Erythrocyte binding activity of the recombinant PkTRAgs by Cell-ELISA.

<p><b>(A)</b> Cell-ELISA. Each well of a microtiter plate containing ~ 1 million of erythrocytes was allowed to interact with different concentration (0–2 μM) of histidine-tagged recombinant proteins. Bacterial thioredoxin and PvTRAg35.2 were used as negative and positive controls, respectively. Plate was developed with monoclonal anti-His₆ antibody as described in the text. Data shown here are mean ± S.D. of at least three experiments. <b>(B)</b> Competition assay. For this assay, 200 nM of each of the recombinant histidine-tagged PkTRAg was mixed with increasing concentration (0–2 μM) of respective untagged PkTRAg. This mixture was then added to the wells of the ELISA plate containing ~ 1 million human erythrocytes. Plate was developed with monoclonal anti-His₆ antibody as described in the text. Data shown here are mean ± S.D. of at least three experiments. <b>(C)</b> Antibody inhibition assay. Different dilutions of polyclonal rabbit sera raised against individual PkTRAg were pre-incubated with the respective recombinant PkTRAg and then added to the well of a microtiter plate containing ~ 1 million human erythrocytes. No antibody i.e. only PBS containing respective histidine-tagged PkTRAg protein was taken as positive control. The plates were developed with mouse anti-His₆ monoclonal antibody as described in the text. Error bar indicates the standard deviation of mean from three experiments.</p

Erythrocyte binding activity of PkTRAgs expressed on the surface of CHO-K1 cells.

<p><b>(A</b>) Expression of PkTRAgs on the surface of CHO-K1 cells. The CHO-K1 cells transfected with pRE4-PkTRAgs, pRE4-PvTRAg53.7, and pRE4-PvRII were stained with mouse monoclonal antibodies DL6 directed against C-terminal of the <i>Herpes simplex</i> glycoprotein D sequences and then stained with Alexa fluor 488 secondary antibodies. Upper panel shows the merged fluorescence images (Blue for DNA counterstained with DAPI, and green for surface expression of proteins). Lower panel shows the binding of human erythrocytes to the transfected CHO-K1 cells. A single transfected CHO-K1 cell attached with more than five RBCs was considered a rosette. The numbers of rosettes were counted in 20 fields at a 200x magnification. CHO-K1 cells transfected with PvTRAg53.7 and PvRII were used as negative and positive controls, respectively. <b>(B)</b> Specificity of PkTRAgs (expressed on surface of CHO-K1 cells) binding to human erythrocytes by competition assay. The human erythrocytes were pre-incubated with different concentrations of PkTRAgs (0–10 μM) before their incubation with transfected CHO-K1 cells to form the rosettes. Erythrocyte binding of CHO cell expressing PkTRAgs with RBCs incubated with PBS only (zero concentration) was taken as positive control. <b>(C)</b> Inhibition of erythrocyte binding to PkTRAgs expressed on surface of CHO-K1 cells by polyclonal antibodies. Transfected CHO-K1 cells were pre-incubated with different dilutions of rabbit sera raised against the respective PkTRAg or with pre-immune sera at 1:10 dilution. The percent binding was determined relative to no antibodies (RPMI only). Data shown are the Mean ± standard deviation of three experiments.</p

Proteomic Identification and Analysis of Arginine-Methylated Proteins of <i>Plasmodium falciparum</i> at Asexual Blood Stages

<i>Plasmodium falciparum</i> undergoes a tightly regulated developmental process in human erythrocytes, and recent studies suggest an important regulatory role of post-translational modifications (PTMs). As compared with <i>Plasmodium</i> phosphoproteome, little is known about other PTMs in the parasite. In the present study, we performed a global analysis of asexual blood stages of <i>Plasmodium falciparum</i> to identify arginine-methylated proteins. Using two different methyl arginine-specific antibodies, we immunoprecipitated the arginine-methylated proteins from the stage-specific parasite lysates and identified 843 putative arginine-methylated proteins by LC–MS/MS. Motif analysis of the protein sequences unveiled that the methylation sites are associated with the previously known methylation motifs such as G<b>R</b>x/<b>R</b>Gx, <b>R</b>xG, Gxx<b>R</b>, or Wxxx<b>R</b>. We identified <i>Plasmodium</i> homologues of known arginine-methylated proteins in trypanosomes, yeast, and human. Hydrophilic interaction liquid chromatography (HILIC) was performed on the immunoprecipitates from the trophozoite stage to enrich arginine-methylated peptides. Mass spectrometry analysis of immunoprecipitated and HILIC fractions identified 55 arginine-methylated peptides having 62 methylated arginine sites. Functional classification revealed that the arginine-methylated proteins are involved in RNA metabolism, protein synthesis, intracellular protein trafficking, proteolysis, protein folding, chromatin organization, hemoglobin metabolic process, and several other functions. Summarily, the findings suggest that protein methylation of arginine residues is a widespread phenomenon in <i>Plasmodium</i>, and the PTM may play an important regulatory role in a diverse set of biological pathways, including host–pathogen interactions

PkTRAgs inhibit <i>P</i>.<i>falciparum</i> growth in in-vitro culture.

<p>Parasite culture at late trophozoite/early schizont stage was incubated with 20 μM of PkTRAg38.3, PkTRAg40.1, PkTRAg67.1 and other controls. After 48 h, parasitemia was determined by ethidium bromide staining and measured by flowcytometry. Representative dot plots are shown in upper panel; <b>a</b>. unstained erythrocytes, <b>b</b>. uninfected erythrocytes, <b>c</b>. infected erythrocytes, <b>d</b>. infected erythrocytes with PBS, <b>e</b>. infected erythrocytes with PkTRAg38.3, <b>f</b>. infected erythrocytes with PkTRAg40.1, and <b>g</b>. infected erythrocytes with PkTRAg67.1. Bar diagram shows the percentage of parasite growth inhibition. The data is depicted as a mean ± SD for two triplicate experiments.</p

Recognition of Human Erythrocyte Receptors by the Tryptophan-Rich Antigens of Monkey Malaria Parasite <i>Plasmodium knowlesi</i>

<div><p>Background</p><p>The monkey malaria parasite <i>Plasmodium knowlesi</i> also infect humans. There is a lack of information on the molecular mechanisms that take place between this simian parasite and its heterologous human host erythrocytes leading to this zoonotic disease. Therefore, we investigated here the binding ability of <i>P</i>. <i>knowlesi</i> tryptophan-rich antigens (PkTRAgs) to the human erythrocytes and sharing of the erythrocyte receptors between them as well as with other commonly occurring human malaria parasites.</p><p>Methods</p><p>Six PkTRAgs were cloned and expressed in <i>E</i>.<i>coli</i> as well as in mammalian CHO-K1 cell to determine their human erythrocyte binding activity by cell-ELISA, and in-vitro rosetting assay, respectively.</p><p>Results</p><p>Three of six PkTRAgs (PkTRAg38.3, PkTRAg40.1, and PkTRAg67.1) showed binding to human erythrocytes. Two of them (PkTRAg40.1 and PkTRAg38.3) showed cross-competition with each other as well as with the previously described <i>P</i>.<i>vivax</i> tryptophan-rich antigens (PvTRAgs) for human erythrocyte receptors. However, the third protein (PkTRAg67.1) utilized the additional but different human erythrocyte receptor(s) as it did not cross-compete for erythrocyte binding with either of these two PkTRAgs as well as with any of the PvTRAgs. These three PkTRAgs also inhibited the <i>P</i>.<i>falciparum</i> parasite growth in in-vitro culture, further indicating the sharing of human erythrocyte receptors by these parasite species and the biological significance of this receptor-ligand interaction between heterologous host and simian parasite.</p><p>Conclusions</p><p>Recognition and sharing of human erythrocyte receptor(s) by PkTRAgs with human parasite ligands could be part of the strategy adopted by the monkey malaria parasite to establish inside the heterologous human host.</p></div

Multiple sequence alignment of <i>P</i>. <i>knowlesi</i> tryptophan rich antigens (PkTRAgs) using GeneDoc software.

<p><i>Plasmodium knowlesi</i> gene sequence retrieval was done at <a href="http://www.plasmodb.org" target="_blank">www.Plasmodb.org</a>. Conserved residues are shaded in grey and tryptophan residues are highlighted in black. Position of amino acid residue numbers is shown on the right hand side of the sequence and Plasmodb ID on left hand side. Dashes represent the gaps generated by multiple sequence alignment.</p

Additional file 2: of Readiness for antimicrobial resistance (AMR) surveillance in Pakistan; a model for laboratory strengthening

Scoring method used including percentage scores for each question in a given category. Total scores and percentage scores for each category of laboratory capacity for both public and private sector are also presented. Presents the scores including total percentage of public and private sector in each category as well as the individual scores in the different components that constituted a particular category. (DOCX 25 kb

Sequence diversity in the central repeat region of PfCSP.

<p>(<b>A</b>) Representation of the variation in sequence repeats in the central region of the CSP in 161 samples. The sequences of eight laboratory-adapted <i>P. falciparum</i> strains [Dd2 (Indochina), K1 (Thailand), MAD20 (Papua New Guinea), Wellcome (West Africa), 7G8 (Brazil), HB3 (Honduras), 3D7 (The Netherlands) and RO33 (Ghana)] are shown here for comparison. The NANP repeats are indicated as “1 with gray shade", NVDP repeats are indicated as “2 with black shade" and all other repeats are un-shaded. Numbers on the right indicate numbers of samples belonging to that particular haplotype. Numbers above the alignment are amino acid position with reference to 3D7 sequence. Dots represent amino acid positions identical to the 3D7 haplotype, whereas those different are indicated. Dashes have been inserted for maximum alignment. C, community cohort; H, hospital cohort; T, total; WC, Wellcome. (<b>B</b>) Distribution of repeats in the central region of the CSP in 161 samples.</p

Global population structure of csp gene.

<p>A minimal spanning tree (MST) generated using BioNumerics software version 6.6 showing the relationship among all the 117 haplotypes based on the Th2R/Th3R sequences of the CSP from worldwide isolates [Asia, n = 974; South America, n = 181 and Africa, n = 184)]. Each circle represents an individual haplotype and the size of the circle is proportional to the number of isolates belonging to that haplotype (also shown as pie). The lines connecting the circles are branch length and are red if two haplotypes differ by only one mutation, blue if differ by 2 mutations, solid black if differ by 3 mutations, dashed black if differ by 4 mutations and gray if they differ by more than 4 mutations. Numbers outside the circles indicate haplotypes H1 to H117. The Dd2, 3D7, HB3, 7G8 and MAD20 type sequences are highlighted in bold. The haplotype pairs H55 & H58; H57 & H87; H60 & H106 and H97 & H98 are identical at amino acid level; but have one synonymous mutation. H1 to H24 are the same haplotypes we observed in our study sites and shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043430#pone-0043430-g003" target="_blank"><b>Fig </b><b>3B</b></a>. Please refer to <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043430#pone.0043430.s004" target="_blank">Table S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043430#pone.0043430.s006" target="_blank">Table S3</a></b> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043430#pone.0043430.s001" target="_blank">Fig S1</a></b> for more details on these sequence haplotypes and their country-wise distributions.</p

Sequence diversity in the N- and C-terminal non-repeat region of PfCSP.

<p>(<b>A</b>) Sequence alignment showing polymorphisms in the non-repeat N-terminal T cell epitope region (amino acid residue 84 to 104) of CSP in 161 samples. The shaded region (amino acid residue 93 to 97) is a conserved motif involved in sporozoite invasion of mosquito salivary gland as well as in binding to hepatocytes prior to invasion. (<b>B</b>) Sequence alignment showing polymorphisms in the non-repeat C-terminal T cell epitope regions (Th2R spanning from amino acid residues 311 to 327 and Th3R from amino acid residues 341 to 364) of CSP in 179 samples. The highly conserved sequences flanking the Th2R and Th3R domains are shaded grey. The eight laboratory-adapted strains are also included in this alignment. Numbers on the right indicate numbers of samples belonging to that particular haplotype. Dots represent amino acid positions identical to the 3D7 haplotype, whereas those different are indicated. C, community cohort; H, hospital cohort; T, total; WC, Wellcome.</p