Rifin might be sequentially regulated by PfArf1 and PfRab1b., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Taku, Hirai, Makiuchi, Shinzawa, Iwanaga, Annoura, Nagamune, Nozaki and Saito-Nakano.)

In the present study, we demonstrate that the Japanese macaque (Macaca fuscata) can be used as an effective alternative in vivo model for investigating hypnozoite-induced relapsing infection caused by Plasmodium cynomolgi B strain, and that this model is comparable to the rhesus macaque model. Two female Japanese macaques (JM-1 and JM-2; aged 5 years; weighing about 4.0 kg) were used for the experiment. To produce sporozoites in mosquitoes, blood infected with P. cynomolgi B strain was collected from the donor monkey JM-1 and fed to approximately 200 mosquitoes using the standard artificial membrane feeding method. The isolated sporozoites (2 × 10 5 ) were intravenously inoculated into the JM-2 monkey, and the blood stage of the parasite was detected on day 8 after the infection. Chloroquine sulfate (CQ) was intramuscularly administered at a dosage of 6.0 mg/kg into the JM-2 monkey for 6 consecutive days from day 12 onward, after which the parasites disappeared from the peripheral blood. The first relapse occurred on day 26, which was treated again with CQ. Then, the second relapse occurred on day 44, which was cured by CQ treatment followed by the administration of primaquine phosphate (PQ) at a dosage of 1.0 mg/kg/day for 15 days. The JM-2 monkey was observed until 69 days after PQ administration, and there was no relapse during the entire follow-up period. We propose that the Japanese macaque model could contribute not only to drug screening for anti-hypnozoite activity, but could also be used as a powerful tool for investigating hypnozoite biology., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The genus Plasmodium is a unicellular eukaryotic parasite that is the causative agent of malaria, which is transmitted by Anopheline mosquito. There are a total of three developmental stages in the production of haploid parasites in the Plasmodium life cycle: the oocyst stage in mosquitoes and the liver and blood stages in mammalian hosts. The Plasmodium oocyst stage plays an important role in the production of the first generation of haploid parasites. Nuclear division is the most important event that occurs during the proliferation of all eukaryotes. However, obtaining the details of nuclear division at the oocyst stage is challenging owing to difficulties in preparation. In this study, we used focused-ion-beam-milling combined with scanning-electron-microscopy to report the 3D architecture during nuclear segregations in oocyst stage. This advanced technology allowed us to analyse the 3D details of organelle segregation inside the oocyst during sporogony formation. It was revealed that multiple nuclei were involved with several centrosomes in one germ nucleus during sporozoite budding (endopolygeny). Our high-resolution 3D analysis uncovered the endopolygeny-like nuclear architecture of Plasmodium in the definitive host. This nuclear segregation was different from that in the blood stage, and its similarity to other apicomplexan parasite nuclear divisions such as Sarcocystis is discussed., Competing Interests: Declaration of Competing Interest The authors have declared that no competing interests exist., (Copyright © 2019. Published by Elsevier B.V.)

Background: Human malaria is a major threat in rural communities of central Vietnam. Anopheles dirus and Anopheles minimus species are critical malaria vectors in Vietnam, which transmit Plasmodium parasites. However, the entomological aspects of malaria transmission in some of the central provinces of Vietnam remain unexplored. Hence, a cross-sectional entomological survey was carried out to identify the malaria vector species and the transmission of Plasmodium parasites in seven endemic provinces of Vietnam., Methods: Mosquitoes were collected from seven provinces, Gia Lai, Khanh Hoa, Phu Yen, Ninh Thuan, Binh Thuan, Dong Nai, and Binh Phuoc. The collection was conducted for four to eight consecutive nights using three established methods, indoor and outdoor human landing catches and light trap method. Nested-PCR analysis was performed to detect the Plasmodium species in the separated thorax and the abdomen of the individual mosquitoes., Results: A total of 2278 mosquitoes belonging to one of the four species of anopheline mosquitoes, An. dirus , An. maculatus , An. aconitus , and An. minimus were collected. Among the collected mosquitoes, 1398 were analysed using nested-PCR, of which, 40 mosquitoes were positive for Plasmodium parasites. Most of these parasites were detected in the samples from the thorax region, followed by the abdominal portion. The parasites were detected in both the thorax and abdomen of An. dirus . Seven species of Plasmodium parasites were detected during the analysis, of which, Plasmodium inui was the most common species, followed by Plasmodium falciparum , Plasmodium vivax , Plasmodium cynomolgi , Plasmodium coatneyi , Plasmodium knowlesi , and Plasmodium fieldi . Out of the 49 positive samples, 12 showed mixed infections. Co-infection of P. inui with human and other non-human primate Plasmodium species was common., Conclusions: This study demonstrated the presence of human and non-human primate Plasmodium infection in An. dirus , a predominant malarial vector. Further, we showed that An. maculatus and An. minimus species also take part in malarial transmission. This might potentially lead to an alarming situation conducive for the emergence of novel zoonotic malaria., Competing Interests: The study was reviewed and approved by the research ethics committee of the National Institute of Malariology, Parasitology and Entomology, Ministry of Health, Vietnam (366/QD-VSR) and the ethics committee of Fujita Health University, Japan (HM17-050). The people involved in mosquito collection and householders were informed about the objectives, processes, and procedures of this study. Oral informed consents were obtained from them.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Transmission of the malaria parasite Plasmodium falciparum involves infection of Anopheles mosquitoes. Here we characterize SOPT, a protein expressed in P. falciparum ookinetes that facilitates infection of the mosquito midgut. SOPT was identified on the basis that it contains a signal peptide, a PEXEL-like sequence and is expressed in asexual, ookinete and sporozoite stages, suggesting it is involved in infecting the human or mosquito host. SOPT is predicted to contain a subtilisin-like fold with a non-canonical catalytic triad and is orthologous to P. berghei PIMMS2. Localization studies reveal that SOPT is not exported to the erythrocyte but is expressed in ookinetes at the parasite periphery. SOPT-deficient parasites develop normally through the asexual and sexual stages and produce equivalent numbers of ookinetes to NF54 controls, however, they form fewer oocysts and sporozoites in mosquitoes. SOPT-deficient parasites were also unable to activate the immune-responsive midgut invasion marker SRPN6 after mosquito ingestion, suggesting they are defective for entry into the midgut. Disruption of SOPT in P. berghei (PIMMS2) did not affect other lifecycle stages or ookinete development but again resulted in fewer oocysts and sporozoites in mosquitoes. Collectively, this study shows that SOPT/PIMMS2 plays a conserved role in ookinetes of different Plasmodium species., (© 2018 John Wiley & Sons Ltd.)

Eukaryotic cells can employ autophagy to defend themselves against invading pathogens. Upon infection by Plasmodium berghei sporozoites, the host hepatocyte targets the invader by labelling the parasitophorous vacuole membrane (PVM) with the autophagy marker protein LC3. Until now, it has not been clear whether LC3 recruitment to the PVM is mediated by fusion of autophagosomes or by direct incorporation. To distinguish between these possibilities, we knocked out genes that are essential for autophagosome formation and for direct LC3 incorporation into membranes. The CRISPR/Cas9 system was employed to generate host cell lines deficient for either FIP200, a member of the initiation complex for autophagosome formation, or ATG5, responsible for LC3 lipidation and incorporation of LC3 into membranes. Infection of these knockout cell lines with P. berghei sporozoites revealed that LC3 recruitment to the PVM indeed depends on functional ATG5 and the elongation machinery, but not on FIP200 and the initiation complex, suggesting a direct incorporation of LC3 into the PVM. Importantly, in P. berghei-infected ATG5 -/- host cells, lysosomes still accumulated at the PVM, indicating that the recruitment of lysosomes follows an LC3-independent pathway., (© 2017 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.)

Chagas disease, caused by Trypanosoma cruzi , although endemic in many parts of Central and South America, is emerging as a global health threat through the potential contamination of blood supplies. Consequently, in the absence of a gold standard assay for the diagnosis of Chagas disease, additional antigens or strategies are needed. A proteomic analysis of the trypomastigote excreted-secreted antigens (TESA) associated with exosomal vesicles shed by T. cruzi identified ∼80 parasite proteins, with the majority being trans -sialidases. Mass spectrometry analysis of immunoprecipitation products performed using Chagas immune sera showed a marked enrichment in a subset of TESA proteins. Of particular relevance for diagnostic applications were the retrotransposon hot spot (RHS) proteins, which are absent in Leishmania spp., parasites that often confound diagnosis of Chagas disease. Interestingly, serological screens using recombinant RHS showed a robust immunoreactivity with sera from patients with clinical stages of Chagas ranging from asymptomatic to advance cardiomyopathy and this immunoreactivity was comparable to that of crude TESA. More importantly, no cross-reactivity with RHS was detected with sera from patients with malaria, leishmaniasis, toxoplasmosis, or African sleeping sickness, making this protein an attractive reagent for diagnosis of Chagas disease., (Copyright © 2017 American Society for Microbiology.)

Many variant proteins encoded by Plasmodium-specific multigene families are exported into red blood cells (RBC). P. falciparum-specific variant proteins encoded by the var, stevor and rifin multigene families are exported onto the surface of infected red blood cells (iRBC) and mediate interactions between iRBC and host cells resulting in tissue sequestration and rosetting. However, the precise function of most other Plasmodium multigene families encoding exported proteins is unknown. To understand the role of RBC-exported proteins of rodent malaria parasites (RMP) we analysed the expression and cellular location by fluorescent-tagging of members of the pir, fam-a and fam-b multigene families. Furthermore, we performed phylogenetic analyses of the fam-a and fam-b multigene families, which indicate that both families have a history of functional differentiation unique to RMP. We demonstrate for all three families that expression of family members in iRBC is not mutually exclusive. Most tagged proteins were transported into the iRBC cytoplasm but not onto the iRBC plasma membrane, indicating that they are unlikely to play a direct role in iRBC-host cell interactions. Unexpectedly, most family members are also expressed during the liver stage, where they are transported into the parasitophorous vacuole. This suggests that these protein families promote parasite development in both the liver and blood, either by supporting parasite development within hepatocytes and erythrocytes and/or by manipulating the host immune response. Indeed, in the case of Fam-A, which have a steroidogenic acute regulatory-related lipid transfer (START) domain, we found that several family members can transfer phosphatidylcholine in vitro. These observations indicate that these proteins may transport (host) phosphatidylcholine for membrane synthesis. This is the first demonstration of a biological function of any exported variant protein family of rodent malaria parasites., Competing Interests: The authors have declared that no competing interests exist.

Multidrug resistance (MDR) proteins belong to the B subfamily of the ATP Binding Cassette (ABC) transporters, which export a wide range of compounds including pharmaceuticals. In this study, we used reverse genetics to study the role of all seven Plasmodium MDR proteins during the life cycle of malaria parasites. Four P. berghei genes (encoding MDR1, 4, 6 and 7) were refractory to deletion, indicating a vital role during blood stage multiplication and validating them as potential targets for antimalarial drugs. Mutants lacking expression of MDR2, MDR3 and MDR5 were generated in both P. berghei and P. falciparum, indicating a dispensable role for blood stage development. Whereas P. berghei mutants lacking MDR3 and MDR5 had a reduced blood stage multiplication in vivo, blood stage growth of P. falciparum mutants in vitro was not significantly different. Oocyst maturation and sporozoite formation in Plasmodium mutants lacking MDR2 or MDR5 was reduced. Sporozoites of these P. berghei mutants were capable of infecting mice and life cycle completion, indicating the absence of vital roles during liver stage development. Our results demonstrate vital and dispensable roles of MDR proteins during blood stages and an important function in sporogony for MDR2 and MDR5 in both Plasmodium species., (© 2016 John Wiley & Sons Ltd.)

Multidrug resistance-associated proteins (MRPs) belong to the C-family of ATP-binding cassette (ABC) transport proteins and are known to transport a variety of physiologically important compounds and to be involved in the extrusion of pharmaceuticals. Rodent malaria parasites encode a single ABC transporter subfamily C protein, whereas human parasites encode two: MRP1 and MRP2. Although associated with drug resistance, their biological function and substrates remain unknown. To elucidate the role of MRP throughout the parasite life cycle, Plasmodium berghei and Plasmodium falciparum mutants lacking MRP expression were generated. P. berghei mutants lacking expression of the single MRP as well as P. falciparum mutants lacking MRP1, MRP2 or both proteins have similar blood stage growth kinetics and drug-sensitivity profiles as wild type parasites. We show that MRP1-deficient parasites readily invade primary human hepatocytes and develop into mature liver stages. In contrast, both P. falciparum MRP2-deficient parasites and P. berghei mutants lacking MRP protein expression abort in mid to late liver stage development, failing to produce mature liver stages. The combined P. berghei and P. falciparum data are the first demonstration of a critical role of an ABC transporter during Plasmodium liver stage development., (© 2015 John Wiley & Sons Ltd.)

Most studies on malaria-parasite digestion of hemoglobin (Hb) have been performed using P. falciparum maintained in mature erythrocytes, in vitro. In this study, we examine Plasmodium Hb degradation in vivo in mice, using the parasite P. berghei, and show that it is possible to create mutant parasites lacking enzymes involved in the initial steps of Hb proteolysis. These mutants only complete development in reticulocytes and mature into both schizonts and gametocytes. Hb degradation is severely impaired and large amounts of undigested Hb remains in the reticulocyte cytoplasm and in vesicles in the parasite. The mutants produce little or no hemozoin (Hz), the detoxification by-product of Hb degradation. Further, they are resistant to chloroquine, an antimalarial drug that interferes with Hz formation, but their sensitivity to artesunate, also thought to be dependent on Hb degradation, is retained. Survival in reticulocytes with reduced or absent Hb digestion may imply a novel mechanism of drug resistance. These findings have implications for drug development against human-malaria parasites, such as P. vivax and P. ovale, which develop inside reticulocytes., (© 2015 Lin et al.)

Little is known about stage-specific gene regulation in Plasmodium parasites, in particular the liver stage of development. We have previously described in the Plasmodium berghei rodent model, a liver stage-specific (lisp2) gene promoter region, in vitro. Using a dual luminescence system, we now confirm the stage specificity of this promoter region also in vivo. Furthermore, by substitution and deletion analyses we have extended our in vitro characterization of important elements within the promoter region. Importantly, the dual luminescence system allows analyzing promoter constructs avoiding mouse-consuming cloning procedures of transgenic parasites. This makes extensive mutation and deletion studies a reasonable approach also in the malaria mouse model. Stage-specific expression constructs and parasite lines are extremely valuable tools for research on Plasmodium liver stage biology. Such reporter lines offer a promising opportunity for assessment of liver stage drugs, characterization of genetically attenuated parasites and liver stage-specific vaccines both in vivo and in vitro, and may be key for the generation of inducible systems.

A highly efficacious pre-erythrocytic stage vaccine would be an important tool for the control and elimination of malaria but is currently unavailable. High-level protection in humans can be achieved by experimental immunization with Plasmodium falciparum sporozoites attenuated by radiation or under anti-malarial drug coverage. Immunization with genetically attenuated parasites (GAP) would be an attractive alternative approach. In this study, we present data on safety and protective efficacy using sporozoites with deletions of two genes, that is the newly identified b9 and slarp, which govern independent and critical processes for successful liver-stage development. In the rodent malaria model, PbΔb9ΔslarpGAP was completely attenuated showing no breakthrough infections while efficiently inducing high-level protection. The human PfΔb9ΔslarpGAP generated without drug resistance markers were infective to human hepatocytes in vitro and to humanized mice engrafted with human hepatocytes in vivo but completely aborted development after infection. These findings support the clinical development of a PfΔb9ΔslarpSPZ vaccine.

Model antigens are frequently introduced into pathogens to study determinants that influence T-cell responses to infections. To address whether an antigen's subcellular location influences the nature and magnitude of antigen-specific T-cell responses, we generated Plasmodium berghei parasites expressing the model antigen ovalbumin (OVA) either in the parasite cytoplasm or on the parasitophorous vacuole membrane (PVM). For cytosolic expression, OVA alone or conjugated to mCherry was expressed from a strong constitutive promoter (OVAhsp70 or OVA::mCherryhsp70); for PVM expression, OVA was fused to HEP17/EXP1 (OVA::Hep17hep17). Unexpectedly, OVA expression in OVAhsp70 parasites was very low, but when OVA was fused to mCherry (OVA::mCherryhsp70), it was highly expressed. OVA expression in OVA::Hep17hep17 parasites was strong but significantly less than that in OVA::mCherryhsp70 parasites. These transgenic parasites were used to examine the effects of antigen subcellular location and expression level on the development of T-cell responses during blood-stage infections. While all OVA-expressing parasites induced activation and proliferation of OVA-specific CD8(+) T cells (OT-I) and CD4(+) T cells (OT-II), the level of activation varied: OVA::Hep17hep17 parasites induced significantly stronger splenic and intracerebral OT-I and OT-II responses than those of OVA::mCherryhsp70 parasites, but OVA::mCherryhsp70 parasites promoted stronger OT-I and OT-II responses than those of OVAhsp70 parasites. Despite lower OVA expression levels, OVA::Hep17hep17 parasites induced stronger T-cell responses than those of OVA::mCherryhsp70 parasites. These results indicate that unconjugated cytosolic OVA is not stably expressed in Plasmodium parasites and, importantly, that its cellular location and expression level influence both the induction and magnitude of parasite-specific T-cell responses. These parasites represent useful tools for studying the development and function of antigen-specific T-cell responses during malaria infection., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

The 10 Plasmodium 6-Cys proteins have critical roles throughout parasite development and are targets for antimalaria vaccination strategies. We analyzed the conserved 6-cysteine domain of this family and show that only the last 4 positionally conserved cysteine residues are diagnostic for this domain and identified 4 additional "6-Cys family-related" proteins. Two of these, sequestrin and B9, are critical to Plasmodium liver-stage development. RT-PCR and immunofluorescence assays show that B9 is translationally repressed in sporozoites and is expressed after hepatocyte invasion where it localizes to the parasite plasma membrane. Mutants lacking B9 expression in the rodent malaria parasites P. berghei and P. yoelii and the human parasite P. falciparum developmentally arrest in hepatocytes. P. berghei mutants arrest in the livers of BALB/c (100%) and C57BL6 mice (>99.9%), and in cultures of Huh7 human-hepatoma cell line. Similarly, P. falciparum mutants while fully infectious to primary human hepatocytes abort development 3 d after infection. This growth arrest is associated with a compromised parasitophorous vacuole membrane a phenotype similar to, but distinct from, mutants lacking the 6-Cys sporozoite proteins P52 and P36. Our results show that 6-Cys proteins have critical but distinct roles in establishment and maintenance of a parasitophorous vacuole and subsequent liver-stage development.

One of the bottlenecks in the development of a whole sporozoite malaria vaccine is the route and method of sporozoite administration. Immunization and challenge of human volunteers by mosquito bites is effective, but cannot be used as a vaccine. Intravenous immunization with sporozoites is effective in rodents and non-human primates, and being studied in humans, but is not yet used for licensed vaccines for infectious diseases. Intradermal and subcutaneous immunization regimens show a strong decrease in protective efficacy, which in rodents, is associated with a decreased degree of parasite liver infection during immunization. The objective of this study was to explore alternative routes of sporozoite administration to increase efficiency of liver infection. Using in vivo imaging, we found that IM injection of sporozoites resulted in a greater parasite liver load compared to ID and SC injection. The use of small inoculation volumes and multiple injections further increased the subsequent liver load. These observations were corroborated in a Plasmodium yoelii model using cryopreserved sporozoites administered ID. Our findings provide a rationale for the design of clinical trials to optimize needle and syringe administration of Plasmodium falciparum sporozoites., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Rhomboid-like proteases cleave membrane-anchored proteins within their transmembrane domains. In apicomplexan parasites substrates include molecules that function in parasite motility and host cell invasion. While two Plasmodium rhomboids, ROM1 and ROM4, have been examined, the roles of the remaining six rhomboids during the malaria parasite's life cycle are unknown. We present systematic gene deletion analyses of all eight Plasmodium rhomboid-like proteins as a means to discover stage-specific phenotypes and potential functions in the rodent malaria model, P. berghei. Four rhomboids (ROM4, 6, 7 and 8) are refractory to gene deletion, suggesting an essential role during asexual blood stage development. In contrast ROM1, 3, 9 and 10 were dispensable for blood stage development and exhibited no, subtle or severe defects in mosquito or liver development. Parasites lacking ROM9 and ROM10 showed no major phenotypic defects. Parasites lacking ROM1 presented a delay in blood stage patency following liver infection, but in contrast to a previous study blood stage parasites had similar growth and virulence characteristics as wild type parasites. Parasites lacking ROM3 in mosquitoes readily established oocysts but failed to produce sporozoites. ROM3 is the first apicomplexan rhomboid identified to play a vital role in sporogony., (© 2013 Blackwell Publishing Ltd.)

We describe simple and sensitive in vitro and in vivo assays to analyze Plasmodium liver stage development using transgenic P. berghei parasites (PbGFP-Luccon), which express the bioluminescent reporter protein, luciferase. In these assays, parasite development in hepatocytes is visualized and quantified by real-time bioluminescence imaging both in culture and in live mice. We also describe quantification of in vitro liver-stage development by measuring luminescence using a microplate reader. Reporter-parasite based quantification of liver-stage development is faster and correlates very well with established quantitative RT-PCR methods currently used to assess parasite development inside hepatocytes, both in live mice and in culture.

The critical first step in the clinical development of a malaria vaccine, based on live-attenuated Plasmodium falciparum sporozoites, is the guarantee of complete arrest in the liver. We report on an approach for assessing adequacy of attenuation of genetically attenuated sporozoites in vivo using the Plasmodium berghei model of malaria and P. falciparum sporozoites cultured in primary human hepatocytes. We show that two genetically attenuated sporozoite vaccine candidates, Δp52+p36 and Δfabb/f, are not adequately attenuated. Sporozoites infection of mice with both P. berghei candidates can result in blood infections. We also provide evidence that P. falciparum sporozoites of the leading vaccine candidate that is similarly attenuated through the deletion of the genes encoding the proteins P52 and P36, can develop into replicating liver stages. Therefore, we propose a minimal set of screening criteria to assess adequacy of sporozoite attenuation necessary before advancing into further clinical development and studies in humans., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: The flagellate protozoan parasite, Trypanosoma cruzi, is a causative agent of Chagas disease that is transmitted by reduviid bugs to humans. The parasite exists in multiple morphological forms in both vector and host, and cell differentiation in T. cruzi is tightly associated with stage-specific protein synthesis and degradation. However, the specific molecular mechanisms responsible for this coordinated cell differentiation are unclear., Methodology/principal Findings: The SUMO conjugation system plays an important role in specific protein expression. In T. cruzi, a subset of SUMOlylated protein candidates and the nuclear localization of SUMO have been shown. Here, we examined the biological roles of SUMO in T. cruzi. Site-directed mutagenesis analysis of SUMO consensus motifs within T. cruzi SUMO using a bacterial SUMOylation system revealed that T. cruzi SUMO can polymerize. Indirect fluorescence analysis using T. cruzi SUMO-specific antibody showed the extra-nuclear localization of SUMO on the flagellum of epimastigote and metacyclic and bloodstream trypomastigote stages. In the short-flagellate intracellular amastigote, an extra-nuclear distribution of SUMO is associated with basement of the flagellum and becomes distributed along the flagellum as amastigote transforms into trypomastigote. We examined the flagellar target protein of SUMO and show that a paraflagellar rod protein, PFR1, is SUMOylated., Conclusions: These findings indicate that SUMOylation is associated with flagellar homeostasis throughout the parasite life cycle, which may play an important role in differentiation of T. cruzi.

Glycosomes are peroxisome-related organelles containing glycolytic enzymes that have been found only in kinetoplastids. We show here that a glycolytic enzyme is compartmentalized in diplonemids, the sister group of kinetoplastids. We found that, similar to kinetoplastid aldolases, the fructose 1,6-bisphosphate aldolase of Diplonema papillatum possesses a type 2-peroxisomal targeting signal. Western blotting showed that this aldolase was present predominantly in the membrane/organellar fraction. Immunofluorescence analysis showed that this aldolase had a scattered distribution in the cytosol, suggesting its compartmentalization. In contrast, orotidine-5'-monophosphate decarboxylase, a non-glycolytic glycosomal enzyme in kinetoplastids, was shown to be a cytosolic enzyme in D. papillatum. Since euglenoids, the earliest diverging branch of Euglenozoa, do not possess glycolytic compartments, these findings suggest that the routing of glycolytic enzymes into peroxisomes may have occurred in a common ancestor of diplonemids and kinetoplastids, followed by diversification of these newly established organelles in each of these euglenozoan lineages., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Background: The lung is one of the sites of granulomatous responses, which are characterized by the recruitment and organization of activated macrophages and lymphocytes. There have been several reports that have shown that some pulmonary granulomatous diseases, such as sarcoidosis and nontuberculous mycobacterial disease, are likely to be characterized by a preponderance in postmenopausal females. Although sex hormones have been shown to play an important role in the regulation of the immune system, the influence of sex hormones on pulmonary granuloma formation is still unclear., Objectives: The purpose of this study was to assess whether sex hormones are involved in granulomatous inflammation and to evaluate how sex hormones modulate this response in the lung., Methods: Ovariectomized rats were used as an experimental postmenopausal model in which chronic pulmonary granulomatous inflammation was induced by intravenous injection of complete Freund's adjuvant., Results: Histological analysis of lung tissues demonstrated enhancement of granuloma formation in the ovariectomized group. Such enhanced granuloma formation was significantly associated with generalized Th1-biased cytokine production in the bronchoalveolar lavage fluid., Conclusion: These results indicate that sex hormones play an important role in pulmonary granuloma formation by altering the Th1 responses., (Copyright © 2011 S. Karger AG, Basel.)

Research on the biology of malaria parasites has greatly benefited from the application of reverse genetic technologies, in particular through the analysis of gene deletion mutants and studies on transgenic parasites that express heterologous or mutated proteins. However, transfection in Plasmodium is limited by the paucity of drug-selectable markers that hampers subsequent genetic modification of the same mutant. We report the development of a novel 'gene insertion/marker out' (GIMO) method for two rodent malaria parasites, which uses negative selection to rapidly generate transgenic mutants ready for subsequent modifications. We have created reference mother lines for both P. berghei ANKA and P. yoelii 17XNL that serve as recipient parasites for GIMO-transfection. Compared to existing protocols GIMO-transfection greatly simplifies and speeds up the generation of mutants expressing heterologous proteins, free of drug-resistance genes, and requires far fewer laboratory animals. In addition we demonstrate that GIMO-transfection is also a simple and fast method for genetic complementation of mutants with a gene deletion or mutation. The implementation of GIMO-transfection procedures should greatly enhance Plasmodium reverse-genetic research., (© 2011 Lin et al.)

A unique feature of the genome architecture in the parasitic trypanosomatid protists is large-scale synteny. We addressed the evolutionary trait of synteny in the eukaryotic group, Euglenozoa, which consists of euglenoids (earliest branching), diplonemids, and kinetoplastids (trypanosomatids and bodonids). Synteny of the pyrimidine biosynthetic (pyr) gene cluster, which constitutes part of a large syntenic cluster in trypanosomatids and includes four separate genes (pyr1-pyr4) and one fused gene (pyr6/pyr5 fusion), was conserved in the bodonid, Parabodo caudatus. In the diplonemid, Diplonema papillatum, we identified pyr4 and pyr6 genes. Phylogenetic analyses of pyr4 and pyr6 showed the separate origin of each in kinetoplastids and euglenoids/diplonemids and suggested that kinetoplastids have acquired these genes via lateral gene transfer (LGT). Because replacement of genes by non-orthologs within the syntenic cluster is highly unlikely, we concluded that, after separation of the line leading to diplonemids, the syntenic pyr gene cluster was established in the common ancestor of kinetoplastids, preceded by their acquisition via LGT. Notably, we found that diplonemid pyr6 is a stand-alone gene, inconsistent with both euglenoid pyr5/pyr6 and kinetoplastid pyr6/pyr5 fusions. Our findings provide insights into the evolutionary gaps within Euglenozoa and the evolutionary trait of rearrangement of gene fusion in this lineage.

The ACT-DHOD gene in the kinetoplastid Bodo saliens encodes aspartate carbamoyltransferase and dihydroorotate dehydrogenase, the second and fourth enzymes of pyrimidine biosynthesis. Although the single mRNA species yielded a 70-kDa ACT-DHOD protein, Western blotting with anti-DHOD-peptide antibody showed a major band of 35-kDa and minor bands. In-gel digestion and liquid chromatography-tandem mass (MS/MS) spectrometry showed that the 35-kDa band contained DHOD-specific polypeptides and an ACT-specific polypeptide, suggesting the occurrence of independent DHOD and ACT. Immunoprecipitation and MS/MS analysis identified a 70-kDa ACT-DHOD and a 35-kDa DHOD independently, and the N-terminal amino acid of 35-kDa DHOD was blocked. In vitro processing assay showed that recombinant ACT-DHOD was decreased by the B. saliens lysate, accompanying the appearance of 35-kDa DHOD and 35-kDa ACT. These results indicate that fused ACT-DHOD is the precursor to mature DHOD. Large amount of 35-kDa DHOD in B. saliens is discussed from a viewpoint of its physiological roles.