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Immunoprofiling of the tryptophan-rich antigen family in Plasmodium vivax.

ABSTRACT: Tryptophan-rich antigens (TRAgs) are an antigen family that has been identified in human and rodent malaria parasites. TRAgs have been proposed as candidate antigens for potential vaccines. The Plasmodium vivax TRAg (PvTRAg) family includes 36 members. Each PvTRAg contains a tryptophan-rich (TR) domain in the C-terminal region. In this study, we recombinantly expressed all 36 PvTRAgs using a cell-free expression system, and, for the first time, profiled the IgG antibody responses against all PvTRAgs in the sera from 96 vivax malaria patients and 40 healthy individuals using protein microarray technology. The mean seropositive rate for all PvTRAgs was 60.3%. Among them, nine PvTRAgs were newly identified in this study and showed a seropositive rate of >50%. Five of them, PvTRAg_13, PvTRAg_15, PvTRAg_16, PvTRAg_26, and PvTRAg_29, produced higher levels of IgG antibody, even in low-endemicity countries. In addition, the results of an immunofluorescence analysis suggest that PvTRAgs are, at least in part, associated with caveola-vesicle complexes, a unique structure of P. vivax-infected erythrocytes. The mechanism of formation and the function of these abundant membrane structures are not known. Further investigation aimed at determining the functions of these proteins would lead to a better understanding of the blood-stage biology of P. vivax.

SUBMITTER: Wang B

PROVIDER: S-EPMC4496608 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Immunoprofiling of the tryptophan-rich antigen family in Plasmodium vivax.

Wang Bo B Lu Feng F Cheng Yang Y Chen Jun-Hu JH Jeon Hye-Yoon HY Ha Kwon-Soo KS Cao Jun J Nyunt Myat Htut MH Han Jin-Hee JH Lee Seong-Kyun SK Kyaw Myat Phone MP Sattabongkot Jetsumon J Takashima Eizo E Tsuboi Takafumi T Han Eun-Taek ET

Infection and immunity 20150518 8

Tryptophan-rich antigens (TRAgs) are an antigen family that has been identified in human and rodent malaria parasites. TRAgs have been proposed as candidate antigens for potential vaccines. The Plasmodium vivax TRAg (PvTRAg) family includes 36 members. Each PvTRAg contains a tryptophan-rich (TR) domain in the C-terminal region. In this study, we recombinantly expressed all 36 PvTRAgs using a cell-free expression system, and, for the first time, profiled the IgG antibody responses against all PvT ...[more]

PMID: 25987709

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Project description:BackgroundPlasmodium falciparum dramatically alters the morphology and properties of the infected red blood cells (iRBCs). A large group of exported proteins participate in these parasite-host interactions occurring at the iRBC membrane skeleton. SURFIN4.2 is one of iRBC surface protein that belongs to surface-associated interspersed protein (SURFIN) family. Although the intracellular tryptophan-rich domain (WRD) was proposed to be important for the translocation of SURFINs from Maurer's clefts to iRBC surface, the molecular basis of this observation has yet to be defined. The WRDs of P. falciparum SURFIN proteins and their orthologous Plasmodium vivax subtelomeric transmembrane proteins (PvSTPs) show homology to the intracellular regions of PfEMP1 and Pf332, both of which are involved in RBC membrane skeleton interactions, and contribute to malaria pathology.MethodsTwo transfected lines expressing recombinant SURFINs (NTC-GFP and NTC-4.2WRD2-GFP) of the 3D7 sequence were generated by transfection in P. falciparum. In vitro binding assays were performed by using recombinant WRDs of SURFIN4.2/PvSTP2 and inside-out vesicles (IOVs). The interactions between the recombinant WRDs of SURFIN4.2/PvSTP2 with actin and spectrin were evaluated by the actin spin down assay and an enzyme-linked immunosorbent assay based binding assays, respectively.ResultsThe recombinant SURFINs (NTC-4.2WRD2-GFP), in which the second WRD from SURFIN4.2 was added back to NTC-GFP, show diffused pattern of fluorescence in the iRBC cytosol. Furthermore, WRDs of SURFIN4.2/PvSTP2 were found to directly interact with the IOVs of RBC, with binding affinities ranging from 0.26 to 0.68 μM, values that are comparable to other reported parasite proteins that bind to the RBC membrane skeleton. Further experiments revealed that the second WRD of SURFIN4.2 bound to F-actin (K d = 5.16 μM) and spectrin (K d = 0.51 μM).ConclusionsBecause PfEMP1 and Pf332 also bind to actin and/or spectrin, the authors propose that the interaction between WRD and RBC membrane skeleton might be a common feature of WRD-containing proteins and may be important for the translocation of these proteins from Maurer's clefts to the iRBC surface. The findings suggest a conserved mechanism of host-parasite interactions and targeting this interaction may disrupt the iRBC surface exposure of Plasmodium virulence-related proteins.

Project description:BACKGROUND: Rhoptries are specialized organelles from parasites belonging to the phylum Apicomplexa; they secrete their protein content during invasion of host target cells and are sorted into discrete subcompartments within rhoptry neck or bulb. This distribution is associated with these proteins' role in tight junction (TJ) and parasitophorous vacuole (PV) formation, respectively. METHODS: Plasmodium falciparum RON2 amino acid sequence was used as bait for screening the codifying gene for the homologous protein in the Plasmodium vivax genome. Gene synteny, as well as identity and similarity values, were determined for ron2 and its flanking genes among P. falciparum, P. vivax and other malarial parasite genomes available at PlasmoDB and Sanger Institute databases. Pvron2 gene transcription was determined by RT-PCR of cDNA obtained from the P. vivax VCG-1 strain. Protein expression and localization were assessed by Western blot and immunofluorescence using polyclonal anti-PvRON2 antibodies. Co-localization was confirmed using antibodies directed towards specific microneme and rhoptry neck proteins. RESULTS AND DISCUSSION: The first P. vivax rhoptry neck protein (named here PvRON2) has been identified in this study. PvRON2 is a 2,204 residue-long protein encoded by a single 6,615 bp exon containing a hydrophobic signal sequence towards the amino-terminus, a transmembrane domain towards the carboxy-terminus and two coiled coil ?-helical motifs; these are characteristic features of several previously described vaccine candidates against malaria. This protein also contains two tandem repeats within the interspecies variable sequence possibly involved in evading a host's immune system. PvRON2 is expressed in late schizonts and localized in rhoptry necks similar to what has been reported for PfRON2, which suggests its participation during target cell invasion. CONCLUSIONS: The identification and partial characterization of the first P. vivax rhoptry neck protein are described in the present study. This protein is homologous to PfRON2 which has previously been shown to be associated with PfAMA-1, suggesting a similar role for PvRON2.

Project description:BackgroundPlasmodium ovale has two different subspecies: P. ovale curtisi and P. ovale wallikeri, which may be distinguished by the gene potra encoding P. ovale tryptophan-rich antigen. The sequence and size of potra gene was variable between the two P. ovale spp., and more fragment sizes were found compared to previous studies. Further information about the diversity of potra genes in these two P. ovale spp. will be needed.MethodsA total of 110 dried blood samples were collected from the clinical patients infected with P. ovale, who all returned from Africa in Henan Province in 2011-2016. The fragments of potra were amplified by nested PCR. The sizes and species of potra gene were analysed after sequencing, and the difference between the isolates were analysed with the alignment of the amino acid sequences. The phylogenetic tree was constructed by neighbour-joining to determine the genetic relationship among all the isolates. The distribution of the isolates was analysed based on the origin country.ResultsTotally 67 samples infected with P. o. wallikeri, which included 8 genotypes of potra, while 43 samples infected with P. o. curtisi including 3 genotypes of potra. Combination with the previous studies, P. o. wallikeri had six sizes, 227, 245, 263, 281, 299 and 335 bp, and P. o. curtisi had four sizes, 299, 317, 335 and 353 bp, the fragment sizes of 299 and 335 bp were the overlaps between the two species. Six amino acid as one unit was firstly used to analyse the amino acid sequence of potra. Amino acid sequence alignment revealed that potra of P. o. wallikeri differed in two amino acid units, MANPIN and AITPIN, while potra of P. o. curtisi differed in amino acid units TINPIN and TITPIS. Combination with the previous studies, there were ten subtypes of potra exiting for P. o. wallikeri and four subtypes for P. o. curtisi. The phylogenetic tree showed that 11 isolates were divided into two clusters, P. o. wallikeri which was then divided into five sub-clusters, and P. o. curtisi which also formed two sub-clusters with their respective reference sequences. The genetic relationship of the P. ovale spp. mainly based on the number of the dominant amino acid repeats, the number of MANPIN, AITPIN, TINPIN or TITPIS. The genotype of the 245 bp size for P. o. wallikeri and that of the 299 and 317 bp size for P. o. curtisi were commonly exiting in Africa.ConclusionThis study further proved that more fragment sizes were found, P. o. wallikeri had six sizes, P. o. curtisi had four sizes. There were ten subtypes of potra exiting for P. o. wallikeri and four subtypes for P. o. curtisi. The genetic polymorphisms of potra provided complementary information for the gene tracing of P. ovale spp. in the malaria elimination era.

Dataset Information

Immunoprofiling of the tryptophan-rich antigen family in Plasmodium vivax.

Publications

Immunoprofiling of the tryptophan-rich antigen family in Plasmodium vivax.

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