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.

Detection of Loa loa-specific DNA in blood from occult-infected individuals.

Accurate and specific diagnosis of human loiasis is of crucial importance in an endemic area where two-thirds of infected individuals are without circulating microfilariae (occult loiasis). By using the polymerase chain reaction (PCR) and specific primers to the repeat 3 region (15r3) of the gene coding for Loa loa 15-kDa polyprotein antigen, DNA was amplified from total blood lysate of occult-infected subjects. A 396-bp DNA fragment was specifically detected. We tested the specificity of this method by qualitative hybridization to PCR products using blood lysates of the following subjects: (1) from Gabon (80 individuals residing in L. loa endemic area where loiasis exists sympatrically with Mansonella perstans); (2) from Togo (12 individuals infected with Onchocerca volvulus and M. perstans); (3) from Tahiti (12 individuals infected with Wuchereria bancrofti); and (4) from Mali (12 individuals infected with O. volvulus and M. perstans). Samples from Gabon included 60 L. loa amicrofilaremics and 20 L. loa occult-infected subjects. Qualitative hybridization carried out at 50 degrees C on PCR products, using a 15r3-specific oligonucleotide probe, revealed hybridization with L. loa-infected samples from Gabon and four samples from Togo after 2 days exposure to the film. The positive samples from Togo were characterized by the use of nested PCR. Three nested PCR products have been sequenced. No differences were observed between the three sequences and they are 99.72% identical to L. loa 15r3. None of bancroftian-infected individuals from Tahiti, nor O. volvulus- and M. perstans-infected individuals from Mali reacted after 1 week's exposure (overexposure) to the film. This allows us to conclude first that our 15r3 PCR assay is specific for L. loa and secondly that L. loa infections occur in Togo. The sensitivity of this 15r3 PCR assay was further investigated with occult patients and field-collected amicrofilaremic samples. We found that 19 of the 20 occult-infected individuals were positive on Southern hybridization, whereas 35/60 amicrofilaremics were positive. These results have shown that the sensitivity of this assay in detecting unequivocal, parasitologically proven occult loiasis was 95%, while the specificity with regard to the sympatric M. perstans was 100%.

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.

Immunoglobulin G subclass responses of children during infection with Onchocerca volvulus.

To characterize the patterns of immunoglobulin G (IgG) subclass and IgE reactivity during the early stages of onchocerciasis, sera were collected from 224 children (age groups, 2 to 5, 6 to 10, and 11 to 15 years) residing in a region of Sierra Leone where Onchocerca volvulus is endemic, and these samples were tested by enzyme-linked immunosorbent assay for their reactivity to adult antigens (OvAg) and against four recombinant proteins (OV11, OV27, OV29, and OV16). Over 88% of the samples contained detectable levels of anti-OvAg IgG. In samples from microfilaria (MF)-positive children, IgG4 responses were significantly elevated and constituted on average 39, 35 and 28% of the total IgG responses for the age groups of 2 to 5, 6 to 10, and 11 to 15 years, respectively. For MF-negative individuals, the mean contributions of IgG4 to the total IgG response were 11% (2 to 5 years), 27% (6 to 10 years), and 56% (11 to 15 years). OvAg-specific IgE was detectable in the sera from both MF-negative and MF-positive individuals. To increase the specificity of the response, recombinant antigens OV11, OV27, and OV29 were tested individually or as a cocktail. Nearly 50% of the MF-negative children and 85% of the MF-positive children had detectable levels of IgG against at least one of the recombinant antigens. Only a small portion of the IgG against the recombinant peptides was IgG4. The prevalence of IgG against OV16 in samples from MF-negative children was 51%, and that for MF-positive children was 75%. The general profile of the humoral immune responses mounted by both MF-positive and a large percentage of the MF-negative children during the initial phases of infection with O. volvulus is similar to the profile reported for adults harboring chronic O. volvulus infections. These results suggest that very quickly after infection, the interactions between parasite and host result in an immunological environment that may contribute to the maintenance of a long-term, chronic infection.

The polyprotein allergens of nematodes.

Symptoms of infection with a number of parasitic nematodes may be associated with IgE antibody and associated immunopathologies. This has drawn attention to parasite allergens and provoked discussion on the wisdom of their inclusion in recombinant vaccines. Here, Larry McReynolds, Malcolm Kennedy and Murray Selkirk review work on a prominent set of allergens recently characterized in several nematode species.

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.

Brugia pahangi and Brugia malayi: a surface-associated glycoprotein (gp15/400) is composed of multiple tandemly repeated units and processed from a 400-kDa precursor.

The cuticle of filarial nematode parasites contains distinct and separable sets of soluble and structural proteins. Surface-labeling techniques have previously identified a soluble protein complex in adult stage Brugia which ranges in molecular weight from 15 to 200 kDa. Using an antiserum directed to the 15-kDa basal subunit of this complex, we show here that this complex is synthesized and processed from a single, very large precursor protein with a molecular weight of approximately 400 kDa. Molecular cloning, sequencing, and Southern analysis indicates that the protein is encoded by a single gene composed predominantly of approximately 20 tandemly repeated segments of 396 bp. The two complete copies of these repeated segments in our cDNA sequence are identical. Each subunit of 132 amino acids bears a consensus site for N-linked glycosylation, and glycosidase treatment indicates that this corresponds to an oligosaccharide side chain of 2 kDa. The protein displays no significant homology to sequences lodged in databases corresponding to molecules of known function. Nevertheless, a significant similarity (19/41 residues) is observed with the N-terminal sequence of a protein termed ABA-1, an allergen from Ascaris.

Recent advances in the application of molecular biology in filariasis.

Monitoring of filarial parasites in the host and vector has traditionally depended on morphological identification. Recently, species-specific DNA probes have been developed for Brugia malayi, Brugia pahangi and Wuchereria bancrofti. Repeated DNA sequences are useful in developing DNA probes because they evolve more rapidly then coding sequences and their high copy number increases the sensitivity of detection. The Hhal repeated DNA family represents 12% of the total B. malayi DNA. This DNA family is present in species of Brugia (B. malayi, B. timori and B. pahangi) but not W. bancrofti. Sequence analysis of the repeated DNA in B. malayi and B. pahangi has allowed construction of two species-specific DNA probes. These probes were used in a double blind field study in Indonesia. Microfilariae (mf) from infected cats and humans were identified by classical morphological methods and DNA probes. Agreement was found in 98.6% of the 642 samples tested by the two different techniques. Besides mf identification DNA probes can be used to determine the species of infective larvae (L3s) in infected mosquitos. This is useful because the L3s have similar morphology. DNA probes for the identification of W. bancrofti have recently been developed and are in the initial stages of testing in China (Piessens, personal communication) and Egypt (Williams, personal communication). An alternative approach for identification of infected individuals is to detect specific parasite antigens in circulation. A WHO initiative to use either an antigen or antibody assay to replace night blood is presently underway. This approach, if successful would not require the presence of microfilariae, but could detect occult infections.

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.

A recombinant clone of Wuchereria bancrofti with DNA specificity for human lymphatic filarial parasites.

In order to understand the immune response to Wuchereria bancrofti and to aid in the diagnosis of W. bancrofti infections, recombinant antigens were identified from a W. bancrofti genomic expression library made in lambda gt11 using a pool of sera from infected Indian patients. One of the recombinant clones, lambda WbN1, containing a 2.5-kb insert, reacted strongly to a pool of sera from patients with lymphatic filariasis but not to normal human sera. In addition, this clone showed restricted specificity at the genomic level to the major lymphatic filarial parasites W. bancrofti and Brugia malayi but not to the closely related filarial parasite Brugia pahangi or to other filarial and non-filarial species tested. Nucleotide sequence analysis indicated the cloned DNA to have homology to myosin-like myofibrillar proteins. Polymerase chain reaction amplification initiated by specific synthetic oligomers amplified DNA in a species-specific manner from as little as 16 pg of isolated DNA or from one microfilaria.

B cell responses to paramyosin. Isotypic analysis and epitope mapping of filarial paramyosin in patients with onchocerciasis.

To examine the fine specificity of the human immune response to filarial paramyosin, the antigenicity of an expressed rcDNA (2.55 kb) of Dirofilaria immitis paramyosin was detailed by ELISA. Using sera from patients infected with Onchocerca volvulus, we analyzed both the entire paramyosin molecule and six subcloned fragments for their IgG, IgG subclasses, and IgE responses. Patients from both Guatemala (64% positive) and Ghana (100% positive) reacted to paramyosin with specific IgG levels above normal controls. Although there was no anti-paramyosin subclass restriction common to all patients, the IgG3 response in the Ghananians was significantly greater than that of Guatemalans (p less than 0.001). IgE anti-paramyosin responses showed positive correlations with IgG2 (p less than 0.001), IgG4 (p less than 0.002), and IgG1 (p less than 0.04) responses. Epitope mapping using the IgG response to the six subclones demonstrated preferential recognition of the amino terminal end of the molecule (nucleotides 1 to 360). IgG2 reactivity was clearly localized to the most amino-terminal 120 amino acids, and the IgG4 antibodies recognized amino acids immediately adjacent to this fragment. These studies examining the fine specificity of anti-filarial immune reactions should provide a method for understanding how parasites either evade or induce host immune responses.

Filarial paramyosin: cDNA sequences from Dirofilaria immitis and Onchocerca volvulus.

The nucleotide sequence of a cDNA copy of the Dirofilaria immitis paramyosin gene was determined. The sequence was 2545 nucleotides in length, consisting of a single open reading frame of 848 amino acids capable of encoding a protein with a calculated molecular weight of 98,000. The cDNA clone was not complete, but probably includes over 97% of the coding region of the gene. We have previously observed that the cloned D. immitis paramyosin is recognized by sera from humans infected with Onchocerca volvulus. To determine the extent of homology at the protein level, we screened a cDNA library of O. volvulus with an antiserum made against D. immitis paramyosin. Ten recombinant clones were partially sequenced, comprising a total of 1186 nucleotides or 389 amino acids. The amino acid sequence of D. immitis paramyosin was 99% identical to the O. volvulus paramyosin. We also compared the amino acid sequence to other cloned paramyosins, and noted that 92% of the amino acids were identical to those of Caenorhabditis elegans, and 34% identical to those of Schistosoma mansoni. Comparison of the paramyosin sequence between different species revealed a hierarchy of similarities: (1) a 7-amino-acid repeat with apolar residues in the a and d position as the most conserved, followed by (2) the amino acid sequence and (3) the DNA sequence.

Use of degenerate oligonucleotides for amplification of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii.

Trichodesmium spp. are marine filamentous, nonheterocystous, nitrogen-fixing cyanobacteria which are an important component of marine ecosystems. This organism has never been maintained in axenic culture, and there has remained some doubt as to the identity of the organism responsible for nitrogen fixation in Trichodesmium aggregates. By using degenerate oligonucleotide primers, it has been possible to amplify, clone, and sequence a segment of the nifH gene from a natural assemblage of Trichodesmium thiebautii. Examination of the DNA and presumed amino acid sequence shows that the gene is most closely related to that of Anabaena spp. and therefore is most likely a cyanobacterial nifH gene. The use of degenerate oligonucleotides, in concert with the polymerase chain reaction, can be a powerful tool for the cloning and sequencing of a variety of genes from microorganisms in the environment.

A lambda gt11 cDNA recombinant that encodes Dirofilaria immitis paramyosin.

The cDNA synthesized from mRNA of Dirofilaria immitis female adult worms was cloned into the expression vector lambda gt11. Screening the library with a hyperimmune rabbit antiserum raised against adult worm homogenates yielded several antigen positive clones. One of these clones, lambda cDi2, was recognized by rabbit antisera raised against either D. immitis L-3, adult, Brugia malayi L-3 or Onchocerca volvulus adult worm antigen, as well as by antisera from humans naturally infected with O. volvulus or Wuchereria bancrofti. Affinity-purified anti-lambda cDi2 antibodies reacted with a 97-kDa protein on Western transfers of adult D. immitis antigen extracts that were reduced with beta-mercaptoethanol. The whole rabbit anti-D. immitis adult antiserum depleted of anti-lambda cDi2 antibodies exhibited decreased reactivity to this 97-kDa band. A monoclonal antibody (IA6) that specifically binds Schistosoma mansoni paramyosin also recognised a 97-kDa protein in D. immitis extracts upon Western transfer. The deduced amino acid sequence of partial DNA sequence from lambda cDi2 showed some similarity to nematode myosin, and gave a stretch of 82 amino acids that is 91.5% identical to Caenorhabditis elegans paramyosin: thus, lambda cDi2 encodes D. immitis paramyosin.

An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein.

A plasmid vector has been constructed that directs the synthesis of high levels (approximately 2% of total cellular protein) of fusions between a target protein and maltose-binding protein (MBP) in Escherichia coli. The MBP domain is used to purify the fusion protein in a one step procedure by affinity chromatography to crosslinked amylose resin. The fusion protein contains the recognition sequence (Ile-Glu-Gly-Arg) for blood coagulation factor Xa protease between the two domains. Cleavage by factor Xa separates the two domains and the target protein domain can then be purified away from the MBP domain by repeating the affinity chromatography step. A prokaryotic (beta-galactosidase) and a eukaryotic (paramyosin) protein have been successfully purified by this method.

Species-specific oligonucleotide probes for the identification of human filarial parasites.

Species-specific oligonucleotide probes have been constructed for the filarial parasites Brugia malayi and Brugia pahangi. Both parasites contain a 322 base pair repeated DNA sequence that is cleaved once by the restriction endonuclease HhaI. A consensus repeat sequence was determined from the DNA sequence of 15 cloned isolates of each species. Although the two repeats have an average homology of 89%, half the differences are clustered in a region of 66 nucleotides that has a homology of only 72%. Within this region, two probes, a 29-mer that is B. malayi specific and a 21-mer that is B. pahangi specific, were constructed. The sequence of both probes was chosen to obtain the maximum difference between the consensus sequences of the two species. The probes were also selected to be GC rich to increase their stability as a DNA hybrid. In a filter hybridization assay, the B. malayi probe has a 500-fold preference for B. malayi DNA versus B. pahangi DNA and a sensitivity of 200 pg. The B. pahangi probe has similar specificity and sensitivity for B. pahangi DNA. A rapid lysis procedure allows the probes to detect 1-2 third stage larvae of either B. malayi or B. pahangi in a filter hybridization assay.

Cloning and comparison of repeated DNA sequences from the human filarial parasite Brugia malayi and the animal parasite Brugia pahangi.

A 320-base-pair repeated sequence was observed when DNA samples from the filarial parasites Brugia malayi and Brugia pahangi were digested with the restriction endonuclease Hha I. A 640-base-pair dimer of the repeated sequence from B. malayi was inserted into the plasmid pBR322. When dot hybridization was used, the copy number of the repeat in B. malayi was found to be about 30,000. The 320-base-pair Hha I repeated sequences are arranged in direct tandem arrays and comprise about 12% of the genome. B. pahangi has a related repeated sequence that cross-hybridizes with the cloned B. malayi Hha I repeat. Dot hybridization with the cloned repeat shows that the sequence is present in B. malayi and in B. pahangi but not in four other species of filarial parasites. The cloned repeated DNA sequence is an extremely sensitive probe for detection of Brugia in blood samples. Hybridization with the cloned repeat permits the detection of DNA isolated from a single parasite in an aliquot of blood from animals infected with B. malayi. There are differences in the restriction sites present in the repeated sequences that can be used to differentiate between the two Brugia species. The B. malayi repeated DNA sequence is cleaved by Alu I and Rsa I but the B. pahangi sequence is not. A comparison of repeated sequences between the two species by DNA sequence analysis indicates that some regions of individual repeats are over 95% homologous, while other short regions are only 60-65% homologous. These differences in DNA sequence will allow the construction of species-specific hybridization probes.

Transfer of a mutant viral thymidine kinase gene results in temperature-sensitive mouse cells.

Temperature-sensitive cell lines were obtained by DNA-mediated transfer of the thymidine kinase (TK) gene from a mutant, ts1117, of herpes simplex virus type 1. The cells died at 39 degrees C in selective medium which contained low levels (1 microgram/ml) of thymidine. In this lethal condition, no revertants were detected among 10(8) cells. It was shown by in vitro analysis of the TK activity that the temperature-sensitive cell line contains an enzyme whose activity is temperature sensitive and relatively unaffected by dTTP. The viral enzyme has these properties. The effect of the lethal growth conditions in the cell line was characterized by cell cycle analysis and rescue experiments which involved a shift to the permissive conditions. The successful transfer of the mutant viral TK activity to cells provides an additional selective marker for gene transfer.