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1.  Analysis of salivary transcripts and antigens of the sand fly Phlebotomus arabicus 
BMC Genomics  2009;10:282.
Sand fly saliva plays an important role in blood feeding and Leishmania 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 Phlebotomus (Adlerius) arabicus, which was recently shown to transmit Leishmania tropica, the causative agent of cutaneous leishmaniasis in Israel.
A cDNA library from salivary glands of P. arabicus 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 P. arabicus to salivary antigens was assessed and many salivary proteins were determined to be antigenic.
This transcriptomic analysis of P. arabicus salivary glands is the first description of salivary proteins of a sand fly in the subgenus Adlerius. Proteomic analysis of P. arabicus 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-Leishmania interactions. Enzymatic and immunological investigations further demonstrate the value of functional transcriptomics in advancing biological and epidemiological research that can impact leishmaniasis.
PMCID: PMC2714351  PMID: 19555500
2.  Detection of genome-wide polymorphisms in the AT-rich Plasmodium falciparum genome using a high-density microarray 
BMC Genomics  2008;9:398.
Genetic mapping is a powerful method to identify mutations that cause drug resistance and other phenotypic changes in the human malaria parasite Plasmodium falciparum. 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.
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.
A large number of mSFP were discovered from the P. falciparum 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 P. falciparum parasite and provide useful resources for mapping important parasite traits.
PMCID: PMC2543026  PMID: 18724869
3.  Insight into the sialome of the castor bean tick, Ixodes ricinus 
BMC Genomics  2008;9:233.
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, Ixodes ricinus.
In the attempt to describe expressed genes and their dynamics throughout the feeding period, we constructed cDNA libraries from four different feeding stages of Ixodes ricinus 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 Ixodes scapularis and I. pacificus transcripts was made.
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.
PMCID: PMC2410133  PMID: 18489795
4.  The midgut transcriptome of Lutzomyia longipalpis: comparative analysis of cDNA libraries from sugar-fed, blood-fed, post-digested and Leishmania infantum chagasi-infected sand flies 
BMC Genomics  2008;9:15.
In the life cycle of Leishmania within the alimentary canal of sand flies the parasites have to survive the hostile environment of blood meal digestion, escape the blood bolus and attach to the midgut epithelium before differentiating into the infective metacyclic stages. The molecular interactions between the Leishmania parasites and the gut of the sand fly are poorly understood. In the present work we sequenced five cDNA libraries constructed from midgut tissue from the sand fly Lutzomyia longipalpis and analyzed the transcripts present following sugar feeding, blood feeding and after the blood meal has been processed and excreted, both in the presence and absence of Leishmania infantum chagasi.
Comparative analysis of the transcripts from sugar-fed and blood-fed cDNA libraries resulted in the identification of transcripts differentially expressed during blood feeding. This included upregulated transcripts such as four distinct microvillar-like proteins (LuloMVP1, 2, 4 and 5), two peritrophin like proteins, a trypsin like protein (Lltryp1), two chymotrypsin like proteins (LuloChym1A and 2) and an unknown protein. Downregulated transcripts by blood feeding were a microvillar-like protein (LuloMVP3), a trypsin like protein (Lltryp2) and an astacin-like metalloprotease (LuloAstacin). Furthermore, a comparative analysis between blood-fed and Leishmania infected midgut cDNA libraries resulted in the identification of the transcripts that were differentially expressed due to the presence of Leishmania in the gut of the sand fly. This included down regulated transcripts such as four microvillar-like proteins (LuloMVP1,2, 4 and 5), a Chymotrypsin (LuloChym1A) and a carboxypeptidase (LuloCpepA1), among others. Upregulated midgut transcripts in the presence of Leishmania were a peritrophin like protein (LuloPer1), a trypsin-like protein (Lltryp2) and an unknown protein.
This transcriptome analysis represents the largest set of sequence data reported from a specific sand fly tissue and provides further information of the transcripts present in the sand fly Lutzomyia longipalpis. This analysis provides the detailed information of molecules present in the midgut of this sand fly and the transcripts potentially modulated by blood feeding and by the presence of the Leishmania parasite. More importantly, this analysis suggests that Leishmania infantum chagasi alters the expression profile of certain midgut transcripts in the sand fly during blood meal digestion and that this modulation may be relevant for the survival and establishment of the parasite in the gut of the fly. Moreover, this analysis suggests that these changes may be occurring during the digestion of the blood meal and not afterwards.
PMCID: PMC2249575  PMID: 18194529
5.  cDNA sequences reveal considerable gene prediction inaccuracy in the Plasmodium falciparum genome 
BMC Genomics  2007;8:255.
The completion of the Plasmodium falciparum 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.
We aimed to obtain cDNA sequences to examine the accuracy of gene prediction in silico. We constructed cDNA libraries from mixed blood stages of P. falciparum 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 versus 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.
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.
PMCID: PMC1978503  PMID: 17662120

Results 1-5 (5)