Insight into the sialome of the castor bean tick, Ixodes ricinus

<p>Abstract</p> <p>Background</p> <p>In recent years, there have been several sialome projects revealing transcripts expressed in the salivary glands of ticks, which are important vectors of several human diseases. Here, we focused on the sialome of the European vector of Lyme disease, <it>Ixodes ricinus</it>.</p> <p>Results</p> <p>In the attempt to describe expressed genes and their dynamics throughout the feeding period, we constructed cDNA libraries from four different feeding stages of <it>Ixodes ricinus </it>females: unfed, 24 hours after attachment, four (partially fed) and seven days (fully engorged) after attachment. Approximately 600 randomly selected clones from each cDNA library were sequenced and analyzed. From a total 2304 sequenced clones, 1881 sequences forming 1274 clusters underwent subsequent functional analysis using customized bioinformatics software. Clusters were sorted according to their predicted function and quantitative comparison among the four libraries was made. We found several groups of over-expressed genes associated with feeding that posses a secretion signal and may be involved in tick attachment, feeding or evading the host immune system. Many transcripts clustered into families of related genes with stage-specific expression. Comparison to <it>Ixodes scapularis </it>and <it>I. pacificus </it>transcripts was made.</p> <p>Conclusion</p> <p>In addition to a large number of homologues of the known transcripts, we obtained several novel predicted protein sequences. Our work contributes to the growing list of proteins associated with tick feeding and sheds more light on the dynamics of the gene expression during tick feeding. Additionally, our results corroborate previous evidence of gene duplication in the evolution of ticks.</p

cDNA sequences reveal considerable gene prediction inaccuracy in the Plasmodium falciparum genome

<p>Abstract</p> <p>Background</p> <p>The completion of the <it>Plasmodium falciparum </it>genome represents a milestone in malaria research. The genome sequence allows for the development of genome-wide approaches such as microarray and proteomics that will greatly facilitate our understanding of the parasite biology and accelerate new drug and vaccine development. Designing and application of these genome-wide assays, however, requires accurate information on gene prediction and genome annotation. Unfortunately, the genes in the parasite genome databases were mostly identified using computer software that could make some erroneous predictions.</p> <p>Results</p> <p>We aimed to obtain cDNA sequences to examine the accuracy of gene prediction <it>in silico</it>. We constructed cDNA libraries from mixed blood stages of <it>P. falciparum </it>parasite using the SMART cDNA library construction technique and generated 17332 high-quality expressed sequence tags (EST), including 2198 from primer-walking experiments. Assembly of our sequence tags produced 2548 contigs and 2671 singletons <it>versus </it>5220 contigs and 5910 singletons when our EST were assembled with EST in public databases. Comparison of all the assembled EST/contigs with predicted CDS and genomic sequences in the PlasmoDB database identified 356 genes with predicted coding sequences fully covered by EST, including 85 genes (23.6%) with introns incorrectly predicted. Careful automatic software and manual alignments found an additional 308 genes that have introns different from those predicted, with 152 new introns discovered and 182 introns with sizes or locations different from those predicted. Alternative spliced and antisense transcripts were also detected. Matching cDNA to predicted genes also revealed silent chromosomal regions, mostly at subtelomere regions.</p> <p>Conclusion</p> <p>Our data indicated that approximately 24% of the genes in the current databases were predicted incorrectly, although some of these inaccuracies could represent alternatively spliced transcripts, and that more genes than currently predicted have one or more additional introns. It is therefore necessary to annotate the parasite genome with experimental data, although obtaining complete cDNA sequences from this parasite will be a formidable task due to the high AT nature of the genome. This study provides valuable information for genome annotation that will be critical for functional analyses.</p

Analysis of salivary transcripts and antigens of the sand fly Phlebotomus arabicus

<p>Abstract</p> <p>Background</p> <p>Sand fly saliva plays an important role in blood feeding and <it>Leishmania </it>transmission as it was shown to increase parasite virulence. On the other hand, immunity to salivary components impedes the establishment of infection. Therefore, it is most desirable to gain a deeper insight into the composition of saliva in sand fly species which serve as vectors of various forms of leishmaniases. In the present work, we focused on <it>Phlebotomus (Adlerius) arabicus</it>, which was recently shown to transmit <it>Leishmania tropica</it>, the causative agent of cutaneous leishmaniasis in Israel.</p> <p>Results</p> <p>A cDNA library from salivary glands of <it>P. arabicus </it>females was constructed and transcripts were sequenced and analyzed. The most abundant protein families identified were SP15-like proteins, ParSP25-like proteins, D7-related proteins, yellow-related proteins, PpSP32-like proteins, antigen 5-related proteins, and 34 kDa-like proteins. Sequences coding for apyrases, hyaluronidase and other putative secreted enzymes were also represented, including endonuclease, phospholipase, pyrophosphatase, amylase and trehalase. Mass spectrometry analysis confirmed the presence of 20 proteins predicted to be secreted in the salivary proteome. Humoral response of mice bitten by <it>P. arabicus </it>to salivary antigens was assessed and many salivary proteins were determined to be antigenic.</p> <p>Conclusion</p> <p>This transcriptomic analysis of <it>P. arabicus </it>salivary glands is the first description of salivary proteins of a sand fly in the subgenus <it>Adlerius</it>. Proteomic analysis of <it>P. arabicus </it>salivary glands produced the most comprehensive account in a single sand fly species to date. Detailed information and phylogenetic relationships of the salivary proteins are provided, expanding the knowledge base of molecules that are likely important factors of sand fly-host and sand fly-<it>Leishmania </it>interactions. Enzymatic and immunological investigations further demonstrate the value of functional transcriptomics in advancing biological and epidemiological research that can impact leishmaniasis.</p

Dissecting the Loci of Low-Level Quinine Resistance in Malaria Parasites

Quinine (QN) remains effective against Plasmodium falciparum, but its decreasing efficacy is documented from different continents. Multiple genes are likely to contribute to the evolution of QN resistance. To locate genes contributing to QN response variation, we have searched a P. falciparum genetic cross for quantitative trait loci (QTL). Results identify additive QTL in segments of chromosomes (Chrs) 13, 7 and 5, and pairwise effects from two additional loci of Chrs 9 and 6 that interact, respectively, with the QTL of Chrs 13 and 7. The mapped segments of Chrs 7 and 5 contain pfcrt, the determinant of chloroquine resistance (CQR), and pfmdr1, a gene known to affect QN responses. Association of pfcrt with a QTL of QN resistance supports anecdotal evidence for an evolutionary relationship between CQR and reduced QN sensitivity. The Chr 13 segment contains several candidate genes, one of which (pfnhe-1) encodes a putative Na+/H+ exchanger. A repeat polymorphism in pfnhe-1 shows significant association with low QN response in a collection of P. falciparum strains from Asia, Africa and Central and South America. Dissection of the genes and modifiers involved in QN response will require experimental strategies that can evaluate multiple genes from different chromosomes in combination

Detection of genome-wide polymorphisms in the AT-rich Plasmodium falciparum genome using a high-density microarray

<p>Abstract</p> <p>Background</p> <p>Genetic mapping is a powerful method to identify mutations that cause drug resistance and other phenotypic changes in the human malaria parasite <it>Plasmodium falciparum</it>. For efficient mapping of a target gene, it is often necessary to genotype a large number of polymorphic markers. Currently, a community effort is underway to collect single nucleotide polymorphisms (SNP) from the parasite genome. Here we evaluate polymorphism detection accuracy of a high-density 'tiling' microarray with 2.56 million probes by comparing single feature polymorphisms (SFP) calls from the microarray with known SNP among parasite isolates.</p> <p>Results</p> <p>We found that probe GC content, SNP position in a probe, probe coverage, and signal ratio cutoff values were important factors for accurate detection of SFP in the parasite genome. We established a set of SFP calling parameters that could predict mSFP (SFP called by multiple overlapping probes) with high accuracy (≥ 94%) and identified 121,087 mSFP genome-wide from five parasite isolates including 40,354 unique mSFP (excluding those from multi-gene families) and ~18,000 new mSFP, producing a genetic map with an average of one unique mSFP per 570 bp. Genomic copy number variation (CNV) among the parasites was also cataloged and compared.</p> <p>Conclusion</p> <p>A large number of mSFP were discovered from the <it>P. falciparum </it>genome using a high-density microarray, most of which were in clusters of highly polymorphic genes at chromosome ends. Our method for accurate mSFP detection and the mSFP identified will greatly facilitate large-scale studies of genome variation in the <it>P. falciparum </it>parasite and provide useful resources for mapping important parasite traits.</p

Short Report: Novel dhps and pfcrt Polymorphisms in Plasmodium falciparum Detected by Heteroduplex Tracking Assay

Several single nucleotide polymorphisms (SNPs) have been linked to antimalarial drug resistance in Plasmodium falciparum . However, standard polymerase chain reaction (PCR) methods to detect these polymorphisms are unable to detect SNPs in variants representing < 20% of the parasites in a mixed infection, nor can they detect polymorphisms at nearby loci. Here we use heteroduplex tracking assays (HTAs) to analyze dhps540 mutations in 96 samples from Peru and pfcrt76 mutations in 70 samples from China. All samples had been previously analyzed by standard PCR. We detected drug-resistant minority variants and two novel non-synonymous pfcrt mutations in China. In Peru, we found no drug-resistant minority variants and a synonymous mutation in dhps. Thus, even in regions of low malaria transmission, HTA assays are more informative than PCR with agarose gel electrophoresis

Recombination Hotspots and Population Structure in Plasmodium falciparum

Understanding the influences of population structure, selection, and recombination on polymorphism and linkage disequilibrium (LD) is integral to mapping genes contributing to drug resistance or virulence in Plasmodium falciparum. The parasite's short generation time, coupled with a high cross-over rate, can cause rapid LD break-down. However, observations of low genetic variation have led to suggestions of effective clonality: selfing, population admixture, and selection may preserve LD in populations. Indeed, extensive LD surrounding drug-resistant genes has been observed, indicating that recombination and selection play important roles in shaping recent parasite genome evolution. These studies, however, provide only limited information about haplotype variation at local scales. Here we describe the first (to our knowledge) chromosome-wide SNP haplotype and population recombination maps for a global collection of malaria parasites, including the 3D7 isolate, whose genome has been sequenced previously. The parasites are clustered according to continental origin, but alternative groupings were obtained using SNPs at 37 putative transporter genes that are potentially under selection. Geographic isolation and highly variable multiple infection rates are the major factors affecting haplotype structure. Variation in effective recombination rates is high, both among populations and along the chromosome, with recombination hotspots conserved among populations at chromosome ends. This study supports the feasibility of genome-wide association studies in some parasite populations

Multiple Transporters Associated with Malaria Parasite Responses to Chloroquine and Quinine

Mutations and/or overexpression of various transporters are known to confer drug resistance in a variety of organisms. In the malaria parasite Plasmodium falciparum, a homologue of P-glycoprotein, PfMDR1, has been implicated in responses to chloroquine (CO), quinine (ON) and other drugs, and a putative transporter, PfCRT, was recently demonstrated to be the key molecule in CO resistance. However, other unknown molecules are probably involved, as different parasite clones carrying the same pfcrt and pfmdr1 alleles show a wide range of quantitative responses to CO and ON. Such molecules may contribute to increasing incidences of ON treatment failure, the molecular basis of which is not understood. To identify additional genes involved in parasite CO and ON responses, we assayed the in vitro susceptibilities of 97 culture-adapted cloned isolates to CO and ON and searched for single nucleotide polymorphisms (SNPs) in DNA encoding 49 putative transporters (total 113 kb) and in 39 housekeeping genes that acted as negative controls. SNPs in 11 of the putative transporter genes, including pfcrt and pfmdr1, showed significant associations with decreased sensitivity to CQ and/or ON in P. faliparum. Significant linkage disequilibria within and between these genes were also detected, suggesting interactions among the transporter genes. This study provides specific leads for better understanding of complex drug resistances in malaria parasite

Lack of allele-specific efficacy of a bivalent AMA1 malaria vaccine

<p>Abstract</p> <p>Background</p> <p>Extensive genetic diversity in vaccine antigens may contribute to the lack of efficacy of blood stage malaria vaccines. Apical membrane antigen-1 (AMA1) is a leading blood stage malaria vaccine candidate with extreme diversity, potentially limiting its efficacy against infection and disease caused by <it>Plasmodium falciparum </it>parasites with diverse forms of AMA1.</p> <p>Methods</p> <p>Three hundred Malian children participated in a Phase 2 clinical trial of a bivalent malaria vaccine that found no protective efficacy. The vaccine consists of recombinant AMA1 based on the 3D7 and FVO strains of <it>P. falciparum </it>adjuvanted with aluminum hydroxide (AMA1-C1). The gene encoding AMA1 was sequenced from <it>P. falciparum </it>infections experienced before and after immunization with the study vaccine or a control vaccine. Sequences of <it>ama1 </it>from infections in the malaria vaccine and control groups were compared with regard to similarity to the vaccine antigens using several measures of genetic diversity. Time to infection with parasites carrying AMA1 haplotypes similar to the vaccine strains with respect to immunologically important polymorphisms and the risk of infection with vaccine strain haplotypes were compared.</p> <p>Results</p> <p>Based on 62 polymorphic AMA1 residues, 186 unique <it>ama1 </it>haplotypes were identified among 315 <it>ama1 </it>sequences that were included in the analysis. Eight infections had <it>ama1 </it>sequences identical to 3D7 while none were identical to FVO. Several measures of genetic diversity showed that <it>ama1 </it>sequences in the malaria vaccine and control groups were comparable both at baseline and during follow up period. Pre- and post-immunization <it>ama1 </it>sequences in both groups all had a similar degree of genetic distance from FVO and 3D7 <it>ama1</it>. No differences were found in the time of first clinical episode or risk of infection with an AMA1 haplotype similar to 3D7 or FVO with respect to a limited set of immunologically important polymorphisms found in the cluster 1 loop of domain I of AMA1.</p> <p>Conclusion</p> <p>This Phase 2 trial of a bivalent AMA1 malaria vaccine found no evidence of vaccine selection or strain-specific efficacy, suggesting that the extreme genetic diversity of AMA1 did not account for failure of the vaccine to provide protection.</p