Correction: The Secreted Triose Phosphate Isomerase of Brugia malayi Is Required to Sustain Microfilaria Production In Vivo
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PROVIDER: S-EPMC4002453 | biostudies-literature | 2014 Apr
REPOSITORIES: biostudies-literature
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Project description:Human lymphatic filariasis is a major tropical disease transmitted through mosquito vectors which take up microfilarial larvae from the blood of infected subjects. Microfilariae are produced by long-lived adult parasites, which also release a suite of excretory-secretory products that have recently been subject to in-depth proteomic analysis. Surprisingly, the most abundant secreted protein of adult Brugia malayi is triose phosphate isomerase (TPI), a glycolytic enzyme usually associated with the cytosol. We now show that while TPI is a prominent target of the antibody response to infection, there is little antibody-mediated inhibition of catalytic activity by polyclonal sera. We generated a panel of twenty-three anti-TPI monoclonal antibodies and found only two were able to block TPI enzymatic activity. Immunisation of jirds with B. malayi TPI, or mice with the homologous protein from the rodent filaria Litomosoides sigmodontis, failed to induce neutralising antibodies or protective immunity. In contrast, passive transfer of neutralising monoclonal antibody to mice prior to implantation with adult B. malayi resulted in 60-70% reductions in microfilarial levels in vivo and both oocyte and microfilarial production by individual adult females. The loss of fecundity was accompanied by reduced IFNγ expression by CD4⁺ T cells and a higher proportion of macrophages at the site of infection. Thus, enzymatically active TPI plays an important role in the transmission cycle of B. malayi filarial parasites and is identified as a potential target for immunological and pharmacological intervention against filarial infections.
| S-EPMC3937304 | biostudies-literature
Project description:The refolding and reactivation of the glycolytic enzyme triose phosphate isomerase (EC 5.3.1.1) has been studied. The enzyme, which is a dimer, is disaggregated and unfolded in solutions of guanidinium chloride. Unfolding, followed by changes in E(233), took place quite rapidly in 3m-guanidinium chloride (i.e. with a half-life of about 1 min). Refolding also took place rapidly when the solution was diluted about tenfold; two first-order processes could be resolved. Regain of enzymic activity was followed by diluting the solution of the denatured enzyme in guanidinium chloride into assay mixture. The half-life (i.e. the time when the activity was half the final activity) depended markedly on the concentration of protein at low concentrations (about 100ng/ml), but at higher concentrations the half-life became independent of concentration. Thus at low concentrations dimerization was a rate-determining step and this is taken to indicate that the monomers showed little or no activity under these conditions. The rate of regain of enzymic activity was the same as the rate of the slower process of refolding, which was detected spectroscopically. The native enzyme was resistant to proteolysis; high concentrations of subtilisin prevented regain of activity, but at lower concentrations refolding competed with proteolysis.
| S-EPMC1165801 | biostudies-literature
Project description:We have previously shown that the microfilarial (mf) stage of Brugia malayi can inhibit the mammalian target of rapamycin (mTOR; a conserved serine/threonine kinase critical for immune regulation and cellular growth) in human dendritic cells (DC) and we have proposed that this mTOR inhibition is associated with the DC dysfunction seen in filarial infections. Extracellular vesicles (EVs) contain many proteins and nucleic acids including microRNAs (miRNAs) that might affect a variety of intracellular pathways. Thus, EVs secreted from mf may elucidate the mechanism by which the parasite is able to modulate the host immune response during infection. EVs, purified from mf of Brugia malayi and confirmed by size through nanoparticle tracking analysis, were assessed by miRNA microarrays and shown to be enriched (>2fold, p-value<0.05, FDR=0.05) for miR100, miR71, miR34, and miR7. The microarray analysis compared mf-derived EVs and mf supernatant. After confirming their presence in EVs using qPCR for these miRNA targets, web-based target predictions (using MIRPathv3, TarBAse and MicroT-CD) predicted that miR100 targeted mTOR and its downstream regulatory protein 4E-BP1 respectively. Our previous data with live parasites demonstrated that mf downregulate the phosphorylation of mTOR and its downstream effectors. Additionally, our proteomic analysis of the mf-derived EVs revealed the presence of proteins commonly found in these vesicles. We confirmed internalization of mf-derived EVs by human DCs and monocytes using confocal microscopy and flow cytometry, and further demonstrated through flow cytometry, that mf-derived EVs downregulate the phosphorylation of mTOR in human monocytes (THP-1 cells) to the same degree that rapamycin (a known mTOR inhibitor) does. Our data collectively suggest that mf release EVs that interact with host cells, such as DC, to modulate host responses.
2021-01-12 | PXD021844 | Pride
Project description:Filarial parasites like Brugia pahangi and Brugia malayi can infect dogs. Adults of Brugia genus resides in the lymphatic system and microfilariae, in blood. There are increasing reports of detection of B. malayi microfilariae in dogs. A study was undertaken to compare the efficacy of repeated oral dosing of ivermectin (IVT) and diethylcarbamazine (DEC), individually and in combination against naturally infected B. malayi microfilariae in dogs. The species of the microfilariae was confirmed by acid phosphatase staining and polymerase chain reaction. The three treatment groups were 200 mcg/kg body weight IVT daily for 14 days (I), 6.6 mg/kg body weight DEC daily for 14 days (II) and IVT and DEC together in the same dose for a period of 5 days (III). Microfilarial status of the peripheral blood was assessed on the 0th, 7th, 14th and 21st day. Haematological parameters were measured on day zero and on the 21st day. Though, all the three treatment groups showed a reduction in the microfilarial concentration through the study period, complete absence of detectable microfilaremia was not noticed in any of the three groups by 21st day. Among the haematological parameters, statistically significant difference was observed in the post-treatment means of haemoglobin levels of group III when compared with group II. Since group III regime (IVT + DEC) was shorter and just as effective as the longer ones, it is considered superior to the other two.
| S-EPMC6841773 | biostudies-literature
Project description:As compared with plant system, triose phosphate isomerase (TPI), a crucial enzyme of glycolysis, has been well studied in animals. In order to characterize TPI in plants, a full-length cDNA encoding OscTPI was cloned from rice and expressed in E. coli. The recombinant OscTPI was purified to homogeneity and it showed Km value of 0.1281 ± 0.025 µM, and the Vmax value of 138.7 ± 16 µmol min (-1) mg (-1) which is comparable to the kinetic values studied in other plants. The OscTPI was found to be exclusively present in the cytoplasm when checked with the various methods. Functional assay showed that OscTPI could complement a TPI mutation in yeast. Real time PCR analysis revealed that OscTPI transcript level was regulated in response to various abiotic stresses. Interestingly, it was highly induced under different concentration of methylglyoxal (MG) stress in a concentration dependent manner. There was also a corresponding increase in the protein and the enzyme activity of OscTPI both in shoot and root tissues under MG stress. Our result shows that increases in MG leads to the increase in TPI which results in decrease of DHAP and consequently decrease in the level of toxic MG.
| S-EPMC3493422 | biostudies-literature
Project description:Visceral leishmaniasis (VL) is one of the most important parasitic diseases with approximately 350 million people at risk. Due to the non availability of an ideal drug, development of a safe, effective, and affordable vaccine could be a solution for control and prevention of this disease. In this study, a potential Th1 stimulatory protein- Triose phosphate isomerase (TPI), a glycolytic enzyme, identified through proteomics from a fraction of Leishmania donovani soluble antigen ranging from 89.9-97.1 kDa, was assessed for its potential as a suitable vaccine candidate. The protein- L. donovani TPI (LdTPI) was cloned, expressed and purified which exhibited the homology of 99% with L. infantum TPI. The rLdTPI was further evaluated for its immunogenicity by lymphoproliferative response (LTT), nitric oxide (NO) production and estimation of cytokines in cured Leishmania patients/hamster. It elicited strong LTT response in cured patients as well as NO production in cured hamsters and stimulated remarkable Th1-type cellular responses including IFN-ã and IL-12 with extremely lower level of IL-10 in Leishmania-infected cured/exposed patients PBMCs in vitro. Vaccination with LdTPI-DNA construct protected naive golden hamsters from virulent L. donovani challenge unambiguously (∼90%). The vaccinated hamsters demonstrated a surge in IFN-ã, TNF-á and IL-12 levels but extreme down-regulation of IL-10 and IL-4 along with profound delayed type hypersensitivity and increased levels of Leishmania-specific IgG2 antibody. Thus, the results are suggestive of the protein having the potential of a strong candidate vaccine.
| S-EPMC3454378 | biostudies-literature
Project description:Lymphatic filariasis results in an altered lymphatic system and the abnormal enlargement of body parts, causing pain, serious disability and social stigma. Effective vaccines are still not available nowadays, drugs against the disease is required. Protein disulfide isomerase (PDI) is an essential catalyst of the endoplasmic reticulum which is involved in folding and chaperone activities in different biological systems. Here, we report the enzymatic characterization of a Brugia malayi Protein disulfide isomerase (BmPDI), which was expressed and purified from Escherichia coli BL21 (DE3). Western blotting analysis showed the recombinant BmPDI could be recognized by anti-BmPDI Rabbit serum. The rBmPDI exhibited an optimum activity at pH 8 and 40 °C. The enzyme was inhibited by aurin and PDI inhibitor. Recombinant BmPDI showed interaction with recombinant Brugia malayi calreticulin (rBmCRT). The three-dimensional model for BmPDI and BmCRT was generated by homology modelling. A total of 25 hydrogen bonds were found to be formed between two interfaces. There are 259 non-bonded contacts present in the BmPDI-BmCRT complex and 12 salt bridges were formed in the interaction.
| S-EPMC5547380 | biostudies-literature
Project description:Vector-borne, filarial nematode diseases cause significant disease burdens in humans and domestic animals worldwide. Although there is strong direct evidence of parasite-driven immunomodulation of mammalian host responses, there is less evidence of parasite immunomodulation of the vector host. We have previously reported that all life stages of Brugia malayi, a filarial nematode and causative agent of Lymphatic filariasis, secrete extracellular vesicles (EVs). Here we investigate the immunomodulatory effects of microfilariae-derived EVs on the vector host Aedes aegypti. RNA-seq analysis of an Ae. aegypti cell line treated with B. malayi microfilariae EVs showed differential expression of both mRNAs and miRNAs. AAEL002590, an Ae. aegypti gene encoding a serine protease, was shown to be downregulated when cells were treated with biologically relevant EV concentrations in vitro. Injection of adult female mosquitoes with biologically relevant concentrations of EVs validated these results in vivo, recapitulating the downregulation of AAEL002590 transcript. This gene was predicted to be involved in the mosquito phenoloxidase (PO) cascade leading to the canonical melanization response and correspondingly, both suppression of this gene using RNAi and parasite EV treatment reduced PO activity in vivo. Our data indicate that parasite-derived EVs interfere with critical immune responses in the vector host, including melanization.
| S-EPMC10232515 | biostudies-literature
Project description:Heat stress causes dysfunction of the carbon-assimilation metabolism. As a member of Calvin-Benson-Bassham (CBB) cycle, the chloroplast triose phosphate isomerases (TPI) catalyse the interconversion of glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP). The tomato (Solanum lycopersicum) genome contains two individual SlTPI genes, Solyc10g054870 and Solyc01g111120, which encode the chloroplast-located proteins SlTPI1 and SlTPI2, respectively. The tpi1 and tpi2 single mutants had no visible phenotypes, but the leaves of their double mutant lines tpi1tpi2 had obviously reduced TPI activity and displayed chlorotic variegation, dysplasic chloroplasts and lower carbon-assimilation efficiency. In addition to altering carbon metabolism, proteomic data showed that the loss of both SlTPI1 and SlTPI2 severely affected photosystem proteins, reducing photosynthetic capacity. None of these phenotypes was evident in the tpi1 or tpi2 single mutants, suggesting that SlTPI1 and SlTPI2 are functionally redundant. However, the two proteins differed in their responses to heat stress; the protein encoded by the heat-induced SlTPI2 showed a higher level of thermotolerance than that encoded by the heat-suppressed SlTPI1. Notably, heat-induced transcription factors, SlWRKY21 and SlHSFA2/7, which negatively regulated SlTPI1 expression and positively regulated SlTPI2 expression, respectively. Our findings thus reveal that SlTPI1 and SlTPI2 have different thermostabilities and expression patterns in response to heat stress, which have the potential to be applied in thermotolerance strategies in crops.
| S-EPMC10893939 | biostudies-literature
Project description:Relatively little is known about the filarial proteins that interact with the human host. Although the filarial genome has recently been completed, protein profiles have been limited to only a few recombinants or purified proteins of interest. Here, we describe a large-scale proteomic analysis using microcapillary reverse-phase liquid chromatography-tandem-mass spectrometry to identify the excretory-secretory (ES) products of the L3, L3 to L4 molting ES, adult male, adult female, and microfilarial stages of the filarial parasite Brugia malayi. The analysis of the ES products from adult male, adult female, microfilariae (Mf), L3, and molting L3 larvae identified 852 proteins. Annotation suggests that the functional and component distribution was very similar across each of the stages studied; however, the Mf contributed a higher proportion to the total number of identified proteins than the other stages. Of the 852 proteins identified in the ES, only 229 had previous confirmatory expressed sequence tags (ESTs) in the available databases. Moreover, this analysis was able to confirm the presence of 274 "hypothetical" proteins inferred from gene prediction algorithms applied to the B. malayi (Bm) genome. Not surprisingly, the majority (160/274) of these "hypothetical" proteins were predicted to be secreted by Signal IP and/or SecretomeP 2.0 analysis. Of major interest is the abundance of previously characterized immunomodulatory proteins such as ES-62 (leucyl aminopeptidase), MIF-1, SERPIN, glutathione peroxidase, and galectin in the ES of microfilariae (and Mf-containing adult females) compared to the adult males. In addition, searching the ES protein spectra against the Wolbachia database resulted in the identification of 90 Wolbachia-specific proteins, most of which were metabolic enzymes that have not been shown to be immunogenic. This proteomic analysis extends our knowledge of the ES and provides insight into the host-parasite interaction.
| S-EPMC2659452 | biostudies-literature