Draft genome of Brugia pahangi: high similarity between B. pahangi and B. malayi.

BACKGROUND: Efforts to completely eradicate lymphatic filariasis from human population may be challenged by the emergence of Brugia pahangi as another zoonotic lymphatic filarial nematode. In this report, a genomic study was conducted to understand this species at molecular level. METHODS: After blood meal on a B. pahangi-harbouring cat, the Aedes togoi mosquitoes were maintained to harvest infective third stage larvae, which were then injected into male Mongolian gerbils. Subsequently, adult B. pahangi were obtained from the infected gerbil for genomic DNA extraction. Sequencing and subsequently, construction of genomic libraries were performed. This was followed by genomic analyses and gene annotation analysis. By using archived protein sequences of B. malayi and a few other nematodes, clustering of gene orthologs and phylogenetics were conducted. RESULTS: A total of 9687 coding genes were predicted. The genome of B. pahangi shared high similarity to that B. malayi genome, particularly genes annotated to fundamental processes. Nevertheless, 166 genes were considered to be unique to B. pahangi, which may be responsible for the distinct properties of B. pahangi as compared to other filarial nematodes. In addition, 803 genes were deduced to be derived from Wolbachia, an endosymbiont bacterium, with 44 of these genes intercalate into the nematode genome. CONCLUSIONS: The reporting of B. pahangi draft genome contributes to genomic archive. Albeit with high similarity to B. malayi genome, the B. pahangi-unique genes found in this study may serve as new focus to study differences in virulence, vector selection and host adaptability among different Brugia spp.

Rapid Differentiation of Filariae in Unstained and Stained Paraffin-Embedded Sections by a High-Resolution Melting Analysis PCR Assay.

BACKGROUND: Apart from infection with human filariae, zoonotic filariasis also occurs worldwide, and the numbers of cases have been increasing steadily. Diagnosis of intact filariae in tissues or organs depends on histological identification. The morphology of parasites in tissue-embedded sections is poor and shows high levels of homoplasy. Thus, the use of morphological characteristics in taxonomic studies is difficult and may not allow a specific diagnosis. METHODS: Here we report the use of real-time PCR with high-resolution melting analysis (HRM) to detect and identify Brugia malayi, Brugia pahangi, Wuchereria bancrofti, and Dirofilaria immitis in paraffin-embedded sections. Assay specificity was determined using other tissue-dwelling parasites, Angiostrongylus cantonensis, Gnathostoma spinigerum, and Cysticercus cellulosae. We also developed a quick paraffin removal protocol. RESULTS: Both human and animal filariae in formalin-fixed paraffin-embedded sections (FFPES) were diagnosed and identified rapidly, whereas other parasites were negative. There was no difference in the melting temperature of products amplified from filarial DNA obtained from unstained FFPES and Hematoxylin & Eosin-stained sections. Therefore, the DNA extraction protocols developed in this study could be used for real-time PCR with HRM. CONCLUSIONS: We report the successful application of a HRM-PCR assay to differentiate four filarial parasites in FFPES, thus providing the pathologist with an effective alternative diagnostic procedure. Furthermore, the quick paraffin removal protocol developed could shorten the duration and number of steps required for paraffin removal using a standard protocol.

A gene family of cathepsin L-like proteases of filarial nematodes are associated with larval molting and cuticle and eggshell remodeling.

Cysteine proteinases are involved in a variety of important biological processes and have been implicated in molting and tissue remodeling in free living and parasitic nematodes. We show that in the lymphatic filarial nematode Brugia pahangi molting of third-stage larvae (L3) to fourth-stage larvae is dependent on the activity of a cathepsin L-like cysteine protease (CPL), which can be detected in the excretory/secretory (ES) products of molting L3. Directed cloning of a cysteine protease gene in B. pahangi and analysis of the expressed sequence tag (EST) and genomic sequences of the closely related human lymphatic filarial nematode Brugia malayi have identified a family of CPLs. One group of these enzymes, Bm-cpl-1, -4, -5 and Bp-cpl-4, is highly expressed in the B. malayi and B. pahangi infective L3 larvae. Immunolocalization indicates that the corresponding enzymes are synthesized and stored in granules of the glandular esophagus of L3 and released during the molting process. Functional analysis of these genes in Brugia and closely related CPL genes identified in the filarial nematode Onchocerca volvulus and the free living model nematode Caenorhabditis elegans indicate that these genes are also involved in cuticle and eggshell remodeling.

Differential diagnosis of human lymphatic filariasis using PCR-RFLP.

Filariasis is still a public health problem in tropical countries. The most common causative agents of human filariasis are Wuchereria bancrofti and Brugia malayi. Traditional methods used to detect filarial parasites in human, animal and vector populations are tedious, time consuming, and confer little guarantee of sensitivity and species specificity. We have developed a rapid and specific method to detect filarial parasite DNAs in blood and mosquito samples using the polymerase chain reaction (PCR) technique. The primers used are MF/F and MF/R which amplify a 1.5 kb glutathione peroxidase gene of filarial worms. Using the restriction fragment length polymorphism (RFLP) technique, these PCR products will be further digested with restriction enzymes either Hpa I, Pst I, Alu I or Hinf I to differentiate the genus of filaria. This PCR-RFLP technique can be apply to use in diagnosis and to differentiate between species of filaria in humans the reservoir host and the mosquito vector in endemic areas

A novel member of the transforming growth factor-beta (TGF-beta) superfamily from the filarial nematodes Brugia malayi and B. pahangi.

Transforming growth factor-beta (TGF-beta) superfamily genes encode products controlling pattern formation, cell differentiation, and immune-mediated inflammation. Members of this superfamily are known in multicellular organisms from mammals to the model nematode Caenorhabditis elegans. Using PCR with oligonucleotides complementary to highly conserved motifs in the TGF-beta superfamily, we first isolated a genomic clone from the filarial nematode Brugia malayi. This gene, termed Bm-tgh-1 (TGF-beta homolog-1), spans 2.5 kb of genomic DNA and contains seven exons. Transcripts of this gene are poorly represented in cDNA libraries, but a full-length cDNA was isolated by RACE from B. pahangi (Bp-tgh-1). The tgh-1 genes from the two species are >98% identical at the nucleotide and amino acid levels, differing at 18/1576 base pairs and 5/428 amino acids; all nonsynonymous substitutions are in the long N-terminal propeptide. They show a high level of similarity throughout all seven exons to a C. elegans gene on cosmid T25F10. Homology to other members of the TGF-beta superfamily is restricted to the C-terminal domain which contains the mature active protein. Key features shared with other members of the superfamily include the tetrabasic proteolytic cleavage site to release an active C-terminal peptide, seven cysteines arrayed in identical fashion, and conserved sequence motifs. tgh-1 is most similar to the BMP-1 subfamily involved in developmental signaling in nematodes, insects, and vertebrates. RT-PCR on first-strand cDNA from both Brugia species, with primers specific to the 3' end, showed that tgh-1 is not expressed in the microfilarial stage, but is detectable in the mosquito-derived infective larvae and is maximal in maturing parasites around the time of molting in the mammalian host. Adult parasites show a relatively low level of expression. The identification of tgh-1, and its preferential expression in developing parasites, suggests that it may be involved in key developmental events in the complex filarial life cycle.

A member of the TGF-beta receptor gene family in the parasitic nematode Brugia pahangi.

The full length cDNA sequence of a Type I transforming growth factor-beta (TGF-beta) receptor has been isolated from the filarial parasitic nematode Brugia pahangi. This new gene, designated Bp-trk-1, encodes a predicted 645 amino acid sequence with an N-terminal hydrophobic stretch which may act as a signal peptide. The extracellular portion (residues 15-187) is cysteine-rich and has three potential N-glycosylation sites. At positions 250-255 the protein contains the glycine-serine rich motif characteristic of Type I receptors. The closest homologue is a Caenorhabditis elegans gene (Q09488) in cosmid C32D5.2 which shares 67% amino acid identity with Bp-trk-1 in the most conserved kinase domain (aa 259-482). Other type I receptors such as C. elegans daf-1 and Drosophila tkv show 38-53% identity in the same region. Some residues conserved in Drosophila and vertebrates are not present in the B. pahangi sequence. RT-PCR amplification has been used to show that the transcript is expressed in the three main stages of the B. pahangi life cycle: microfilariae, infective larvae and adults. The ligand remains unknown at this time but is likely to be most similar to that for C. elegans Q09488.

Expression of recombinant microfilarial chitinase and analysis of domain function.

A family of chitinase isozymes was previously characterized from the microfilariae of Brugia malayi and Brugia pahangi. The expression of these enzymes correlates with the onset of microfilarial infectivity for the mosquito vector. To study the role of chitinase activity in filarial transmission, the p70 chitinase from Brugia malayi was cloned and expressed in two forms: a full-length product of approximately 62 kDa and a truncated product of 43 kDa containing only the N-terminal catalytic domain. Two epitopes defined by monoclonal antibodies were preserved only in the full-length recombinant enzyme. It was found that deletion of the cysteine-rich C-terminal domain increased the yield of the recombinant expression product, and did not affect the K(m) for di- or trisaccharide substrates. However, affinity for high molecular weight chitin was specific to the full-length molecule, and is apparently mediated by the cysteine-rich domain, suggesting a role for this part of the protein in targeting the secreted enzyme to its substrate.

Heterologous expression and enzymatic properties of a selenium-independent glutathione peroxidase from the parasitic nematode Brugia pahangi.

A full-length cDNA from the parasitic nematode Brugia pahangi encoding a secreted homolog of glutathione peroxidase in which the codon for the active site selenocysteine is substituted naturally by a cysteine codon has been expressed in Spodoptera frugiperda (insect) cells via Autographa californica nuclear polyhedrosis virus (baculovirus). The recombinant protein was glycosylated and secreted from the cells in tetrameric form. The purified protein showed glutathione peroxidase activity with a range of organic hydroperoxides, including L-alpha-phosphatidylcholine hydroperoxide, but no significant activity against hydrogen peroxide. Glutathione was the only thiol tested that served as a substrate for the enzyme, which showed no activity with the thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH). No glutathione-conjugating activity was detected against a range of electrophilic compounds that are common substrates for glutathione S-transferases. The apparent (pseudo)m for glutathione was determined as 4.9 mM at a fixed concentration of linolenic acid hydroperoxide (3 microM). The enzyme showed low affinity for hydroperoxide substrates (apparent Km for linolenic acid hydroperoxide and L-alpha-phosphatidylcholine hydroperoxide of 3.8 and 9.7 mM, respectively at a fixed glutathione concentration of 3 mM).