In Silico Identification of Novel Biomarkers and Development of New Rapid Diagnostic Tests for the Filarial Parasites Mansonella perstans and Mansonella ozzardi.

Mansonelliasis is a widespread yet neglected tropical infection of humans in Africa and South America caused by the filarial nematodes, Mansonella perstans, M. ozzardi, M. rodhaini and M. streptocerca. Clinical symptoms are non-distinct and diagnosis mainly relies on the detection of microfilariae in skin or blood. Species-specific DNA repeat sequences have been used as highly sensitive biomarkers for filarial nematodes. We have developed a bioinformatic pipeline to mine Illumina reads obtained from sequencing M. perstans and M. ozzardi genomic DNA for new repeat biomarker candidates which were used to develop loop-mediated isothermal amplification (LAMP) diagnostic tests. The M. perstans assay based on the Mp419 repeat has a limit of detection of 0.1 pg, equivalent of 1/1000th of a microfilaria, while the M. ozzardi assay based on the Mo2 repeat can detect as little as 0.01 pg. Both LAMP tests possess remarkable species-specificity as they did not amplify non-target DNAs from closely related filarial species, human or vectors. We show that both assays perform successfully on infected human samples. Additionally, we demonstrate the suitability of Mp419 to detect M. perstans infection in Culicoides midges. These new tools are field deployable and suitable for the surveillance of these understudied filarial infections.

Filarial and Wolbachia genomics.

Filarial nematode parasites, the causative agents for a spectrum of acute and chronic diseases including lymphatic filariasis and river blindness, threaten the well-being and livelihood of hundreds of millions of people in the developing regions of the world. The 2007 publication on a draft assembly of the 95-Mb genome of the human filarial parasite Brugia malayi- representing the first helminth parasite genome to be sequenced - has been followed in rapid succession by projects that have resulted in the genome sequencing of six additional filarial species, seven nonfilarial nematode parasites of animals and nearly 30 plant parasitic and free-living species. Parallel to the genomic sequencing, transcriptomic and proteomic projects have facilitated genome annotation, expanded our understanding of stage-associated gene expression and provided a first look at the role of epigenetic regulation of filarial genomes through microRNAs. The expansion in filarial genomics will also provide a significant enrichment in our knowledge of the diversity and variability in the genomes of the endosymbiotic bacterium Wolbachia leading to a better understanding of the genetic principles that govern filarial-Wolbachia mutualism. The goal here is to provide an overview of the trends and advances in filarial and Wolbachia genomics.

Alternative splicing creates sex-specific transcripts and truncated forms of the furin protease in the parasite Dirofilaria immitis.

Many extracellular proteins are activated by specific cleavage with an endoprotease. In nematodes, several proteins are cleaved after RX(K/R)R, the recognition site for the subtilisin-like proprotein convertases, furin and blisterase. To characterize furin in the parasitic nematode Dirofilaria immitis, we determined the sequence of the difur gene and its multiple transcripts. The gene spans 11 kb; encodes 16 exons and has a complex pattern of alternative splicing which generates at least 16 distinct mRNAs. The major transcript is a 4.4 kb mRNA which codes for a protein of 834 aa with an unusually long prodomain of 254 aa. Sex-specific splice variants of difur were observed by RT-PCR. The three female-specific and five male-specific transcripts are the first reported examples of sex-specific splicing in parasitic nematodes. This suggests that nematodes have sex-specific factors which regulate RNA splicing. Other splice variants are predicted to alter the phosphorylation and localization of the protease. Alternative splicing after the prodomain encodes a truncated protein that may be an inhibitor and/or substrate of Difurin.

Carboxy-terminal sequence divergence and processing of the polyprotein antigen from Dirofilaria immitis.

A polyprotein composed of multiple units arranged in direct tandem arrays has been identified in parasitic and free living nematodes. Analysis of previously cloned units from the Dirofilaria immitis polyprotein antigen (DiPA) indicated the units were nearly identical but here we demonstrate that they segregate into two related families. The consensus repeats, DiPA-CR1 and CR2, derived for each family are 80% identical. However, the repeats at the C-terminus of the polyprotein have diverged from DiPA-CR1 and CR2. This was shown by DNA sequence and Southern blot analysis of a 1.9 kb cDNA clone that encodes 4.4 C-terminal repeats (DiPA-TR1 through TR5). DiPA-TR3 through TR5 show 27-52% amino acid identity with the consensus repeats and 31-35% amino acid identity with one another. Metabolic labeling studies have shown that cleavage of DiPA generates a protein "ladder' from 14 to > 200 kDa. RRKR, a cleavage motif of subtilisin-like proprotein convertases, was identified as the natural cleavage site. In vitro digestion experiments with proteinase K suggest a structural model for DiPA consisting of protease resistant cores joined by protease sensitive linkers containing the RRKR site. This motif is absent between DiPA-TR3 and TR4 and has been altered to KR between DiPA-TR4 and TR5. An immunoblot of D. immitis extract probed with anti-DiPA-TR4/5 serum demonstrates the absence of cleavage at these sites. These divergent repeats provide an opportunity to investigate processing of the D. immitis polyprotein in vivo.

Brugia malayi and Brugia pahangi: synthetic biotin labeling of oligonucleotide probes for use in species-specific detection assays.

We have developed a novel, high-yield synthetic approach for the incorporation of multiple biotin residues into a series of species-specific oligonucleotide probes for the detection of filarial parasites. The probes are designed to detect species-specific regions of a highly repeated DNA sequence (HhaI repeat) found in all species of Brugia. The synthetic method described in this paper was used to construct oligomer probes tailed on the 5' end with 1 to 46 biotinylated uridine residues. Probes with 46 biotins were found to be more sensitive than probes with 30 or fewer biotins. We also found that alternating the biotinylated uridine residues with nonbiotinylated thymidine residues improved the sensitivity of the probes. Melting temperature studies indicated that the long tails (up to 91 nucleotides) had only a minimal effect on the Tm of the probes. Conditions were found that optimized the sensitivity of the probes while maintaining their species specificity. Using these conditions, the probes were shown to be sensitive enough to detect single parasites in blood using a chemiluminescent detection system. This method of nonradioactively labeling oligonucleotides for the detection of infectious agents will enable the use of such probes in endemic regions in developing countries.

Cloning of a cuticular antigen that contains multiple tandem repeats from the filarial parasite Dirofilaria immitis.

An unusual antigen composed of tandemly repeated protein units was cloned from the filarial parasite Dirofilaria immitis. The antigen was initially identified by screening a lambda gt11 cDNA library with serum from dogs immunized with irradiated D. immitis third-stage larvae. DNA sequence analysis of the cDNA clone, Di5, revealed a continuous open reading frame composed of two 399-base-pair repeats arranged in tandem. Southern blot analysis of genomic D. immitis DNA showed that the gene coding for Di5 is composed of a tandem array of 25-50 copies of this same 399-base-pair repeat. Antiserum raised against recombinant Di5 protein detected a protein "ladder," from about 14 to greater than 200 kDa with steps approximately 15 kDa apart, on immunoblots of D. immitis extract. Metabolic labeling of adult parasites with [35S]methionine showed that Di5 is synthesized as a large precursor that is subsequently cleaved to produce the ladder-like array. These results suggest that the characteristic ladder is created by proteolytic cleavage of the precursor at the same site in each monomer. The Di5 antigen was localized to the cuticle and hypodermis of adult D. immitis by immunoelectron microscopy. Both male and female parasites were found to release Di5 when cultured in vitro. DNA hybridization analysis demonstrated that Di5 is a member of a gene family present in many filarial parasites that infect both animal and human populations.

Protein organization in mouse liver peroxisomes.

Peroxisomes from mouse liver were fractionated with Triton X-114, a procedure which yields a detergent phase consisting of proteins containing hydrophobic binding sites, and a nondetergent, or aqueous, phase containing hydrophilic proteins. When this method was applied to peroxisomes from control mice, catalase and fatty acyl-CoA oxidase distributed to the aqueous phase, whereas the integral membrane protein, PMP68, and the bifunctional protein were recovered exclusively in the detergent phase. Urate oxidase distributed intermediate between these two phases. With peroxisomes from mice treated with the peroxisome proliferator clofibrate, the bifunctional protein was recovered in both the detergent and the aqueous phases, and urate oxidase was shifted toward the aqueous phase. Other analyses of the subperoxisomal distribution of the bifunctional protein were consistent with a proportion of this protein being tightly associated with the peroxisomal membrane, or with some other uncharacterized, poorly soluble, component. Sucrose gradient centrifugation of the aqueous phase resulting from Triton X-114 fractionation of peroxisomes revealed that a major proportion of catalase, fatty acyl-CoA oxidase, the bifunctional protein, and other unidentified proteins behaved as if associated under these conditions. In this respect, use of a higher concentration of Triton X-114 for peroxisome fractionation led to the partitioning of some catalase and fatty acyl-CoA oxidase to the detergent phase, indicating the presence of some detergent-accessible hydrophobic binding sites even on these proteins. These data have been interpreted as indicating matrix protein associations in vivo, associations which may be responsive to proliferator treatment.

Nucleotide sequence of the phage lambda gt11 SacI-KpnI lacZ region.

The nucleotide sequence of the lambda gt11 SacI-KpnI region, surrounding the unique EcoRI cloning site, was directly determined. This sequence previously had to be compiled from several diverse sources. The direct sequence confirms the sequence predicted from the compilation and pinpoints other unique restriction enzyme targets in the region for use in subcloning.

A rapid DNA assay for the species-specific detection and quantification of Brugia in blood samples.

This report describes a new assay for detecting filarial parasites of the genus Brugia in blood samples using labeled DNA probes. The sequences of these DNA probes are based on the HhaI repeat DNA family found in the genus Brugia. These DNA probes are species-specific and can detect the DNA from a single microfilaria in hybridization assays. To adapt this test for use on blood samples collected in the field, complex steps to separate microfilariae from blood cells and to purify parasite DNA were eliminated. We found that the most effective method was to filter blood samples through 5.0 microns pore nitrocellulose membranes, lyse the microfilariae on the membranes by proteinase K digestion, denature the parasite DNA with sodium hydroxide, and hybridize with the DNA probe. With this method, individual microfilariae can be visualized and counted on autoradiograms. The assay was evaluated in a mock field study using Brugia malayi-infected jirds and was found to be an efficient and accurate method for quantifying microfilariae in blood samples.

Production and characterization of antibody probes directed at constant regions of the alpha and beta subunit of the human T cell receptor.

To generate antibodies directed at constant regions of the human T cell receptor, purified alpha and beta subunits of a human T cell antigen/major histocompatibility complex receptor from the REX tumor (Ti-REX) were isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and utilized to immunize rabbits. H36 (anti-alpha subunit) and H38 (anti-beta subunit) antisera were strongly reactive with the denatured subunits and also immunoprecipitated the Ti heterodimer from 125I surface-labeled lysates of REX, inducer, suppressor and cytotoxic T cell clones, peripheral T lymphocytes and thymocytes. Moreover, immunodepletion experiments showed that such antisera recognized antigenic determinant(s) shared by all Ti molecules expressed in the thymus. Several observations were made with these anticonstant region antibodies. First, peptide map analysis showed that the T cell receptor molecules recognized by the anti-clonotype and the anti-constant region heteroantisera on a given T cell clone are identical, thus supporting the view that the T cell receptor undergoes allelic exclusion. Second, since the individual antisera were weakly cross-reactive with the other denatured subunit, such subunits probably share conserved sequences. Third, the absence of antisera reactivity with intact cells implies that most of these constant region epitopes must be obscured by associated molecules, perhaps including one or more of the 20-25-kDa T3 subunits. Fourth, the extensive difference in two-dimensional peptide maps of Ti alpha subunits from clones of differing specificities makes it likely that the subunit contributes in a major way to antigen/major histocompatibility complex binding.

Molecular analysis of T cell receptor (Ti) variable region (V) gene expression. Evidence that a single Ti beta V gene family can be used in formation of V domains on phenotypically and functionally diverse T cell populations.

We examine the rules governing Ti beta variable (V) gene segment usage in the formation of T cell antigen-MHC receptors in diverse regulatory and effector T lymphoid subpopulations. To this end, a single Ti beta V gene family and its products were analyzed. A monoclonal antibody, termed anti-Ti3A, which was shown to be reactive with an epitope encoded by members of the REX cell line Ti beta V gene family, and which was expressed on 2% of human T lymphocytes was used in selection of clones from unprimed peripheral T lymphocytes. Both T4+, as well as T8+ T cell clones with inducer, suppressor, and/or cytotoxic function were defined. Southern analysis, isoelectric focusing and two-dimensional peptide mapping indicated that individual members of the REX V gene family were linked to different Ti beta diversity and/or joining and constant region segments. Moreover, the Ti alpha chains of such clones were distinct. These results imply that Ti beta V gene usage is not restricted to any functionally or phenotypically defined T cell subsets, and there is presumably little, if any, restriction on the mechanisms that generate combinational, junctional or chain association-mediated diversity.

Purification and NH2-terminal amino acid sequencing of the beta subunit of a human T-cell antigen receptor.

To obtain information about the structural basis for T-cell antigen recognition, a T3-associated Ti receptor molecule was isolated from crude membranes of the REX human thymic tumor line and purified by affinity chromatography with an anti- clonotypic monoclonal antibody in conjunction with preparative gel electrophoresis. NH2-terminal amino acid sequencing of the beta subunit unambiguously identified the amino acids in positions 2-12. Comparative protein sequence analysis by computer search demonstrated that this Ti beta sequence bore weak, but definite, homology to the first framework of the variable region of human lambda light chain. Anti-sera to a synthetic peptide corresponding to positions 2-11 precipitated the denatured Ti beta subunit from REX, thus confirming the above sequence. This information suggests that the Ti beta subunit is distantly related to human immunoglobulin lambda light chain and, moreover, should be of use in the molecular cloning of the Ti beta gene.