Unnasch TR et al. (1988) J Clin Invest "Isolation and characterization of expression cDNA clones encoding antigens of Onchocerca volvulus infective larvae."

Gallin MY, Soboslay PT, Greene BM, Erttmann KD, Unnasch TR

[J Clin Invest, 1988]

The isolation of recombinant cDNA clones expressing antigens found in Onchocerca volvulus infective larvae is described. To isolate such clones, an expression cDNA library constructed from adult O. volvulus RNA was screened with antiserum raised against infective larvae. One clone, designated lambda RAL-1 was characterized further. The recombinant antigen produced by lambda RAL-1 stimulates proliferation of peripheral blood mononuclear cells from O. volvulus infected humans. Lambda RAL-1 is derived from a 1450 bases message that encodes a protein with an apparent molecular weight of 42,000 in adult O. volvulus. The inserted DNA of lambda RAL-1 contains an open reading frame of 1008 bp. The amino acid sequence predicted by this open reading frame contains three repeats of the sequence KKPEDWD. The identification of clones such as lambda RAL-1 will provide quantities of purified antigens sufficient to begin to study the immune response to and explore the development of immunity against the infectious form of the parasite.

Gladyshev VN et al. (1999) Biochem Biophys Res Commun "Selenocysteine-containing thioredoxin reductase in C. elegans."

Wootton JC, Hatfield DL, Lee BJ, Korotkov KV, Krause MW, Xu XM, Sun QA, Gladyshev VN, Kryukov GV

[Biochem Biophys Res Commun, 1999]

Mammalian thioredoxin reductases contain a TGA-encoded C-terminal penultimate selenocysteine (Sec) residue, and show little homology to bacterial, yeast, and plant thioredoxin reductases. Here we show that the nematode, Caenorhabditis elegans, contains two homologs related to the mammalian thioredoxin reductase family. The gene for one of these homologs contains a cysteine codon in place of TGA, and its product, designated TR-S, was previously suggested to function as thioredoxin reductase. The other gene contains TGA and its product is designated TR-Se. This Sec-containing thioredoxin reductase lacks a canonical Sec insertion sequence element in the 3'-untranslated area of the gene. TR-Se shows greater sequence similarity to mammalian thioredoxin reductase isozymes TR1 and TR2, whereas TR-S is more similar to TR3. TR-Se was identified as a thioredoxin reductase selenoprotein by labeling C. elegans with 75Se and characterizing the resulting 75Se-labeled protein by affinity and other column chromatography and gel-electrophoresis. TR-Se was expressed in Escherichia coli as a selenoprotein when a bacterial SECIS element was introduced downstream of the Sec TGA codon. The data show that TR-Se is the major naturally occurring selenoprotein in C. elegans, and suggest an important role for selenium and the thioredoxin system in this organism.

Higazi TB et al. (2013) Methods Mol Biol "Biolistic transformation of Brugia malayi."

Higazi TB, Unnasch TR

[Methods Mol Biol, 2013]

Biolistics has become a versatile tool for direct gene transfer to various cell and tissue types. Following its successful use on the parasitic nematode Ascaris suum, we developed and evaluated biolistics in the transfection of the model filarial parasite Brugia malayi. Biolistics was proven to be an efficient strategy for transfection of all life stages of the parasite and paved the way for studies on elements essential for promoter function and gene regulation of filarial parasites. Here we present a biolistics protocol for the transfection of B. malayi based on the Biolistics PDS 1000/He system and gold microcarriers.

Toe L et al. (1994) Am J Trop Med Hyg "DNA probe-based classification of Simulium damnosum s. l.-borne and human-derived filarial parasites in the onchocerciasis control program area."

Toe L, Merriweather A, Unnasch TR

[Am J Trop Med Hyg, 1994]

The development of polymerase chain reaction-based methods using strain- and species-specific DNA probes for Onchocerca volvulus has permitted classification of individual parasites from every stage of the parasite's life cycle. This technology has been applied on a large scale basis by Onchocerciasis Control Program (OCP) in West Africa. The primary objective of the OCP in using the DNA probes was to obtain accurate estimates of the annual transmission potential of the blinding strain of O. volvulus. The DNA probe classification of larvae collected throughout the OCP area demonstrated that larvae of less pathogenic strains of O. volvulus and other filarial parasites carried by Simulium damnosum s.l. have resulted in a significant overestimation of the annual transmission potential for blinding onchocerciasis. This effect is particularly pronounced along the southern border of the OCP, where the blinding and less pathogenic strains of O. volvulus coexist, and in the north of the control area, where animal parasites, particularly O. ochengi, may even predominate. A second objective of the OCP in applying the DNA probe technology was to determine the distribution of blinding and less pathogenic O. volvulus in infected individuals along the southern border of the control area. Results obtained from these studies have generally confirmed the distribution pattern established by previous epidemiologic studies. In addition, DNA probe classifications have demonstrated that in areas where the blinding and less pathogenic strains of O. volvulus coexist, a single individual may simultaneously be infected with both strains of the parasite.

Keddie EM et al. (1998) Mol Biochem Parasitol "The mitochondrial genome of Onchocerca volvulus: sequence, structure and phylogenetic analysis."

Unnasch TR, Keddie EM, Higazi T

[Mol Biochem Parasitol, 1998]

The complete DNA sequence of the mitochondrial genome of Onchocerca volvulus is described. The O. volvulus mitochondrial genome is 13747 bp, slightly smaller than the mitochondrial genomes of the nematodes Ascaris suum and Caenorhabditis elegans, and the smallest metazoan mitochondrial DNA molecule reported to date. The O. volvulus mitochondrial genome contains genes for two ribosomal RNAs, 22 transfer RNAs and 12 proteins. Consistent with the small size of the genome, four gene pairs overlap and eight contain no intergenic regions. Only 17 intergenic regions are found, ranging in size from 1 to 46 bp. As in C. elegans and A. suum, the O. volvulus mitochondrial genome lacks an open reading frame encoding ATPase subunit 8, and all genes are apparently transcribed in the same direction. However, the mitochondrial gene order of O. volvulus differs from that of A. suum, C. elegans and other metazoan mitochondrial genomes. A total of 20 of the 22 transfer RNAs encoded in the O. volvulus mitochondrial genome have the potential to fold into secondary structures lacking the TpsiC arm, as has been reported in other nematodes. The genome exhibits a striking codon bias, with 15/20 amino acids having a single codon preference of > 70%.

Tsukamoto T et al. (2016) J Biol Chem "X-ray Crystallographic Structure of Thermophilic Rhodopsin: IMPLICATIONS FOR HIGH THERMAL STABILITY AND OPTOGENETIC FUNCTION."

Shimono K, Honda N, Hasegawa T, Takahashi M, Hashimoto N, Sudo Y, Hayashi S, Mizutani K, Miyauchi S, Yamamoto M, Takagi S, Yamashita K, Tsukamoto T, Murata T

[J Biol Chem, 2016]

Thermophilic rhodopsin (TR) is a photoreceptor protein with an extremely high thermal stability and the first characterized light-driven electrogenic proton pump derived from the extreme thermophile Thermus thermophilus JL-18. In this study, we confirmed its high thermal stability compared with other microbial rhodopsins and also report the potential availability of TR for optogenetics as a light-induced neural silencer. The x-ray crystal structure of TR revealed that its overall structure is quite similar to that of xanthorhodopsin, including the presence of a putative binding site for a carotenoid antenna; but several distinct structural characteristics of TR, including a decreased surface charge and a larger number of hydrophobic residues and aromatic-aromatic interactions, were also clarified. Based on the crystal structure, the structural changes of TR upon thermal stimulation were investigated by molecular dynamics simulations. The simulations revealed the presence of a thermally induced structural substate in which an increase of hydrophobic interactions in the extracellular domain, the movement of extracellular domains, the formation of a hydrogen bond, and the tilting of transmembrane helices were observed. From the computational and mutational analysis, we propose that an extracellular LPGG motif between helices F and G plays an important role in the thermal stability, acting as a "thermal sensor." These findings will be valuable for understanding retinal proteins with regard to high protein stability and high optogenetic performance.

Rokeach LA et al. (1994) Infect Immun "Epitopes of the Onchocerca volvulus RAL1 antigen, a member of the calreticulin family of proteins, recognized by sera from patients with onchocerciasis."

Zimmerman PA, Unnasch TR, Rokeach LA

[Infect Immun, 1994]

RAL1 is an antigen (Ag) encoded by the filarial nematode Onchocerca volvulus, the parasite causing onchocerciasis (river blindness). RAL1 shares 64.4% identity with the autoantigen calreticulin. The striking similarity of the parasite Ag and the human autoantigen has led to the hypothesis that RAL1 may induce a cross-reactive immune response to calreticulin, which in turn may be involved in the pathogenesis of onchocerciasis. To test this hypothesis, we explored the immune response to RAL1 recombinant Ag (RAL1 rAg) and human calreticulin in patients with O. volvulus infection. A total of 86% of the O. volvulus-infected individuals produced antibodies recognizing RAL1 rAg. Antibody reactivity to RAL1 rAg in patient sera was confined primarily to the central and carboxyl-terminal parts of the molecule. No significant correlations were found to associate recognition of RAL1 rAg, or any particular portion thereof, with a particular disease state. Antibodies against RAL1 thus appear to be produced as a general immune reaction to O. volvulus infection and do not necessarily lead to a cross-reacting response with the host protein. In contrast, 33% of the patient sera tested bound recombinant human calreticulin. All of these sera also recognized a polypeptide encompassing the carboxyl-terminal portion of the RAL1 rAg. These results suggest that recognition of an epitope encoded in the carboxyl-terminal portion of RAL1 is at least in part responsible for inducing a cross-reacting immune response to the host protein.

Liu C et al. (2009) Mol Biochem Parasitol "The splice leader addition domain represents an essential conserved motif for heterologous gene expression in B. malayi."

Katholi CR, Unnasch TR, Chauhan C, Liu C

[Mol Biochem Parasitol, 2009]

Two promoters from the human filarial parasite Brugia malayi have been mapped in detail. The essential domains of both promoters lacked canonical eukaryotic core promoter motifs. However, the largest contiguous essential domain in both promoters flanked and included the splice leader addition site. These findings suggested that the region flanking the trans-splicing addition site might represent a conserved core domain in B. malayi promoters. To test this hypothesis, the putative promoters of 12 trans-spliced genes encoding ribosomal protein homologues from B. malayi were isolated and tested for activity in a B. malayi transient transfection system. Of the 12 domains examined, 11 produced detectable reporter gene activity. Mutant constructs of the six most active promoters were prepared in which the spliced leader acceptor site and the 10 nt upstream and downstream of the site were deleted. All deletion constructs exhibited >90% reduction in reporter gene activity relative to their respective wild type sequences. A conserved pyrimidine-rich tract was located directly upstream from the spliced leader splice acceptor site which contained a conserved T residue located at position -3. Mutation of the entire polypyrimidine tract or the conserved T individually resulted in the loss of over 90% of reporter gene activity. In contrast, mutation of the splice acceptor site did not significantly reduce promoter activity. These data suggest that the region surrounding the splice acceptor site in the ribosomal promoters represents a conserved essential domain which functions independently of splice leader addition.

Liu C et al. (2012) Mol Biochem Parasitol "Identification of genes containing ecdysone response elements in the genome of Brugia malayi."

Tzertzinis G, Unnasch TR, Liu C, Enright T

[Mol Biochem Parasitol, 2012]

Recent studies have demonstrated that filarial parasites contain a functional homologue of the insect ecdysone receptor (EcR). As a first step in deciphering the physiological role that ecdysteroids play in filarial parasites, adult female parasites cultured in the presence and absence of 20-OH ecdysone were metabolically labeled. Gel electrophoretic analysis of proteins extracted from the cultured parasites revealed changes in the level of expression of several proteins, indicating that adult female parasites contained an ecdysone-responsive gene network. A bioinformatic analysis was then conducted to identify putative ecdysone response elements (EcREs) in the Brugia malayi genome. A total of 18 genes were identified that contained putative EcREs located in the 4 kbp upstream from the start of their open reading frames. The most common functional classifications of the encoded proteins were factors involved in transcription and metabolism. These genes revealed a number of different developmental patterns of transcription. The promoter of one EcRE-containing gene was cloned into a luciferase reporter vector and transfected into B. malayi embryos. Reporter gene expression from embryos transfected with this construct was up-regulated by 20-OH ecdysone. Deletion and substitution mutations in the canonical EcRE resulted in a loss of the ecdysone response. These results demonstrate the presence of functional EcREs in the B. malayi genome.

Liu C et al. (2020) PLoS Negl Trop Dis "In vivoimaging of transgenic Brugia malayi."

Unnasch TR, Liu C, De SL, Miley K

[PLoS Negl Trop Dis, 2020]

BACKGROUND: Studies of the human filarial parasite have been hampered by the fact that they are obligate parasites with long life cycles. In other pathogenic infections, in vivo imaging systems (IVIS) have proven extremely useful in studying pathogenesis, tissue tropism and in vivo drug efficacy. IVIS requires the use of transgenic parasites expressing a florescent reporter. Developing a method to produce transgenic filarial parasites expressing a florescent reporter would permit IVIS to be applied to the study of tissue tropism and provide a non-invasive way to screen for in vivo drug efficacy against these parasites. METHODOLOGY/PRINCIPAL FINDINGS: We report the development of a dual luciferase reporter construct in a piggyBac backbone that may be used to stably transfect Brugia malayi, a causative agent of human filariasis. Parasites transfected with this construct were visible in IVIS images obtained from infected gerbils. The signal in these infected animals increased dramatically when the transgenic parasites matured to the adult stage and began to produce transgenic progeny microfilaria. We demonstrate that the IVIS system can be used to develop an effective method for cryopreservation of transgenic parasites, to non-invasively monitor the effect of treatment with anti-filarial drugs, and to rapidly identify transgenic F1 microfilariae. CONCLUSIONS: To our knowledge, this represents the first application of IVIS to the study of a human filarial parasite. This method should prove useful in studies of tissue tropism and as an efficient in vivo assay for candidate anti-filarial drugs.