Heterologous expression, chaperone mediated solubilization and purification of parasitic nematode-specific growth factor-like protein of Setaria digitata

OBJECTIVE@#To clone, express and purify a putative parasitic nematode specific protein of Setaria digitata (S. digitata), filarial nematode that infects livestock and cause significant economic losses in Far East and Asia to be used for structural and functional analyses.@*METHODS@#To characterize uncharacterized gene of S. digitata (SDUG), the herterologous expression of SDUG was carried out in the pET [cloned into pET45b(+)] expression system initially and co-expression of SDUG using chaperone plasmids pG-KJE8, pGro 7, pKJE7, pG-Tf2 and pTf16 containing chaperone proteins of dnaK-dnaJ-grpE-groES-gro-E, groES-groEL, dnaK-dnaJ-grpE, groES-groEL-tig, and tig respectively, was carried out subsequently.@*RESULTS@#Expression of SDUG was seen when Escherichia coli strain BL21(DE3) is used, while concentrating protein largely into the insoluble fraction. The co-expression of SDUG using chaperone plasmid mediated system indicated a significant increase of the protein in the soluble fraction. Of the chaperon plasmid sets, the highest amount of recombinant SDUP in the soluble fraction was seen when pGro7 was used in the presence of 2 mg/mL L-arabinose and 0.6M IPTG concentration in the culture medium and for 3 h of incubation at the temperature of 28 °C. Recombinant SDUG was purified both from soluble and insoluble fractions using Ni affinity chromatography. SDS-PAGE and western blot analyses of these proteins revealed a single band having expected size of ∼24 kDa.@*CONCLUSIONS@#SDUG seems to be more aggregate-prone and hydrophobic in nature and such protein can make soluble by correct selecting the inducer concentrations and induction temperature and its duration.

Partial purification and characterization of acetylcholinesterase isozymes from adult bovine filarial parasite Setaria cervi.

Filariasis is a major health problem, affecting millions of people in tropical and sub-tropical regions of the world. The isolation and characterization of parasite-specific enzyme targets is essential for developing effective control measures against filariasis. Acetylcholinesterase (AchE, E.C. 3.1.1.7), an important enzyme of neuromuscular transmission is found in a number of helminths including filarial parasites and may be playing a role in host-parasite interactions. Earlier, we demonstrated the presence of two isozymes of AchE, different from the host enzyme in the human (Brugia malayi) and bovine (Setaria cervi) filarial parasites. In the present study, two isozymes of AchE (pAchE1 and pAchE2) were isolated from S. cervi adults and characterized biochemically and immunochemically. The AchE was partially purified on Con-A Sepharose column and then subjected to preparative polyacrylamide gel electrophoresis (PAGE) for separation of the isozymes. The AchE activity was localized by the staining of gel and the isozymes were isolated from the PAGE strips by electroelution. Both isozymes preferentially utilized acetylcholine iodide as substrate and were strongly inhibited by the true AchE inhibitor (BW284c51), suggesting that they were true AchE. The polyclonal antibodies produced against the isozymes showed significant cross-reactivity with B. malayi AchE, but not against the host enzyme. These findings suggested that both the isozymes were biochemically (in terms of their substrate specificity and inhibitor sensitivity) and immunochemically similar, but different from the host enzyme.

Potential antifilarial activity of fruit extracts of Ficus racemosa Linn. against Setaria cervi in vitro.

Effect of alcoholic and aqueous extracts of the fruits of F. racemosa Linn., on the spontaneous movements of both the whole worm and nerve muscle preparation of Setaria cervi and on the survival of microfilariae in vitro was studied. Alcoholic as well as aqueous extracts caused inhibition of spontaneous motility of whole worm and nerve muscle preparation of Setaria cervi characterized by increase in amplitude and tone of contractions. Initial stimulatory effect was not observed with aqueous extract on whole worm preparation, while effect of alcoholic extract on whole worm and nerve muscle preparation was characterized by an increase in amplitude and tone of contractions followed by paralysis. The concentrations required to inhibit the movement of the whole worm and nerve muscle preparation for alcoholic extract of fruits of F. racemosa were 250 and 50 microg/ml, respectively, whereas aqueous extract caused inhibition of the whole worm and nerve muscle preparation at 350 and 150 microg/ml, respectively, suggesting a cuticular barrier. Both alcoholic and aqueous extracts caused death of microfilariae in vitro. LC50 and LC90 were 21 and 35 ng/ml, respectively for alcoholic, which were 27 and 42 ng/ml for aqueous extracts.

Voltage activated calcium channels in somatic muscle of filarial nematode Setaria cervi.

Acetylcholine (Ach), levamisole and pyrantel pamoate all cause stimulation of spontaneous rhythmic movements of whole worm and nerve muscle preparation of filarial nematode Setaria cervi. These stimulant effects are manifested only in the presence of available Ca2+ or extracellular Ca2+. Electrical stimulation of nerve muscle preparation of Setaria cervi elicited depolarization and increase in amplitude and tone of contractions. Electrical current stimulates Ca2+ entry leading to depolarization and during the phase of depolarization addition of any of the three stimulants viz. Ach, levamisole or pyrantel pamoate fails to elicit any response on nerve muscle preparation. The findings indicate that electrical stimulation, excitatory neurotransmitter Ach and stimulant anthelmintics levamisole and pyrantel pamoate all produce their stimulant effect by triggering entry of Ca2+ into the muscle cell. Further, blocking the calcium channels by nifedepine and thereby the entry of Ca2+ into the cells blocks the stimulant effect of Ach levamisole and pyrantel pamoate.

Wuchereria bancrofti: detection of microfilariae in asymptomatic microfilaremic individuals with Setaria digitata antigens.

A dot-ELISA for detection of microfilariae of Wuchereria bancrofti in an endemic area was developed. This test can differentiate the endemic normals from the microfilaraemic asymptomatic individuals. Antigens of molecular weight 130 and 52 kDa of the cattle filaria worm Setaria digitata were used for this test. It was observed that these two antigens were also present in the serum of asymptomatic microfilaraemic individuals.

Filariosis por Setaria equina en un caballo del estado de Yucatán, México / Filariosis for Setaria equina in a horse from the state of Yucatán, Mexico

Introducción. Setaria equina es un nematodo que viven en la cavidad peritoneal de los equinos. En su ciclo biológico actúa como huésped intermediario los mosquitos de los géneros Anopheles, Aedes y Culex. Caso clínico. Se presentó un caso de un caballo macho de la raza criolla de tres años de edad, procedente del municipio de Río Lagartos, Yucatán, México. El caballo fue sometido a una castración y en el líquido testicular se encontró la presencia de 3 hembras que midieron de largo 83 mm (75-93) y 4 machos de 54.5 mm (43-64) de largo. Los nematodos fueron clasificados como Setaria equina. Discusión. S. equina puede tener localizaciones erráticas, sobre todo en la cavidad pleural, ojos, cerebro, médula espinal y los testículos de los equinos, sin presentar signos clínicos. No obstante, la larva en el globo ocular y en el sistema nervioso central, puede causar serios daños. A pesar de que S. equina tiene distribución mundial, en el sureste mexicano no ha sido reportado como parásito en los equinos.

Succinate oxidase and fumarate reductase systems of filarial parasite Setaria digitata.

Activities of succinate oxidase, fumarate reductase (FR) and succinate dehydrogenase (SDH) under a set of defined conditions were determined in the mitochondrial isolate from Setaria digitata, the filarial parasite from the cattle Bos indicus. Presence of only two activities namely SDH and succinate--UQ reductase of the succinate oxidase system could be detected in S. digitata. In the absence of cytochromes, the 3rd enzyme of the complex namely cytochrome oxidase is absent and it is proposed that an alternative oxidase is responsible for completing the succinate oxidation expressed as succinate oxidase activity. Though SDH and FR catalyse reverse reactions, they responded differently to modulators such as oxaloacetate, aspartate, alanine, pyruvate and fumarate. The degree of response of the two activities against inhibitors of electron transport was also different. Interestingly fumarate caused only 50% inhibition of succinate oxidation, while the effect against FR was more convincing.

5/6/7/8 mono or disubstituted 1H/1-phenyl-9H-pyrido [3,4 b] indoles--a new class of antifilarial agents and their relationship with voltage sensitive calcium channels in Setaria cervi.

Synthetic compounds of 5/6/7/8 mono or disubstituted 1H/1-phenyl-9H pyrido (3,4 b) indole series were screened for their antifilarial activity in vitro by observing their effect on the spontaneous movements of the whole worm and nerve muscle preparation of S. cervi (Nematoda:Filarioidea). Of the 10 compounds tested only compound 87/581 was able to modify the movements of whole worm and nerve muscle preparation. This compound caused inhibition of spontaneous motility of whole worm and nerve muscle preparation of S. cervi characterized by initial short lasting stimulation followed by irreversible paralysis. The concentration required to inhibit the movements of n.m. preparation was 10 micrograms/ml as compared to 25 micrograms/ml for the whole worm preparation, suggesting a cuticular permeability barrier. Prior addition of compound 87/581 to the bath fluid blocked the calcium ion dependent stimulant effect of Ach and pyrantel pamoate. Further the initial stimulant effect of the compound was blocked by prior addition of nifedipine a calcium channel blocker. The findings suggest that the action is due to blockade of voltage sensitive calcium channels.

Immune response of electroeluted detergent soluble 29 kDa antigen from Setaria digitata (Von Linstow).

Filariasis is one of the typical parasitic infections which cause immune suppression during the course of infection in both humans and experimental animals. A 29 kDa protein isolated from detergent soluble antigen of S. digitata showed maximum inhibition of cell mediated immune response. The heat inactivated 29 kDa protein was found to be devoid of property of suppression of immune response in the host. Histological study of spleen of BALB/C mice immunized with 29 kDa protein showed changes in regions of spleen such as follicle, trabeculae, capsule, reticuloendothelial cells and eosinophils. The 29 kDa protein, the most reactive of the detergent soluble proteins produced partial suppression of immune response, thereby contributing to the factors responsible for the survival of filarial parasites in hosts.

Optimisation of immunoaffinity purification of Wuchereria bancrofti specific antibodies from human sera.

Immunoaffinity column using Setaria digitata antigens coupled to cyanogen bromide activated Sepharose 4B beads were developed to purify antibodies from sera of filarial patients. Chaotropic (KSCN) ion elution was more efficient for purifying specific antibodies from the column in comparison to ]c elution. Dot blot analysis indicated that purified antibodies showed a high degree of reactivity with cattle filarial antigen and recombinant filarial protein but not with bacterial proteins of E. coli suggesting that the antibodies are specific.

Antifilarial activity of Mallotus philippensis Lam. on Setaria cervie (Nematoda: Filarioidea) in vitro.

The effect of aqueous and alcoholic extracts of the leaves of Mallotus philippensis (Lam.) Muell. Arg. was studied on the spontaneous movements of the whole worm and nerve-muscle (n.m.) preparation of Setaria cervi and on the survival of microfilariae in vitro. Both the extracts caused inhibition of spontaneous motility of whole worm and the n.m. preparation of S. Cervi characterized by initial stimulation followed by depression in amplitude. The tone and rate of contractions remained visibly unaffected. Aqueous extract at higher concentration showed immediate reduction in tone. The concentration required to inhibit the movements of n.m. preparation was 1/5th for aqueous and 1/11th for alcoholic extract compared to that for the whole worm, suggesting a cuticular permeability barrier. The stimulatory response of acetylcholine was blocked by aqueous extract on whole worm movements. On the microfilariae the LC50 and LC90 were 18 and 20 ng/ml for aqueous and 12 and 15 ng/ml for alcoholic extracts respectively.

Partial characterization of affinity purified excretory-secretory protease from Setaria digitata.

Excretory-secretory protease of S. digitata released along with the microfilariae (mf) during hatching has been purified by affinity chromatography. No other activity could be detected in the affinity purified material. Homogeneity is checked by native PAGE. It has a pH optimum of 5.4 and a molecular weight of 70 kD. The purified material showed positivity against antibodies raised against ES material.

Potential antifilarial activity of roots of Asparagus adscendens Roxb, against Setaria cervi in vitro.

Effect of aqueous and alcoholic extracts of the roots of A. adscendens was studied on the spontaneous movements of whole worm and nerve muscle (n.m.) preparation of S. cervi and on the survival of microfilariae in vitro. Aqueous as well alcoholic extracts caused inhibition of spontaneous motility of whole worm and n.m. preparation of S. cervi characterized by initial, short lasting small increase in amplitude and tone of contractions followed by paralysis. The initial stimulatory effect was not observed by aqueous extract on n.m. preparation. The concentration required to inhibit the movements of n.m. preparation was 1/4th for aqueous and 1/3rd for alcoholic extract suggesting a cuticular permeability barrier. The effect of acetylcholine on n.m. preparation was concentration related being more with a concentration of 5 micrograms/ml as compared to 1 microgram/ml. Both alcoholic as well as aqueous extracts caused death of microfilariae in vitro, LC50 and LC90 being 8 and 16 ng/ml for aqueous, 3 and 12 ng/ml for alcoholic extracts respectively.

Analysis and potential of excretory-secretory antigens of Setaria cervi for immunodiagnosis of human filariasis.

The excretory-secretory (E-LS) products released by the adult Setaria cervi, a bovine filarial parasite, were used to raise polyvalent hyperimmune serum in rabbits. Analysis of E-S products, using anti-E-S serum showed the presence of 10-14 immunogenic proteins, the rabbit anti-E-S serum showed reciprocal antibody titres in the range of 100,000-250,000 by enzyme linked immunosorbent assay. The anti-E-S antibodies could detect circulating antigen in filarial patients sera by Counter immunoelectrophoresis.

Histological changes in the spleen of BALB/C mice caused by excretory-secretory protein(s) of Setaria digitata (Von Linstow).

The hatching associated materials generally called the excretory-secretory material (ES) released from the filarial parasite S. digitata along with the microfilariae (mf) under in vitro conditions showed immunosuppression. However the protein from lysate of embryo zone, from which ES materials were released along with the mf showed immunopotentiation in BALB/C mice. The histological analysis of spleen of immunized BALB/C mice showed changes confirming the in vitro findings. The main changes observed in the suppressed spleen were decrease in size and number of lymph follicles and increase in number of reticuloendothelial (RE) cells and cosinophil cells, whereas the main change in the potentiated spleen was an increase in the number of lymph follicles as compared to control spleen. These results clearly indicate that there are two types of ES materials in S. digitata, the native material present in the mature embryo causes immunopotentiation, and the materials released during the hatching process into the hostile environment along with mf causes immunosuppression. The difference in the biological effect between the materials released by lysis and hatching of the embryo opens up fresh understanding about the survival strategies of filarial parasites.

Identification of voltage-sensitive calcium channels in Setaria cervi (Nematoda: Filarioidea) and their role in regulation of spontaneous motility.

Whole worm of Setaria cervi and its nerve muscle preparation exhibit rhythmical movements when suspended in modified Ringer's solution in an isolated organ bath. Deprivation of calcium from the bathing fluid results in gradual reduction in the amplitude and rate of contraction till the movements ceased completely. Similar results were obtained by adding EDTA (5 micrograms/ml) to the bath. The effect was concentration related and was evident early with higher concentration of EDTA. Acetylcholine which increases spontaneous movements of the whole worm as well as N.M. preparation failed to do so when the movements were inhibited either in calcium free solution or in the presence of EDTA. Addition of calcium channel blocker, Nifedipine to the bathing fluid also resulted in concentration related reduction of movements of the preparation of Setaria. Further, the stimulant response of Ach was also reduced by Nifedipine and this too was related to the concentration of calcium channel blocker in the bath. The evidence clearly indicates that presence of calcium is essential for the stimulant response of Ach on S. cervi, which like mammalian tissues contain calcium channels that can be blocked by specific blocking agents.

Effect of acetylcholine, L-glutamine and diethyl carbamazine citrate on the release of microfilariae from Setaria digitata.

Release of microfilariae from adult filarial parasite S. digitata was reversibly arrested in presence of 2 mM L-glutamine. Diethylcarbamazine also showed a similar effect, but at a lower concentration (0.26 mM). Both compounds inhibited activities of protease and leucine amino peptidase enzymes. The only difference being, different concentrations of the two materials were required for comparable effects. Levels of histamine and acetylcholine in parasite were lowered during incubation under in vitro conditions. Direct effect of L-glutamine on the release of mf was also confirmed.

Immune response in mice against hatching associated materials from the filarial parasite Setaria digitata (Von Linstow).

The hatching associated materials (excretory-secretory materials (ES)) from filarial parasite S. digitata showed immunosuppression in BALB/C mice on immunization. The material released along with microfilariae (mf) in Tyrode medium showed maximum immunosuppression while that released in presence of sublethal concentration of diethyl carbamazine (DEC, 0.25 mM) showed initial potentiation followed by suppression. While, protein from lysate of embryo zone from which ES materials were released along with the release of mf originate, showed generalized immunopotentiation in BALB/C mice. The latter suggest a change in the nature of materials of embryonic origin before and after the release of mf.

Monoclonal antibodies against antigenic epitopes common between Setaria cervi and Brugia malayi.

Several common antigens between the bovine (Setaria cervi) and human (Brugia malayi) filarial parasites have been demonstrated [Immunol Investig, 16 (1987) 139]. Hybridoma cell lines producing monoclonal antibodies against such common antigenic epitopes were obtained by immunizing the BALB/c mice with S. cervi antigen, fusing the spleen cells with Sp2/0 myeloma cells and screening the culture supernatants for antibody against both S. cervi and B. malayi antigens by ELISA. Nine monoclonal antibodies directed against antigenic epitopes common between the bovine and human filarial parasites were identified. Two monoclonal antibodies (I3B4 and I5D6) showed reactivity with the antigen(s) present in filariasis patients serum and thus may have potential for detecting circulating antigen in filaria infected individuals.

Ontogenetic transformation of surface epitope expression, an adaptive immunoevasive strategy of filarial parasites.

Filarial nematodes are highly successful in invading, persisting and propagating in human body and eliciting severe ailments. The exact mechanism by which, filarial nematodes evade the host immunity is still ill-defined. The present investigation on the surface antigens of S. digitata revealed the occurrence of shared antigens in the egg, embryo, mf and adult stages. All these stages showed exposed carbohydrate moieties on their surface. In situ localization studies proved that the egg and embryo have exposed surface epitopes whereas the microfilariae and adults did not have any such epitopes. Based on these observations, a model has been proposed on "the surface epitope hiding", as an immunoevasive strategy of the filarial parasite which explains why the naturally shed surface antigens evoke antifilarial immune response in the host even though the system could not recognize the microfilariae or adult parasite due to lack of exposed surface epitopes, permitting the parasite to escape successfully from immune rejection. As treatment with detergents leads to exposure of surface epitopes of parasites, a safe intervention of parasite surface would be an effective strategy for detection and ultimate control of filariasis.