Plasmodium ookinete-secreted proteins secreted through a common micronemal pathway are targets of blocking malaria transmission

The mosquito midgut ookinete stage of the malaria parasite, Plasmodium, possesses microneme secretory organelles that mediate locomotion and midgut wall egress to establish sporogonic stages and subsequent transmission. The purpose of this study was 2-fold: 1) to determine whether there exists a single micronemal population with respect to soluble and membrane-associated secreted proteins; and 2) to evaluate the ookinete micronemal proteins chitinase (PgCHT1), circumsporozoite and TRAP-related protein (CTRP), and von Willebrand factor A domain-related protein (WARP) as immunological targets eliciting sera-blocking malaria parasite infectivity to mosquitoes. Indirect immunofluorescence localization studies in Plasmodium gallinaceum using specific antisera showed that all three proteins are distributed intracellularly with a similar granular cytoplasmic appearance and with focal concentration of PgCHT1 and PgCTRP, but not PgWARP, at the ookinete apical end. Immunogold double-labeling electron microscopy, using antisera against the membrane-associated protein CTRP and the soluble WARP, showed that these two proteins co-localized to the same micronemal population. Within the microneme CTRP was associated peripherally at the microneme membrane, whereas PgCHT1 and WARP were diffuse within the micronemal lumen. Sera produced against Plasmodium falciparum WARP significantly reduced the infectivity of P. gallinaceum to Aedes aegypti and P. falciparum to Anopheles mosquitoes. Antisera against PgCTRP and PgCHT1 also significantly reduced the infectivity of P. gallinaceum for A. aegypti. These results support the concept that ookinete micronemal proteins may constitute a general class of malaria transmission-blocking vaccine candidates.     

A Unique Protein Phosphatase with Kelch-Like Domains (PPKL) in Plasmodium Modulates Ookinete Differentiation,Motility and Invasion

Protein phosphorylation and dephosphorylation (catalysed by kinases and phosphatases, respectively) are post-translational modifications that play key roles in many eukaryotic signalling pathways, and are often deregulated in a number of pathological conditions in humans. In the malaria parasite Plasmodium, functional insights into its kinome have only recently been achieved, with over half being essential for blood stage development and another 14 kinases being essential for sexual development and mosquito transmission. However, functions for any of the plasmodial protein phosphatases are unknown. Here, we use reverse genetics in the rodent malaria model, Plasmodium berghei, to examine the role of a unique protein phosphatase containing kelch-like domains (termed PPKL) from a family related to Arabidopsis BSU1. Phylogenetic analysis confirmed that the family of BSU1-like proteins including PPKL is encoded in the genomes of land plants, green algae and alveolates, but not in other eukaryotic lineages. Furthermore, PPKL was observed in a distinct family, separate to the most closely-related phosphatase family, PP1. In our genetic approach, C-terminal GFP fusion with PPKL showed an active protein phosphatase preferentially expressed in female gametocytes and ookinetes. Deletion of the endogenous ppkl gene caused abnormal ookinete development and differentiation, and dissociated apical microtubules from the inner-membrane complex, generating an immotile phenotype and failure to invade the mosquito mid-gut epithelium. These observations were substantiated by changes in localisation of cytoskeletal tubulin and actin, and the micronemal protein CTRP in the knockout mutant as assessed by indirect immunofluorescence. Finally, increased mRNA expression of dozi, a RNA helicase vital to zygote development was observed in ppkl⁻ mutants, with global phosphorylation studies of ookinete differentiation from 1.5–24 h post-fertilisation indicating major changes in the first hours of zygote development. Our work demonstrates a stage-specific essentiality of the unique PPKL enzyme, which modulates parasite differentiation, motility and transmission.     

CTRP is essential for mosquito infection by malaria ookinetes

The malaria parasite suffers severe population losses as it passes through its mosquito vector. Contributing factors are the essential but highly constrained developmental transitions that the parasite undergoes in the mosquito midgut, combined with the invasion of the midgut epithelium by the malaria ookinete (recently described as a principal elicitor of the innate immune response in the Plasmodium-infected insect). Little is known about the molecular organization of these midgut-stage parasites and their critical interactions with the blood meal and the mosquito vector. Elucidation of these molecules and interactions will open up new avenues for chemotherapeutic and immunological attack of parasite development. Here, using the rodent malaria parasite Plasmodium berghei, we identify and characterize the first microneme protein of the ookinete: circumsporozoite- and TRAP-related protein (CTRP). We show that transgenic parasites in which the CTRP gene is disrupted form ookinetes that have reduced motility, fail to invade the midgut epithelium, do not trigger the mosquito immune response, and do not develop further into oocysts. Thus, CTRP is the first molecule shown to be essential for ookinete infectivity and, consequently, mosquito transmission of malaria.     

The Plasmodium vivax homolog of the ookinete adhesive micronemal protein, CTRP

The Plasmodium circumsporozoite protein/thrombospondin-related anonymous protein-related protein (CTRP) is expressed at the mosquito midgut ookinete stage and is considered to be a transmission-blocking vaccine candidate. CTRP is composed of multiple von Willebrand factor A (vWA) and thrombospondin type 1 domains in the extracellular portion of the molecule, and a short acidic cytoplasmic domain that interacts with the actomyosin machinery. As a means to predict functionally relevant domains within CTRP we determined the nucleotide sequences of CTRP from the Plasmodium vivax Sall and the Plasmodium yoelii 17XL strains and characterized the conservation of domain architectures and motifs across Plasmodium genera. Sequence alignments indicate that the CTRP 1st to 4th vWA domains exhibit greater conservation, and thereby are perhaps functionally more important than the 5th and 6th domains. This point should be considered for the development of a transmission-blocking vaccine that includes CTRP recombinant subunit. To complement previous cellular studies on CTRP, we further determined the expression and cellular localization of CTRP protein in P. vivax and P. yoelii.     

C1q/TNF-related Protein 4 (CTRP4) Is a Unique Secreted Protein with Two Tandem C1q Domains That Functions in the Hypothalamus to Modulate Food Intake and Body Weight

CTRP4 is a unique member of the C1q family, possessing two tandem globular C1q domains. Its physiological function is poorly defined. Here, we show that CTRP4 is an evolutionarily conserved, ∼34-kDa secretory protein expressed in the brain. In human, mouse, and zebrafish brain, CTRP4 expression begins early in development and is widespread in the central nervous system. Neurons, but not astrocytes, express and secrete CTRP4, and secreted proteins form higher-order oligomeric complexes. CTRP4 is also produced by peripheral tissues and circulates in blood. Its serum levels are increased in leptin-deficient obese (ob/ob) mice. Functional studies suggest that CTRP4 acts centrally to modulate energy metabolism. Refeeding following an overnight fast induced the expression of CTRP4 in the hypothalamus. Central administration of recombinant protein suppressed food intake and altered the whole-body energy balance in both chow-fed and high-fat diet-fed mice. Suppression of food intake by CTRP4 is correlated with a decreased expression of orexigenic neuropeptide (Npy and Agrp) genes in the hypothalamus. These results establish CTRP4 as a novel nutrient-responsive central regulator of food intake and energy balance.     

The role of Pvs28 in sporozoite development in Anopheles sinensis and its longevity in BALB/c mice

To develop a vivax malaria vaccine for blocking malarial transmission, the ookinete surface protein Pvs28 was cloned from Korean malaria patients using polymerase chain reaction. The Pvs28 gene consists of 726 bp and encodes 241 amino acids. It was subcloned into the expression vector pQE30 and expressed in Escherichia coli. The expressed recombinant protein, rPvs28, has a molecular weight of about 28 kDa in SDS–PAGE analysis. A monoclonal antibody against rPvs28 was produced using BALB/c mice. It inhibited sporozoite development in Anopheles sinensis mosquitoes (n = 81) which is one of the malaria vectors in Korea, with relatively high antibody titer against rPv28 persisting for more than 6 months. These results indicate that rPvs28 induces an immune response in mice that effectively blocks sporozoite development in mosquitoes. Therefore it could be a vaccine candidate for preventing vivax malaria in Korea.     

Apical Surface Expression of Aspartic Protease Plasmepsin 4, a Potential Transmission-blocking Target of the Plasmodium Ookinete

To invade its definitive host, the mosquito, the malaria parasite must cross the midgut peritrophic matrix that is composed of chitin cross-linked by chitin-binding proteins and then develop into an oocyst on the midgut basal lamina. Previous evidence indicates that Plasmodium ookinete-secreted chitinase is important in midgut invasion. The mechanistic role of other ookinete-secreted enzymes in midgut invasion has not been previously examined. De novo mass spectrometry sequencing of a protein obtained by benzamidine affinity column of Plasmodium gallinaceum ookinete axenic culture supernatant demonstrated the presence of an ookinete-secreted plasmepsin, an aspartic protease previously only known to be present in the digestive vacuole of asexual stage malaria parasites. This plasmepsin, the ortholog of Plasmodium falciparum plasmepsin 4, was designated PgPM4. PgPM4 and PgCHT2 (the P. gallinaceum ortholog of P. falciparum chitinase PfCHT1) are both localized on the ookinete apical surface, and both are present in micronemes. Aspartic protease inhibitors (peptidomimetic and natural product), calpain inhibitors, and anti-PgPM4 monoclonal antibodies significantly reduced parasite infectivity for mosquitoes. These results suggest that plasmepsin 4, previously known only to function in the digestive vacuole of asexual blood stage Plasmodium, plays a role in how the ookinete interacts with the mosquito midgut interactions as it becomes an oocyst. These data are the first to delineate a role for an aspartic protease in mediating Plasmodium invasion of the mosquito and demonstrate the potential for plasmepsin 4 as a malaria transmission-blocking vaccine target.     

Detection and characterization of the in vitro e3 ligase activity of the human MID1 protein

Human MID1 (midline-1) is a microtubule-associated protein that is postulated to target the catalytic subunit of protein phosphatase 2A for degradation. It binds alpha4 that then recruits the catalytic subunit of protein phosphatase 2A. As a member of the TRIM (tripartite motif) family, MID1 has three consecutive zinc-binding domains—RING (really interesting new gene), Bbox1, and Bbox2—that have similar ββα-folds. Here, we describe the in vitro characterization of these domains individually and in tandem. We observed that the RING domain exhibited greater ubiquitin (Ub) E3 ligase activity compared to the Bbox domains. The amount of autopolyubiquitinated products with RING-Bbox1 and RING-Bbox1-Bbox2 domains in tandem was significantly greater than those of the individual domains. However, no polyubiquitinated products were observed for the Bbox1-Bbox domains in tandem. Using mutants of Ub, we observed that these MID1 domain constructs facilitate Ub chain elongation via Lys63 of Ub. In addition, we observed that the high-molecular-weight protein products were primarily due to polyubiquitination at one site (Lys154) on the Bbox1 domain of the RING-Bbox1 and RING-Bbox1-Bbox2 constructs. We observed that MID1 E3 domains could interact with multiple E2-conjugating enzymes. Lastly, a 45-amino-acid peptide derived from the C-terminus of alpha4 that binds tightly to Bbox1 was observed to be monoubiquitinated in the assay and appears to down-regulate the amount of polyubiquitinated products formed. These studies shed light on MID1 E3 ligase activity and show how its three zinc-binding domains can contribute to MID1's overall function.     

A novel pattern recognition protein of the Chinese oak silkmoth,Antheraea pernyi,is involved in the pro-PO activating system

In this paper, we firstly reported a C-type lectin cDNA clone of 1029 bps from the larvae of A. Pernyi (Ap-CTL) using PCR and RACE techniques. The full-length cDNA contains an open reading frame encoding 308 amino acid residues which has two different carbohydrate-recognition domains (CRDs) arranged in tandem. To investigate the biological activities in the innate immunity, recombinant Ap-CTL was expressed in E. coli with a 6-histidine at the amino-terminus (Ap-rCTL). Besides acted as a broad-spectrum recognition protein binding to a wide range of PAMPs and microorganisms, Ap-rCTL also had the ability to recognize and trigger the agglutination of bacteria and fungi. In the proPO activation assay, Ap-rCTL specifically restored the PO activity of hemolymph blocked by anti-Ap-rCTL antibody in the presence of different PAMPs or microorganisms. In summary, Ap-rCTL plays an important role in insect innate immunity as an pattern recognition protein. [BMB Reports 2013; 46(7): 358-363]     

Pfs 16 pivotal role in Plasmodium falciparum gametocytogenesis: A potential antiplasmodial drug target

Mature gametocytes, the sexual stage of Plasmodium falciparum, ensure the continued transmission of malaria from the human host to the mosquito vector. Even if gametocytes are not implicated in the malaria physiopathology it is crucial to the spread of malaria. Gametocytes are to be a key target for drugs used against Plasmodium in public health. The expression levels of 4 sexual-stage specific genes, Pfs 16, Pfs 25, Pfg 27and S 18S rRNA, during gametocytogenesis of various P. falciparum strains were analyzed by a real time PCR assay. The strains showed different capacities to produce mature gametocytes and in parallel different patterns of sexual gene expression. There was a correlation only between Pfs 16 cDNA overexpression in the first 48 h of the culture and the production of mature gametocytes. Pfs 16 is an early marker of the development of mature gametocytes in cultures and is therefore a potential target for new antimalarial drugs.     

PSOP1, putative secreted ookinete protein 1, is localized to the micronemes of Plasmodium yoelii and P. berghei ookinetes

Plasmodium parasites cause malaria in mammalian hosts and are transmitted by Anopheles mosquitoes. Activated gametocytes in the mosquito midgut egress from erythrocytes followed by fertilization and zygote formation. Zygotes differentiate into motile invasive ookinetes, which penetrate the midgut epithelium before forming oocysts beneath the basal lamina. Ookinete development and traversal across the mosquito midgut wall are major bottlenecks in the parasite life cycle. In ookinetes, surface proteins and proteins stored in apical organelles have been shown to be involved in parasite-host interactions. A group of ookinete proteins that are predicted to have such functions are named PSOPs (putative secreted ookinete protein). PSOP1 is possibly involved in migration through the midgut wall, and here its subcellular localization was examined in ookinetes by immunoelectron microscopy. PSOP1 localizes to the micronemes of Plasmodium yoelii and Plasmodium berghei ookinetes, indicating that it is stored and possibly apically secreted during ookinete penetration through the mosquito midgut wall.     

In silico analysis of antibody triggering biofilm associated protein in Acinetobacter baumannii

Acinetobacter baumannii surface protein, commonly known as biofilm associated protein (Bap), is involved in biofilm formation. A high propensity among the clinical isolates to form biofilm and a significant association of biofilms with multiple drug resistance has been demonstrated. Production of antibodies can be used for inhibition of biofilm and control of the diseases caused by A. baumannii. Large molecular mass of Bap justifies an approach to identifying A. baumannii effective antigens. It has a core domain of seven repeat modules A-G. With the large number of available biofilm gene sequences, bioinformatic tools are needed to identify the genes encoding the antigens. Proteins containing these tandem repeats of Bap domains have high propensities to attach to each other to form biofilm. We hypothesized that conserved and functional domains of tandem repeat could be identified with a search and alignment of the repeats for evaluation of antigenic determinants. Here we demonstrate the results of bioinformatics screening and gene scan of the gene sequence database of homolog sequences to identify conserved domains. Higher scoring hits were found in repeat modules mostly D, B, C and A, respectively. Upon the analysis four regions of highly structural and functional conserved regions from Bap sequence of A. baumannii were selected. 3D structure, antigenicity and solubility predictions revealed that these regions were appropriate candidates for antibody production.     

Plant-like phosphofructokinase from Plasmodium falciparum belongs to a novel class of ATP-dependent enzymes

Malaria parasite-infected erythrocytes exhibit enhanced glucose utilisation and 6-phospho-1-fructokinase (PFK) is a key enzyme in glycolysis. Here we present the characterisation of PFK from the human malaria parasite Plasmodium falciparum. Of the two putative PFK genes on chromosome 9 (PfPFK9) and 11 (PfPFK11), only the PfPFK9 gene appeared to possess all the catalytic features appropriate for PFK activity. The deduced PfPFK proteins contain domains homologous to the plant-like pyrophosphate (PPi)-dependent PFK β and α subunits, which are quite different from the human erythrocyte PFK protein. The PfPFK9 gene β and α regions were cloned and expressed as His₆- and GST-tagged proteins in Escherichia coli. Complementation of PFK-deficient E. coli and activity analysis of purified recombinant proteins confirmed that PfPFK9β possessed catalytic activity. Monoclonal antibodies against the recombinant β protein confirmed that the PfPFK9 protein has β and α domains fused into a 200 kDa protein, as opposed to the independent subunits found in plants. Despite an overall structural similarity to plant PPi-PFKs, the recombinant protein and the parasite extract exhibited only ATP-dependent enzyme activity, and none with PPi. Unlike host PFK, the Plasmodium PFK was insensitive to fructose-2,6-bisphosphate (F-2,6-bP), phosphoenolpyruvate (PEP) and citrate. A comparison of the deduced PFK proteins from several protozoan PFK genome databases implicates a unique class of ATP-dependent PFK present amongst the apicomplexan protozoans.     

The novel heme oxygenase-like protein from Plasmodiumfalciparum converts heme to bilirubin IXα in the apicoplast

The metabolic pathways in apicoplasts of human malaria parasites are promising drug targets. The apicomplexan parasites exhibit delayed cell death when their apicoplast is impaired, but the metabolic pathways within apicoplasts are poorly understood. A nuclear-encoded heme oxygenase (HO)-like protein with an apicoplast-targeted bipartite transit peptide was identified in the Plasmodiumfalciparum genome. Purified mature recombinant PfHO protein converted heme into bilirubin IXα as confirmed by high-performance liquid chromatography. In addition, PfHO required an iron chelator such as deferoxamine for complete activity. These observations lead to the conclusion that a novel enzymatic heme degradation system is present in human malaria parasites.     

Malaria vectors of Papua New Guinea

Understanding malaria transmission in Papua New Guinea (PNG) requires exact knowledge of which Anopheles species are transmitting malaria and is complicated by the cryptic species status of many of these mosquitoes. To identify the malaria vectors in PNG we studied Anopheles specimens from 232 collection localities around human habitation throughout PNG (using CO₂ baited light traps and human bait collections). A total of 22,970 mosquitoes were individually assessed using a Plasmodium sporozoite enzyme-linked immunosorbent assay to identify Plasmodiumfalciparum, Plasmodiumvivax and Plasmodiummalariae circumsporozoite proteins. All mosquitoes were identified to species by morphology and/or PCR. Based on distribution, abundance and their ability to develop sporozoites, we identified five species as major vectors of malaria in PNG. These included: Anophelesfarauti, Anopheleshinesorum (incriminated here, to our knowledge, for the first time), Anophelesfarauti 4, Anopheleskoliensis and Anophelespunctulatus. Anopheleslongirostris and Anophelesbancroftii were also incriminated in this study. Surprisingly, An. longirostris showed a high incidence of infections in some areas. A newly identified taxon within the Punctulatus Group, tentatively called An. farauti 8, was also found positive for circumsporozoite protein. These latter three species, together with Anopheleskarwari and Anophelessubpictus, incriminated in other studies, appear to be only minor vectors, while Anophelesfarauti 6 appears to be the major vector in the highland river valleys (>1500 m above sea level). The nine remaining Anopheles species found in PNG have been little studied and their bionomics are unknown; most appear to be uncommon with limited distribution and their possible role in malaria transmission has yet to be determined.     

Analysis of the Plasmodium vivax dihydrofolate reductase–thymidylate synthase gene sequence

A basis for the intrinsic resistance of some Plasmodium vivax isolates to pyrimethamine is suggested following the isolation of the bifunctional gene encoding dihydrofolate reductase–thymidylate synthase (DHFR-TS) of this human malaria parasite. Malaria parasites are dependent on this enzyme for folate biosynthesis. Specific inhibition of the DHFR domain of the enzyme by pyrimethamine blocks pyrimidine biosynthesis, leading to an inhibition of DNA replication. The gene was isolated by the polymerase chain reaction (PCR) from genomic DNA using degenerate oligonucleotides designed to hybridize on the highly conserved regions of the sequence. The nucleotide sequence was completed by screening P. vivax genomic bank. Sequence analysis revealed an open reading frame (ORF) of 1872 nucleotides encoding a deduced protein of 623 amino acids (aa). Alignment with other malarial DHFR-TS genes showed that a 237-residue DHFR domain and a 286-residue TS domain were separated by a 100-aa linker region. Comparison with other malarial species showed low and essentially no isology in the DHFR and junctional domains, respectively, whereas an extensive isology was observed in the TS domain. The characteristic features of the P. vivax DHFR-TS gene sequence include an insertion of a short repetitive tandem array within the DHFR domain that is absent in another human malaria parasite, P. falciparum, and a GC-biased aa composition, giving rise to highly GC-rich DHFR (50.8%), junctional (58.7%), and TS (40.5%) domains, as compared with other malaria parasites. Analysis of the 5′ noncoding region revealed the presence of a putative TATA box at 116 nucleotides upstream of the ATG start codon as well as a putative GC box at −636. Comparison of the DHFR sequences from pyrimethamine-sensitive and pyrimethamine-resistant P. vivax isolates revealed two residue changes: Ser « Arg-58 and Ser « Asn-117. These aa residues correspond to codons 59 and 108 in the P. falciparum DHFR active site in which similar aa substitutions (Cys « Arg-59 and Ser « Asn-108) are associated with pyrimethamine resistance. These findings may explain the intrinsic resistance of some P. vivax isolates to pyrimethamine.