N-terminal palmitoylation is required for Toxoplasma gondii HSP20 inner membrane complex localization.

Toxoplasma gondii is an obligate intracellular parasite and the causative agent of toxoplasmosis. Protein palmitoylation is known to play roles in signal transduction and in enhancing the hydrophobicity of proteins thus contributing to their membrane association. Global inhibition of protein palmitoylation has been shown to affect T. gondii physiology and invasion of the host cell. However, the proteins affected by this modification have been understudied. This paper shows that the small heat shock protein 20 from T. gondii (TgHSP20) is synthesized as a mature protein in the cytosol and is palmitoylated in three cysteine residues. However, its localization at the inner membrane complex (IMC) is dependent only on N-terminal palmitoylation. Absence or incomplete N-terminal palmitoylation causes TgHSP20 to partially accumulate in a membranous structure. Interestingly, TgHSP20 palmitoylation is not responsible for its interaction with the daughter cells IMCs. Together, our data describe the importance of palmitoylation in protein targeting to the IMC in T. gondii.

Protein palmitoylation inhibition by 2-bromopalmitate alters gliding, host cell invasion and parasite morphology in Toxoplasma gondii.

Protein palmitoylation is the reversible covalent attachment of palmitic acid onto proteins. This post-translational modification has been shown to play a part in diverse processes such as signal transduction, cellular localization and regulation of protein activity. Although many aspects of protein palmitoylation have been identified in mammalian and yeast cells, little is known of this modification in Toxoplasma gondii. In order to determine the functional role of protein palmitoylation in T. gondii, tachyzoites were treated with the palmitoylation inhibitor 2-bromopalmitate (2-BP). Parasites treated with 2-BP displayed a significant increase in non-circular trails which were longer than those trails left by non-treated parasites. Furthermore, 2-BP treatment reduced the invasion process to the host cells. Long-term treatment of intracellular tachyzoites resulted in major changes in parasite morphology and shape in a dose-dependent manner. These results suggest that palmitoylation could be modifying proteins that are key players in gliding, invasion and cytoskeletal proteins in T. gondii.

PCR detection of Tritrichomonas foetus in preputial bull fluid without prior DNA isolation.

Tritrichomonosis is a widespread, economically important venereal disease caused by Tritrichomonas foetus. The traditional diagnosis of this disease, which causes infertility and abortion in cattle, is based on the culture of the parasite. This process is time consuming, has low sensitivity, and is prone to contamination with intestinal or coprophilic trichomonadid protozoa, resulting in false positive diagnostics of T. foetus. In order to avoid the shortcomings of the traditional method, we developed a simple PCR assay based on TFR3 and TFR4 primers, which does not require parasite culturing. The sensitivity of the PCR assay resulted comparable to that of the classical method, being able to detect as few as five T. foetus parasites. In addition the method is highly specific. The analysis of preputial fluid washing samples showed that 58 out of 203 samples were positive by both, the PCR and the culture method (+/+), 9 samples were positive by PCR and negative by the traditional method (+/-) and only one sample resulted negative by PCR and negative by culture (-/+). The samples for the PCR assay can be stored for a week at 4 degrees C or 72h at room temperature. In summary, our study demonstrated that the PCR assay is an effective method for the diagnosis of T. foetus from preputial samples, and that it compares advantageously to the classical method.

High polymorphism in genes encoding antigen B from human infecting strains of Echinococcus granulosus.

Echinococcus granulosus antigen B (AgB) is encoded by a gene family and is involved in the evasion of the host immune response. E. granulosus exists as a number of strains (G1-G10) that differ in biological characteristics. We used PCR-SSCP followed by DNA sequencing to evaluate sequence variation and transcription profile of AgB in 5 E. granulosus strains. Twenty-four genomic sequences were isolated and clustered in 3 groups related to 2 of the 5 reported AgB genes. AgB4 genes were present in almost all strains, whereas AgB2 were present as functional genes exclusively in G1/G2 cluster, and as non-functional genes in G5 and the G6/G7 cluster, suggesting inter-strain variation. The AgB transcription patterns, analysed by RT-PCR, showed that AgB2 and AgB4 genes were transcribed in G1, while only the AgB4 gene was transcribed in G7 strain. Cysts from the same strain or cluster shared more genomic and cDNA variants than cysts from different strain or cluster. The level of nucleotide and deduced amino acid sequence variation observed is higher than that reported so far for coding genes of other helminths. Neutrality was rejected for AgB2 genes. These data show the genetic polymorphism of antigen-coding genes among genetically characterized strains of E. granulosus.

Differential subcellular localization of members of the Toxoplasma gondii small heat shock protein family.

The results of this study describe the identification and characterization of the Toxoplasma gondii alpha-crystallin/small heat shock protein (sHsp) family. By database (www.toxodb.org) search, five parasite sHsps (Hsp20, Hsp21, Hsp28, Hsp29, and the previously characterized Hsp30/Bag1) were identified. As expected, they share the homologous alpha-crystallin domain, which is the key characteristic of sHsps. However, the N-terminal segment of each protein contains unique characteristics in size and sequence. Most T. gondii sHsps are constitutively expressed in tachyzoites and fully differentiated bradyzoites, with the exception of Hsp30/Bag1. Interestingly, by subcellular localization we observed that T. gondii sHsps are located in different compartments. Hsp20 is located at the apical end of the cell, Hsp28 is located inside the mitochondrion, Hsp29 showed a membrane-associated labeling, and Hsp21 appeared throughout the cytosol of the parasites. These particular differences in the immunostaining patterns suggest that their targets and functions might be different.

Leishmania infantum heat shock protein 83 for the serodiagnosis of tegumentary leishmaniasis.

The serologic assay is an important tool in the diagnosis of leishmaniasis. One of the most commonly used tests is enzyme-linked immunosorbent assay (ELISA). Since total Leishmania promastigotes are used as antigen in the routine assay, false-positive reactions are frequent due to cross-reaction with sera from other diseases, mainly Chagas' disease. Therefore, an antigen that determines less cross-reactivity has been pursued for the serodiagnosis of leishmaniasis. In the present study we analyzed the use of recombinant Leishmania infantum heat shock protein (Hsp) 83 in ELISA for the serodiagnosis of cutaneous (N = 12) and mucocutaneous leishmaniasis (N = 14) and we observed the presence of anti-L. infantum Hsp 83 antibodies in all samples as well as anti-Leishmania total antigen antibodies. When cross-reactivity was tested, chronic Chagas' disease patients (N = 10) did not show any reactivity. Therefore, we consider this L. infantum Hsp 83 to be a good antigen for routine use for serodiagnosis of tegumentary leishmaniasis.

Leishmania infantum heat shock protein 83 for the serodiagnosis of tegumentary leishmaniasis

The serologic assay is an important tool in the diagnosis of leishmaniasis. One of the most commonly used tests is enzyme-linked immunosorbent assay (ELISA). Since total Leishmania promastigotes are used as antigen in the routine assay, false-positive reactions are frequent due to cross-reaction with sera from other diseases, mainly Chagas' disease. Therefore, an antigen that determines less cross-reactivity has been pursued for the serodiagnosis of leishmaniasis. In the present study we analyzed the use of recombinant Leishmania infantum heat shock protein (Hsp) 83 in ELISA for the serodiagnosis of cutaneous (N = 12) and mucocutaneous leishmaniasis (N = 14) and we observed the presence of anti-L. infantum Hsp 83 antibodies in all samples as well as anti-Leishmania total antigen antibodies. When cross-reactivity was tested, chronic Chagas' disease patients (N = 10) did not show any reactivity. Therefore, we consider this L. infantum Hsp 83 to be a good antigen for routine use for serodiagnosis of tegumentary leishmaniasis.

Immunodiagnosis of fasciolosis using recombinant procathepsin L cystein proteinase.

Cathepsin L1, a cysteine protease secreted by the gastrodermis of juvenile and adult Fasciola hepatica, was expressed in Escherichia coli as a fusion protein containing the proregion, supplied with six histidyl residues at the N-terminal end (rproCL1). In this study we tested its potential as antigen for the serologic diagnosis of F. hepatica infections by enzyme-linked immunosorbent assay (ELISA). The analyzed human sera included 16 positive samples, 99 negative controls and 111 from individuals affected by other parasitic and non parasitic diseases. The sensitivity and specificity of the rproCL1-ELISA were 100%. We also assessed the ability to detect antibodies in sera from 10 experimentally infected sheep, obtaining preliminary results that shown a response since the third week post infection in all the studied animals. Therefore, the recombinant rproCL1-based ELISA could be a standardized test for the accurate diagnosis of fasciolosis.

High level of expression of the Toxoplasma gondii-recombinant Rop2 protein in Escherichia coli as a soluble form for optimal use in diagnosis.

The rhoptry 2 protein (Rop2) is an interesting protein of Toxoplasma gondii that is involved in the parasite invasion of host cell, it has three T-cell epitopes and high antigenic value. However, the expression of Rop2 as a recombinant protein in Escherichia coli is not an easy task, showing low levels of expression or degradation and solubility problems. Using a recombinant Rop2(196-561) fused to 6 histidine residues, we showed high levels of expression in bacteria growing in terrific broth. rRop2(196-561) was purified mainly as a soluble product and in high concentrations (approx 1 mg/mL) under native conditions (40 mM imidazol in phosphate buffer). However, after a cycle of freezing-thawing rRop2(196-561) became insoluble. When glycerol was added to 26%, immediately after purification, the protein stayed soluble after cycles of freezing-thawing. Finally, it was demonstrated that under these conditions soluble rRop2(196-561) keeps its diagnostic value in contrast with the insoluble protein.

Analysis of the adjuvant effect of recombinant Leishmania infantum Hsp83 protein as a tool for vaccination.

The properties of Leishmania infantum hsp83 (LiHsp83) to elicit an immune response against a fused reporter antigen, maltose binding protein (MBP), was studied. CF1 mice were immunized with different purified recombinant proteins: MBP, LiHsp83 and MBP fused to LiHsp83 (MBP-LiHsp83). Serum samples were obtained at days 0, 21, 28, 60, 90, 120 and 150 post-immunization. MBP-LiHsp83 fusion protein elicited a strong humoral response against MBP, higher than that one obtained in mice immunized with MBP alone or MBP mixed with LiHsp83, showing the secretion of both anti-MBP IgG2a and IgG1 isotypes (IgG2a/IgG1 ratio: 2:1). This response was specific for recombinant proteins and was maintained for at least 150 days, whereas the reactivity in mice immunized with MBP alone dissapeared at day 90. After in vitro stimulation with MBP, spleen cells from MBP-LiHsp83 immunized mice showed higher proliferation indices and produced higher secretion of IFN-gamma than spleen cells from either control or MBP-immunized mice. In all groups of mice IL-4 was undetectable. Thus we consider that LiHsp83 may be a promising candidate to be used as carrier of fused antigens for adjuvant-free vaccination.

Expression of a cDNA encoding a Toxoplasma gondii protein belonging to the heat-shock 90 family and analysis of its antigenicity.

A cDNA clone (Tgzy85d11.r1) obtained from the Toxoplasma Expressed Sequence Tag project was chosen due to its homology with proteins of the heat shock 90 family. The cDNA encodes 137 amino acids of the C-terminal portion of the Toxoplasma Hsp90 protein (TgHsp90). Serum samples obtained from orally infected BALB/c and C57BL/6 mice showed reactivity against a recombinant TgHsp90 (rTgHsp90) after 8 weeks postinfection. Isotype analysis showed an anti-rTgHsp90 IgG2a/IgG3 response in infected BALB/c and anti-rTgHsp90 IgG1/IgG2a/IgG2b response in infected C57BL/6 mice. Serum samples from individuals chronically and putative acutely infected with T. gondii showed a similar anti-rTgHsp90 IgG response. Our work identifies TgHsp90 as a novel parasite antigen that seems to elicit a higher relation of anti-TgHsp90/anti-T. gondii IgGs during chronic infection in comparison with the acute stage.

Molecular cloning, sequencing and expression of a serine proteinase inhibitor gene from Toxoplasma gondii.

A cDNA clone from a Toxoplasma gondii tachyzoite cDNA library encoding a serine proteinase inhibitor (serpin) was isolated. The 1376 bp cDNA sequence encodes a 294 amino acid protein with a putative signal peptide of 23 amino acids resulting in a mature protein with a predicted mass of 30,190 Da and a pI of 4.86. This protein has internal sequence similarity of residues 30-66, 114-150, 181-217 and 247-283 indicating a four-domain structure. The four domains exhibit high identity to serine proteinase inhibitors belonging to the non-classical Kazal-type family. The gene is single copy in the tachyzoite haploid genome of RH strain and was amplified by polymerase chain reaction (PCR). Several introns were identified. The sequence encoding the mature protein was amplified by PCR, cloned into the pQE30 vector and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was used in immunoblot assay and two bands of 38 and 42 kDa were detected in a whole parasite homogenate. The recombinant protein showed trypsin-inhibitory activity, one of the two potential specificities. We discuss the possible roles that T. gondii serpin(s) may play in the survival of the tachyzoites in the host.

Characterisation of a novel interspersed Toxoplasma gondii DNA repeat with potential uses for PCR diagnosis and PCR-RFLP analysis.

A novel Toxoplasma gondii interspersed repeat element (TgIRE), present in most of the tachyzoite chromosomes, was characterised. Two regions on the TgIRE sequence showed high identity to two different T. gondii expressed sequence tag cDNAs of unknown function, which seems to be TgIRE pseudogenes. Two set of primers were designed, 2-2' and 2-3, that amplify products of 1.02 and 0.62 kb, respectively. T. gondii DNA from RH and Me49 strains was amplified with TgIRE 2-2' primers, and the respective 1.02 kb products were digested with several endonucleases. Different fragment patterns by gel electrophoresis were found only with MboI. Sensitivity analysis revealed that the set 2-3 was more sensitive than 2-2', detecting by gel visualisation the amount of DNA equivalent to 1 and 10 parasites, respectively.

Arrays of repetitive DNA elements in the largest chromosomes of Toxoplasma gondii.

A novel tandemly repeated DNA structure of Toxoplasma gondii that meets the requirements assigned for satellital DNA was characterized. A DNA fragment of 1002 bp contains two different elements of repetitive DNA families named ABGTg7 and ABGTg8.2. Both repeats are members of a more complex tandem structure where ABGTg7-like monomers can be arranged either as direct tandems or flanked by other related or non-related repeats. Pulse-field gel electrophoresis analysis showed that these repeats hybridize with the largest T. gondii chromosomes. Bal31 sensitivity assays indicated that these elements are located near the telomeres and along other regions too. Five genomic lambda phages were isolated and two different completed clusters of the repeated structure were analyzed.

Immunostimulatory properties of the Leishmania infantum heat shock proteins HSP70 and HSP83.

Emerging evidence indicates that the heat shock proteins (HSPs), a set of highly evolutionary conserved proteins, are playing essential roles in both normal processes of the immune system and specific immune responses. In a previous work, we demonstrated that the Leishmania infantum HSP70 possesses remarkable immunostimulatory properties. In the present work, we have extended the study to another HSP from this parasite, the HSP83. We show that this protein also has an adjuvant effect to an accompanying protein by stimulation of the humoral response when both proteins are fused and co-administered to BALBjc mice. The analysis of the IgG isotypes, IgG1 and IgG2a, indicated that the immunisations with the Leishmania HSPs, mainly the HSP70, potentiate a Thl-type response. It was found that the amino-terminal domain of the HSP70, the most evolutionary conserved region of the molecule, maintains the ability to stimulate the humoral response, whereas the carboxyl-terminal domain does not have a similar effect. Unexpectedly, we found that the L. infantum HSP70 and HSP83 recombinant proteins stimulated the proliferation of spleen cells from unprimed BALB/c mice. Remarkably, this proliferation was abolished either by thermal denaturing of the proteins or by using specific antibodies. The use of the T-cell inhibitor cyclosporin A in the splenocytes proliferation assays suggested that both T- and non-T-cells are stimulated by the Leishmania HSPs. These findings may be relevant for therapeutic and prophylactic applications.

Detection of human Toxoplasma-specific immunoglobulins A, M, and G with a recombinant Toxoplasma gondii rop2 protein.

The Toxoplasma gondii rhoptry protein Rop2 was expressed in Escherichia coli as a fusion protein containing 44 kDa of the 55-kDa mature Rop2, supplied with six histidyl residues at the N-terminal end (Rop2196-561). Humoral response during Toxoplasma infection of humans was analyzed by immunoglobulin G (IgG), IgA, and IgM enzyme-linked immunosorbent assay with Rop2196-561 as the antigen substrate. The analyzed sera were divided according to T. gondii-specific serological tests (IgG, IgA, or IgM indirect immunofluorescence and IgA or IgM immunosorbent agglutination assay) as group A (IgG+ IgA- IgM-; n = 35), group B (IgG+ IgA+ IgM+; n = 21), group C (IgG+ IgA+ IgM-; n = 5), and group D (IgG+ IgA- IgM+; n = 16). Twenty-six T. gondii-seronegative sera from individuals with other infections were also included (group E). Anti-Rop2 IgG antibodies were detected in 82.8% of group A sera and in 97.6% of the sera with acute-phase marker immunoglobulins (groups B, C, and D). The percentage of IgA antibody reactivity against Rop2196-561 was 17.1% in group A, 50% in group D, and 80.8% in groups B and C. The percentage of IgM antibody reactivity was 0% in groups A and C and 62% in groups B and D. Sera from group E failed to show IgA, IgM, or IgG antibody reactivity. Since T. gondii Rop2 elicits a strong humoral response from an early stage of infection, it is suggested that recombinant Rop2196-561 would be suitable for use in diagnostic systems, in combination with other T. gondii antigens, to detect specific IgG, IgA, and IgM antibodies.

Screening for active toxoplasmosis in patients by DNA hybridization with the ABGTg7 probe in blood samples.

We report the potential use of a specific Toxoplasma gondii DNA probe (ABGTg7). We applied a dot blot hybridization assay to blood samples for the diagnosis of cerebral toxoplasmosis (CT), acute toxoplasmic lymphadenopathy (ATL), and disseminated toxoplasmosis in transplant recipients (TRs). We studied a total of 84 individuals: 38 patients and 46 controls. We found positive hybridization signals for 12 (66.7%) of 18 patients with confirmed CT, 9 (52.9%) of 17 patients with ATL, and 2 (66.7%) of 3 TRs. PCR assays were performed in parallel for patients with ATL, resulting in T. gondii DNA detection for 10 patients (58.8%). A comparative study between dot blot and PCR assays performed with the blood of mice that had been experimentally infected with tachyzoites gave similar results: 60 and 70% positive results, respectively. Finally, the sum of positive values obtained by both DNA tests (dot blot assay plus PCR) increased the rate of positivity for ATL patients to 76.4%. These results demonstrate that the T. gondii ABGTg7 repetitive DNA element is an additional useful resource for diagnosing Toxoplasma parasitemia in patients with CT and ATL and in TRs. Thus, our ABGTg7-based dot blot test may lead to an improvement in T. gondii detection methods in patients with acute toxoplasmosis.

During canine leishmaniasis a protein belonging to the 83-kDa heat-shock protein family elicits a strong humoral response.

By screening of a Leishmania infantum expression library with the serum from a dog affected with visceral leishmaniasis, a cDNA clone with sequence homology to the Hsp83 gene family was isolated. From analysis of the genomic distribution of the cDNA sequence, it was estimated that the L. infantum genome contains 7 Hsp83 genes tandemly organized. The full-length coding region of the Hsp83 gene located at the 5'-end of the cluster was determined. The deduced amino acid sequence of the L. infantum Hsp83 shows a high level of sequence identity with members of the Hsp83's protein family from other eukaryotic organisms. The complete protein (LiHsp83) and 4 subfragments (LiA1, LiB1, LiC1 and LiD1) were expressed in Escherichia coli as recombinant proteins and used as target antigens in FAST-ELISA assays against a collection of sera from dogs with visceral leishmaniasis. Ninety percent of the sera recognized the recombinant LiHsp83, indicating that L. infantum Hsp83 is a potent immunogen during canine leishmaniasis. Serological analysis of the recombinant subfragments identified the LiB1 subfragment, from amino acid 156 to 283, as the immunodominant region of the protein. This region, which is the less evolutionary conserved region of the protein, was recognized by 88% of the visceral leishmaniasis sera. The results suggest that L. infantum Hsp83 and particular protein subfragments may be useful in serodiagnostic assays for canine leishmaniasis.

During active viscerocutaneous leishmaniasis the anti-P2 humoral response is specifically triggered by the parasite P proteins.

In this work we show that in the sera from dogs naturally infected with the protozoan parasite Leishmania infantum there are antibodies that react specifically against the parasite acidic ribosomal proteins LiP2a and LiP2b, and that each one of the Leishmania P proteins elicits a specific humoral immune response. Using synthetic peptides, the antigenic epitope of these proteins has been mapped in a single region located adjacent to the C-terminal domain highly conserved among the eukaryotic P proteins. The anti-P antibodies elicited during the Leishmania infection do not recognize the conserved C-terminal domain of the parasite P proteins, in contrast with the findings reported in Chagas' disease or systemic lupus erythematosus. The antigenic epitopes of the LiP2a and LiP2b are almost identical in amino acid sequence. No reactivity against Trypanosoma cruzi and human P proteins was found in sera from L. infantum-infected dogs.

Use of a monoclonal antibody and a DNA probe for diagnosing acute toxoplasmosis in ambiguous cases.

An unusual case of cerebral toxoplasmosis in an HIV negative 11 year old child is reported. Central nervous system disease was assessed immunohistochemically in a brain biopsy specimen with TgP8--a specific monoclonal antibody against Toxoplasma gondii antigens--thus confirming IgG and IgM serology, technetium scan findings, and clinical data. In addition, an active parasitaemia was confirmed by DNA in situ hybridisation assay in white cells using an ABGTg4 probe. The child recovered after specific T gondii treatment. Follow up six months later showed that he was immunodeficient.