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At least 19 recordsLinked to original sources
PubMed2005

Crystal structures and proposed structural/functional classification of three protozoan proteins from the isochorismatase superfamily.

We have determined the crystal structures of three homologous proteins from the pathogenic protozoans Leishmania donovani, Leishmania major, and Trypanosoma cruzi. We propose that these proteins represent a new subfamily within the isochorismatase superfamily (CDD classification cd004310). Their overall fold and key active site residues are structurally homologous both to the biochemically well-characterized N-carbamoylsarcosine-amidohydrolase, a cysteine hydrolase, and to the phenazine biosynthesis protein PHZD (isochorismase), an aspartyl hydrolase. All three proteins are annotated as mitochondrial-associated ribonuclease Mar1, based on a previous characterization of the homologous protein from L. tarentolae. This would constitute a new enzymatic activity for this structural superfamily, but this is not strongly supported by the observed structures. In these protozoan proteins, the extended active site is formed by inter-subunit association within a tetramer, which implies a distinct evolutionary history and substrate specificity from the previously characterized members of the isochorismatase superfamily. The characterization of the active site is supported crystallographically by the presence of an unidentified ligand bound at the active site cysteine of the T. cruzi structure.

Amino Acid Sequence↗
PubMed2006

Large-scale survey for potentially targetable indels in bacterial and protozoan proteins.

Our previous results demonstrated that some essential, housekeeping proteins from pathogenic microorganisms may contain sizable insertions-deletions in their sequences (compared to close human homologs) that can be responsible for unexpected virulence properties. For example, we found that indel-bearing elongation factor-1alpha from several pathogenic protozoa can activate a human tyrosine phosphatase SHP-1 leading to deactivation of macrophages. On the one hand, these findings allowed development of a strategy for targeting some indel-containing pathogen proteins that have similar human counterparts. On the other hand, the results raised numerous questions regarding the nature and implications of sequence indels in pathogen proteins. In the present study, we conducted a large-scale survey of indels in proteins from 136 bacterial and protozoan genomes. It has been established that sizable insertions and deletions occur in approximately 5-10% of bacterial proteins with close human homologs, while proteins from the protozoan pathogens such as Trypanosoma cruzi, Plasmodium falciparum, and Leishmania donovani exhibit elevated indel content that can reach up to 25%. The finding suggested that the occurrence of sequence indels may be involved in the evolution of pathogenic mechanisms in these protozoa.

Animals↗
PubMed2006

Recombinant Eimeria protozoan protein elicits resistance to acute phlebovirus infection in mice but not hamsters.

A protein antigen from an Eimeria protozoan has recently been reported to induce antitumor activity in mice. This activity most likely results from the strong induction of interkeukin-12 (IL-12) and gamma interferon (IFN-gamma), which are also essential factors in the establishment of protective immunity against viral infection. We evaluated recombinant Eimeria antigen (rEA) as a potential immunotherapeutic agent in mouse and hamster models of acute phleboviral disease. Punta Toro virus (PTV) was highly sensitive to a single dose of nanogram quantities of rEA in the mouse infection model. Intraperitoneal treatment with rEA also reduced virus load and liver damage associated with PTV infection. IL-12 was elicited following exposure of uninfected mice to quantities of rEA of 10 ng or greater, and the levels peaked at between 3 and 8 h postexposure. IFN-gamma release was induced more slowly and required less rEA (1 ng) to produce a significant rise in systemic levels. The induction of IL-12 and IFN-gamma involved in the coordination of innate and adaptive immune responses to microbial pathogens required myeloid differentiation factor 88, a signaling adaptor shared by most members of the Toll-like receptor (TLR) family. Despite encouraging results in the murine system, rEA failed to protect hamsters challenged with PTV. Our findings suggest that hamsters may lack functional TLR11, which has recently been shown to recognize a profilin-like protein homologous to rEA from the protozoan Toxoplasma gondii. Further investigation into the immunostimulatory capacity of rEA in other mammalian systems is necessary.

Acute Disease↗
PubMed2005

PROTOGIM: a novel tool to search motifs and domains in hypothetical proteins of protozoan genomes.

Whole sequencing of protozoan trypanosomatid genomes revealed the presence of several predicted unknown genes coding for hypothetical proteins. Pairwise, alignment-based, computational methods available online are unable to identify the function of these sequences. To detect clues to identify the function of hypothetical proteins, a user-friendly, bioinformatic tool named PROTOzoan Gene Identification Motifs (PROTOGIM, available on http://www.biowebdb.org/protogim ) was developed, which allows the user to search functional patterns of hypothetical proteins through the screening of regular expression in the sequences. The analysis of 1,194 trypanosomatid hypothetical proteins through PROTOGIM resulted in an identification of motifs and domains in 98% of the cases, demonstrating the reliability and accuracy of the employed method. The added value of this tool is the possibility to modify or insert new regular expressions to perform an analysis against either one or several sequences at the same time. An in silico strategy along with biochemical and molecular characterizations creates new possibilities to find the functions of hypothetical proteins at the postgenome era.

Amino Acid Motifs↗
PubMed2000

A novel protein kinase from Brassica juncea stimulated by a protozoan calcium binding protein. Purification and partial characterization.

A novel protein kinase (BjCCaBPk) from etiolated Brassica juncea seedlings has been purified and partially characterized. The purified enzyme migrated on SDS/PAGE as a single band with an apparent molecular mass of 43 kDa. The optimum pH for the kinase activity was 8.0. It was stimulated more than sixfold by the protozoa Entamoeba histolytica calcium binding protein EhCaBP (10.5 nM) but not by calmodulin (CaM) when used at equimolar concentration. Moreover the kinase also did not bind CaM-Sepharose. There was neither inhibition of the kinase activity in the presence of W-7 (a CaM antagonist), KN-62 (a specific calcium/CaM kinase inhibitor) and anti-CaM Ig, nor any effect on BjCCaBPk activity of staurosporine (a protein kinase C inhibitor). Furthermore a CaM-kinase specific substrate, syntide-2, proved to be a poor substrate for the BjCCaBPk compared with histone III-S. The phosphorylation of histone III-S involved serine residues. Southern and Northern blot analysis showed the presence of EhCaBP homologues in Brassica. The data suggest that BjCCaBPk may be a novel protein kinase with an affinity towards a calcium binding protein like EhCaBP.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Pore-forming proteins in pathogenic protozoan parasites.

Pore-forming proteins (PFPs) may play important roles in pathogenesis by protozoan parasites by either directly damaging the plasma membrane of the host cells or ensuring intracellular survival of the parasites by promoting their exit from lysosomal vacuoles. The Leishmania amazonensis pore-forming cytolysin, leishporin, may play a crucial role in the pathogenesis of leishmaniasis.

Animals↗
PubMed2003

Characterization of factors favoring the expression of soluble protozoan tubulin proteins in Escherichia coli.

The alpha- and beta-tubulin genes of the parasitic protozoa Giardia duodenalis, Cryptosporidium parvum, and Encephalitozoon intestinalis have been overexpressed in soluble form using Escherichia coli-based expression systems. Several expression systems were compared in terms of the amount of soluble protein produced with different fusion partners, strains of E. coli BL21, and expression temperatures. The cleavability of the fusion partner was also assessed in terms of post-expression applications of the recombinant protein. The maltose-binding protein (MBP) and glutathione S-transferase (GST) fusion partners produced the highest expression levels for all six proteins without the formation of inclusion bodies. The expression system also provided a means of purifying the soluble protein using affinity and anion-exchange chromatography while minimizing protein losses. The yield and purity were therefore very high for both the MBP and GST systems. The tubulin monomers were demonstrated to be assembly-competent using a standard dimerization assay and also retained full antigenicity with monoclonal antibodies. This study presents several methods which are suitable for producing soluble tubulin monomers and, thus, circumventing the formation of inclusion bodies which necessitates re-folding of the tubulin.

Animals↗
PubMed1999

Identification and characterization of a protozoan uncoupling protein in Acanthamoeba castellanii.

An uncoupling protein (UCP) has been identified in mitochondria from Acanthamoeba castellanii, a nonphotosynthetic soil amoeboid protozoon that, in molecular phylogenesis, appears on a branch basal to the divergence points of plants, animals, and fungi. The existence of UCP in A. castellanii (AcUCP) has been revealed using antibodies raised against plant UCP. Its molecular mass (32,000 Da) was similar to those of plant and mammalian UCPs. The activity of AcUCP has been investigated in mitochondria depleted of free fatty acids. Additions of linoleic acid stimulated state 4 respiration and decreased transmembrane electrical potential (DeltaPsi) in a manner expected from fatty acid cycling-linked H(+) reuptake. The half-maximal stimulation by linoleic acid was reached at 8.1 +/- 0.4 microM. Bovine serum albumin (fatty acid-free), which adsorbs linoleic acid, reversed the respiratory stimulation and correspondingly restored DeltaPsi. AcUCP was only weakly inhibited by purine nucleotides like UCP in plants. A single force-flow relationship has been observed for state 4 respiration with increasing concentration of linoleic acid or of an uncoupler and for state 3 respiration with increasing concentration of oligomycin, indicating that linoleic acid has a pure protonophoric effect. The activity of AcUCP in state 3 has been evidenced by ADP/oxygen atom determination. The discovery of AcUCP indicates that UCPs emerged, as specialized proteins for H(+) cycling, early during phylogenesis before the major radiation of phenotypic diversity in eukaryotes and could occur in the whole eukaryotic world.

Acanthamoeba↗
PubMed1999

Prediction of potential GPI-modification sites in proprotein sequences.

Glycosylphosphatidylinositol (GPI) lipid anchoring is a common posttranslational modification known mainly from extracellular eukaryotic proteins. Attachment of the GPI moiety to the carboxyl terminus (omega-site) of the polypeptide follows after proteolytic cleavage of a C-terminal propeptide. For the first time, a new prediction technique locating potential GPI-modification sites in precursor sequences has been applied for large-scale protein sequence database searches. The composite prediction function (with separate parametrisation for metazoan and protozoan proteins) consists of terms evaluating both amino acid type preferences at sequence positions near a supposed omega-site as well as the concordance with general physical properties encoded in multi-residue correlation within the motif sequence. The latter terms are especially successful in rejecting non-appropriate sequences from consideration. The algorithm has been validated with a self-consistency and two jack-knife tests for the learning set of fully annotated sequences from the SWISS-PROT database as well as with a newly created database "big-Pi" (more than 300 GPI-motif mutations extracted from original literature sources). The accuracy of predicting the effect of mutations in the GPI sequence motif was above 83 %. Lists of potential precursor proteins which are non-annotated in SWISS-PROT and SPTrEMBL are presented on the WWW-page http://www.embl-heidelberg.de/beisenha/gpi/gpi_p rediction. html The algorithm has been implemented in the prototype software "big-Pi predictor" which may find application as a genome annotation and target selection tool.

Algorithms↗
PubMed2006

Prophylactic treatment with recombinant Eimeria protein, alone or in combination with an agonist cocktail, protects mice from Banzi virus infection.

A recombinant Eimeria protozoan protein antigen (rEA) has been shown to have antitumor and antiviral activity. The purpose of this study was to determine the effect of rEA treatment alone or in combination with an agonist cocktail consisting of granulocyte macrophage colony stimulating factor (GM-CSF), interferon gamma (IFN-gamma), interleukin 4 (IL-4), and anti CD-40 antibody, in the treatment of Banzi virus (BV) disease in BALB/c mice. Treatment with rEA resulted in a significant increase in survival, weight gain, and mean day to death in BV-infected mice and resulted in a significant decrease in brain virus titer. Treatment with rEA, in combination with a 4-agonist cocktail, improved disease parameters to a greater degree than rEA treatment alone. The effect of treatment with a reduced concentration of agonist cocktail or fewer components of the agonist cocktail, in combination with rEA, on disease outcome in BV-infected mice was also investigated. Treatment with rEA, alone or in combination with agonist cocktail, 24h after virus challenge did not improve disease. Treatment with rEA, alone or in combination with an agonist cocktail, is efficacious for the prophylaxis of BV infection in mice.

Animals↗
PubMed1995

Sequence of cDNA coding for a 65 kDa adhesive protein for the specific detection of Trichomonas vaginalis by PCR.

A Trichomonas vaginalis cDNA library was constructed and recombinant plaques were screened using rabbit immunoglobulins specific for P65, a protozoan protein involved in pathogenicity that we identified in a previous study. A 1.38 kilobases cDNA fragment coding for the P65 protein was cloned in E. coli and then sequenced. On the basis of of the sequence obtained, six primers were synthesised and used to set up a Polymerase Chain Reaction. The presence of a specific amplicon in all 30 clinical isolates tested shows that P65 is a conserved and stable gene. The reaction is highly sensitive (as few as 5 to 10 parasites can be detected) and specific for Trichomonas vaginalis; the gene coding P65 adhesin can be therefore considered a very good molecular target for polymerase chain reaction-based diagnostic purposes.

Animals↗
PubMed1999

MPS1: a small, evolutionarily conserved zinc finger protein from the protozoan Toxoplasma gondii.

Within the expressed sequence tag (EST) dataset of Toxoplasma gondii we have identified several ESTs encoding a protein similar to the small zinc finger protein MPS1. In human it is suggested that MPS1 plays a role as a transcriptional mediator in response to various growth factors and it is used as a tumour marker in sera from cancer patients. However, in rat a cDNA sequence homologous to MPS1 encodes ribosomal protein S27. To further characterise MPS1 in T. gondii we transformed tachyzoites with a c-Myc-tagged version of the Toxoplasma MPS1 cDNA, flanked by SAG1 sequences. Western blot analysis showed that the Myc-MPS1 was only poorly expressed in the stable transformants. In contrast, Northern blot analysis demonstrated that the Myc-MPS1 mRNA was abundantly transcribed and that the endogenous level of MPS1 mRNA was not affected.

Amino Acid Sequence↗
PubMed1997

Molecular cloning, characterization and localization of PfPK4, an eIF-2alpha kinase-related enzyme from the malarial parasite Plasmodium falciparum.

PfPK4, a protein kinase gene from the human malarial parasite Plasmodium falciparum, has been cloned utilizing oligonucleotide probing. The gene encodes a protein of a predicted length of 1123 amino acids, and within this amino acid sequence all the conserved regions characteristic of protein kinases can be identified. The catalytic kinase domain possesses highest identities (34-37%) with eukaryotic initiation factor-2alpha (eIF-2alpha) kinases, especially haem-regulated inhibitory (HRI) protein kinases. There are two kinase inserts in PfPK4, located at positions common to eIF-2alpha kinases. The first insert separates kinase subdomains IV and VI by 559 amino acids, and the second subdomains VII and VIII by 41 amino acids. Both inserts are larger than their homologues in eIF-2alpha kinases. The sequence of PfPK4 has one putative haemin-binding site. The recombinant protein, expressed in Escherichia coli, phosphorylates a synthetic peptide representing a substrate of eIF-2alpha kinases. Autophosphorylation and substrate phosphorylation are inhibited by haemin. Thus PfPK4 appears to be the first protozoan protein kinase related to eIF-2alpha kinases and might be the first non-mammalian HRI kinase. Western blots indicated that the protein is expressed as major forms of 80 and 90 kDa. Whereas the 80 kDa form is present throughout the intraerythrocytic development and in merozoites, the two 90 kDa forms are only found in mature parasites. One of the latter is also present in the membrane fraction of erythrocytes harbouring segmenters. Confocal microscopy detected the protein distributed throughout the trophozoite, whereas it was found in discrete foci (punctate distribution) in segmenters. PfPK4 co-localizes with P. falciparum 83 kDa antigen/apical membrane antigen-1 at the apical complex in segmenters and merozoites, but does not co-localize with rhoptry-associated protein-1.

Amino Acid Sequence↗
PubMed2005

Isolation of integral membrane proteins of Leishmania promastigotes and evaluation of their prophylactic potential in hamsters against experimental visceral leishmaniasis.

The integral membrane proteins (IMP's) of promastigotes of two virulent strain of Leishmania (L.) donovani Dd8 and Leishmania (L.) infantum LV9 and one avirulent viscerotropic strain Leishmania tropica UR6 were extracted by phase separation technique using a non-ionic detergent "Triton X-114". This detergent is homogeneous at 0 degrees C but divides in an aqueous phase and a detergent phase at above 20 degrees C. The phase partition resulted in solubilisation of hydrophilic proteins in aqueous phase, and IMP's with an amphiphilic nature were recovered in the detergent phase. The strain wise quantitative recovery rates of IMP's were estimated. These proteins were purified by chill methanol centrifugation and used as vaccinogen, alone or in combination with adjuvant against L. donovani challenge in hamster model. Among all the combinations, hamsters immunised with IMP of L. donovani (Dd8) in combination with CFA resulted in 75% parasite inhibition in spleen (P <0.001), however, 61.14% (P <0.001) and 77.60% (P <0.001) parasitic inhibition was achieved in liver and bone marrow respectively as compared to their unvaccinated counter part. Similar combinations with UR6 and LV9 strain inhibited the parasite establishment up to 65.12% (P <0.001) and 66.87% (P <0.001), respectively on splenic site. The specific IgG level against (Dd8 strain) soluble leishmania promastigote antigen was monitored at different stages by enzyme linked immunosorbent assay (ELISA) corresponds to the level of parasitic establishment. Similar observations were made in cases of LV9 and UR6 strains. However, significant lymphoproliferative response to IMPs of Dd8 strain (SI 3.5-4.9, P <0.001) was noticed in all IMP + CFA vaccinated animals. Thus, this study will provide a lead for more manipulative trials to develop a subunit vaccine against the fatal disease.

Animals↗
PubMed2003

Expression of a recombinant IRP-like Plasmodium falciparum protein that specifically binds putative plasmodial IREs.

Plasmodium falciparum iron regulatory-like protein (PfIRPa, accession AJ012289) has homology to a family of iron-responsive element (IRE)-binding proteins (IRPs) found in different species. We have previously demonstrated that erythrocyte P. falciparum PfIRPa binds a mammalian consensus IRE and that the binding activity is regulated by iron status. In the work we now report, we have cloned a C-terminus histidine-tagged PfIRPa and overexpressed it in a bacterial expression system in soluble form capable of binding IREs. To overexpress PfIRPa, we used the T7 promoter-driven vector, pET28a(+), in conjunction with the Rosetta(DE3)pLysS strain of E. coli, which carries extra copies of tRNA genes usually found in organisms such as P. falciparum whose genome is (A+T)-rich. The histidine-tagged recombinant protein (rPfIRPa) in soluble form was partially purified using His-bind resin. We searched the plasmodial database, plasmoDB, to identify sequences capable of forming IRE loops using a specially developed algorithm, and found three plasmodial sequences matching the search criteria. In gel retardation assays, rPfIRPa bound three 32P-labeled putative plasmodial IREs with affinity exceeding the affinity for the mammalian consensus IRE. The binding was concentration-dependent and was not inhibited by heparin, an inhibitor of non-specific binding. Immunodepletion of rPfIRPa resulted in substantial inhibition of the signal intensity in the gel retardation assays and in Western blot-determinations of rPfIRPa protein levels. Endogenous PfIRPa retained all three putative 32P-IREs at the same position on the gel as the recombinant PfIRPa.

Animals↗
PubMed2003

Perforin and gamma interferon are critical CD8+ T-cell-mediated responses in vaccine-induced immunity against Leishmania amazonensis infection.

Previous studies have demonstrated that protection against New World leishmaniasis caused by Leishmania amazonensis can be elicited by immunization with the developmentally regulated Leishmania amastigote antigen, P-8. In this study, several independent experimental approaches were employed to investigate the protective immunological mechanisms involved. T-cell subset depletion experiments clearly indicate that elicitation of CD8(+) (as well as CD4(+)) effector responses is required for protection. Further, mice lacking beta(2)-microglobulin (and hence deficient in major histocompatibility complex class I antigen presentation) were not able to control a challenge infection after vaccination, indicating an essential protective role for CD8(+) T effector responses. Analysis of the events ongoing at the cutaneous site of infection indicated a changing cellular dynamic involved in protection. Early postinfection in protectively vaccinated mice, a predominance of CD8(+) T cells, secreting gamma interferon (IFN-gamma) and expressing perforin, was observed at the site of infection; subsequently, activated CD4(+) T cells producing IFN-gamma were primarily found. As protection correlated with the ratio of total IFN-gamma-producing cells (CD4(+) and CD8(+) T cells) to macrophages found at the site of infection, a role for IFN-gamma was evident; in addition, vaccination of IFN-gamma-deficient mice failed to provide protection. To further assess the effector mechanisms that mediate protection, mice deficient in perforin synthesis were examined. Perforin-deficient mice vaccinated with the P-8 antigen were unable to control infection. Thus, the elicitation of CD8(+) T cell effector mechanisms (perforin, IFN-gamma) are clearly required in the protective immune response against L. amazonensis infection in vaccinated mice.

Animals↗
PubMed2001

HBV core particles as a carrier for B cell/T cell epitopes.

In the middle 80s, recombinant hepatitis B virus cores (HBc) gave onset to icosahedral virus-like particles (VLPs) as a basic class of non-infectious carriers of foreign immunological epitopes. The recombinant HBc particles were used to display immunodominant epitopes of hepatitis B, C, and E virus, human rhinovirus, papillomavirus, hantavirus, and influenza virus, human and simian immunodeficiency virus, bovine and feline leukemia virus, foot-and-mouth disease virus, murine cytomegalovirus and poliovirus, and other virus proteins, as well as of some bacterial and protozoan protein epitopes. Practical applicability of the HBc particles as carriers was enabled by their ability to high level synthesis and correct self-assembly in heterologous expression systems. The interest in the HBc VLPs was reinforced by the resolution of their fine structure by electron cryomicroscopy and X-ray crystallography, which revealed an unusual alpha-helical organization of dimeric units of HBc shells, alternative packing into icosahedrons with T = 3 and T = 4 symmetry, and the existence of long protruding spikes. The tips of the latter seem to be the optimal targets for the display of foreign sequences up to 238 amino acid residues in length. Combination of numerous experimental data on epitope display with the precise structural information enables a knowledge-based design of diagnostic, and vaccine and gene therapy tools on the basis of the HBc particles.

Amino Acid Sequence↗