Identification and Functional Analysis of <i>Trypanosoma cruzi</i> Genes That Encode Proteins of the Glycosylphosphatidylinositol Biosynthetic Pathway

<div><p>Background</p><p><i>Trypanosoma cruzi</i> is a protist parasite that causes Chagas disease. Several proteins that are essential for parasite virulence and involved in host immune responses are anchored to the membrane through glycosylphosphatidylinositol (GPI) molecules. In addition, <i>T. cruzi</i> GPI anchors have immunostimulatory activities, including the ability to stimulate the synthesis of cytokines by innate immune cells. Therefore, <i>T. cruzi</i> genes related to GPI anchor biosynthesis constitute potential new targets for the development of better therapies against Chagas disease.</p><p>Methodology/Principal Findings</p><p><i>In silico</i> analysis of the <i>T. cruzi</i> genome resulted in the identification of 18 genes encoding proteins of the GPI biosynthetic pathway as well as the inositolphosphorylceramide (IPC) synthase gene. Expression of GFP fusions of some of these proteins in <i>T. cruzi</i> epimastigotes showed that they localize in the endoplasmic reticulum (ER). Expression analyses of two genes indicated that they are constitutively expressed in all stages of the parasite life cycle. <i>T. cruzi</i> genes <i>TcDPM1</i>, <i>TcGPI10</i> and <i>TcGPI12</i> complement conditional yeast mutants in GPI biosynthesis. Attempts to generate <i>T. cruzi</i> knockouts for three genes were unsuccessful, suggesting that GPI may be an essential component of the parasite. Regarding <i>TcGPI8</i>, which encodes the catalytic subunit of the transamidase complex, although we were able to generate single allele knockout mutants, attempts to disrupt both alleles failed, resulting instead in parasites that have undergone genomic recombination and maintained at least one active copy of the gene.</p><p>Conclusions/Significance</p><p>Analyses of <i>T. cruzi</i> sequences encoding components of the GPI biosynthetic pathway indicated that they are essential genes involved in key aspects of host-parasite interactions. Complementation assays of yeast mutants with these <i>T. cruzi</i> genes resulted in yeast cell lines that can now be employed in high throughput screenings of drugs against this parasite.</p></div

MHC Class I Chain-Related Gene A Polymorphisms and Linkage Disequilibrium with HLA-B and HLA-C Alleles in Ocular Toxoplasmosis.

This study investigated whether polymorphisms of the MICA (major histocompatibility complex class I chain-related gene A) gene are associated with eye lesions due to Toxoplasma gondii infection in a group of immunocompetent patients from southeastern Brazil. The study enrolled 297 patients with serological diagnosis of toxoplasmosis. Participants were classified into two distinct groups after conducting fundoscopic exams according to the presence (n = 148) or absence (n = 149) of ocular scars/lesions due to toxoplasmosis. The group of patients with scars/lesions was further subdivided into two groups according to the type of the ocular manifestation observed: primary (n = 120) or recurrent (n = 28). Genotyping of the MICA and HLA alleles was performed by the polymerase chain reaction-sequence specific oligonucleotide technique (PCR-SSO; One Lambda®) and the MICA-129 polymorphism (rs1051792) was identified by nested polymerase chain reaction (PCR-RFLP). Significant associations involving MICA polymorphisms were not found. Although the MICA*002~HLA-B*35 haplotype was associated with increased risk of developing ocular toxoplasmosis (P-value = 0.04; OR = 2.20; 95% CI = 1.05-4.60), and the MICA*008~HLA-C*07 haplotype was associated with protection against the development of manifestations of ocular toxoplasmosis (P-value = 0.009; OR: 0.44; 95% CI: 0.22-0.76), these associations were not statistically significant after adjusting for multiple comparisons. MICA polymorphisms do not appear to influence the development of ocular lesions in patients diagnosed with toxoplasmosis in this study population

Ocular toxoplasmosis with positive polymerase chain reaction in peripheral blood – report of two cases, São Paulo State, Brazil

Aims: To describe the use of polymerase chain reaction (PCR) in peripheral blood and demonstrate its importance in the clinical follow-up of patients with ocular toxoplasmosis. Case description: Two immunocompetent patients were clinically diagnosed with acute ocular toxoplasmosis. The routine clinical evaluation consisted of fundus examination using binocular indirect ophthalmoscopy, color fundus photography, fluorescein angiography, and spectral domain optical coherence tomography. The serological diagnosis was made by ELISA (IgM, IgG) and confirmed by ELFA (IgG, IgM). The molecular diagnosis was made by PCR in peripheral blood using the B1 gene of Toxoplasma gondii as marker. The younger patient was male, had previous lesion in the right eye, complained of low visual acuity in the left eye and was under treatment. The older patient was male, had retinal detachment, and presented with sudden loss of acuity in the right eye. The fundus examination revealed chorioretinal scar in the left eye. IgG was reactive, IgM was non-reactive, and PCR was positive in the peripheral blood of both patients. New blood samples were collected for serological and molecular monitoring and PCR remained positive in both cases. Six weeks after treatment with oral sulfadiazine and pyrimethamine, the PCR yielded negative results. Conclusions: The results show that T. gondii antigens may be found in peripheral blood during ocular reactivations and that PCR may be a good tool for the follow-up of patients with ocular toxoplasmosis

Cell membrane morphology of <i>T. cruzi GPI8</i> mutants.

<p>Transmission electron microscopy showing cellular membranes of wild type <i>T. cruzi</i> epimastigotes (WT), <i>TcGPI8</i> single allele knockout, neomycin resistant (+/− N1) and double resistant <i>TcGPI8</i> epimastigote mutants (N/H1). Although displaying similar morphologies, representative images show that single allele <i>TcGPI8</i> mutants present a thinner layer of parasite glycocalyx, when compared to wild type cells, whereas cell membranes of double resistant parasites present a glycocalyx layer that is slightly thicker than the glycocalyx of wild type parasite membranes (indicated by the arrows).</p

Yeast complementation with <i>T. cruzi</i> genes encoding enzymes of the GPI biosynthetic pathway.

<p>(<b>A</b>) <i>DPM1</i>, <i>GPI10</i> and <i>GPI12</i> yeast conditional lethal mutants (YPH499-HIS-GAL-DPM1, YPH499-HIS-GAL-GPI10 and YPH499-HIS-GAL-GPI12, respectively) were transformed with pRS426Met plasmids carrying either <i>T. cruzi</i> or <i>S. cerevisiae</i> genes encoding DPM1, GPI10 and GPI12 (<i>TcDPM1</i> or <i>ScDPM1</i>, <i>TcGPI10</i> or <i>ScGPI10</i>, and <i>TcGPI12</i> or <i>ScGPI12</i>, respectively). Wild-type (WT), non-transformed mutants and transformed yeast mutants were streaked onto plates with nonpermissive, glucose-containing SD medium lacking histidine, with or without uracil or in galactose-containing medium (with uracil) and incubated at 30°C for 3 days. In the bottom panel, yeast mutants (YPH499-HIS-GAL-GPI14) transformed with pRS426Met plasmid carrying <i>T. cruzi</i> gene (<i>TcGPI14</i>), which could not restore cell growth of GPI14 deficient yeast are shown. (<b>B</b>) GPI-anchored proteins synthesized by the conditional lethal yeast mutants expressing <i>T. cruzi</i> genes were separated by SDS-PAGE and analyzed after fluorography. Wild-type (WT), non-transformed yeast mutants and yeast mutants that were transformed with plasmids containing the corresponding yeast genes (<i>ScDPM1</i> or <i>ScGPI12</i>) or with the <i>T. cruzi</i> genes (<i>TcDPM1</i> or <i>TcGPI12</i>), were cultivated in medium glucose-containing in the presence of [2-³H]<i>myo</i>-inositol for 1 hour. Total protein extract corresponding to 1×10⁸ cells were loaded on each lane of a 10% SDS-PAGE and the labeled proteins were visualized by fluorography (top panels). As a loading control, Coomassie Blue stained gels prepared with equivalents amounts of total proteins are shown in the bottom panels. Untransfected <i>DPM1</i> and <i>GPI12</i> mutants were grown in the presence of galactose for 2 days and then switched to glucose-containing medium for 16 hours before addition of [2-³H]<i>myo</i>-inositol. Molecular weight markers (M) are shown on the left.</p

Structure and the biosynthesis of <i>T. cruzi</i> GPI anchors.

<p>(<b>A</b>) Structure of a <i>T. cruzi</i> GPI anchor, according to Previato et al. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002369#pntd.0002369-Previato1" target="_blank">[3]</a>. (<b>B</b>) Proposed biosynthetic pathway of GPI anchor in the endoplasmic reticulum of <i>T. cruzi</i>. N-acetylglucosamine (GlcNAc) is added to phosphatidylinositol (PI) in step 1 and, during the following steps, deacetylation and addition of four mannose residues occur. The addition of ethanolamine-phosphate on the third mannose (step 7) enables the transferring of the completed GPI anchor to the C-terminal of a protein (step 8). Dolichol-P-mannose acts as a mannose donor for all mannosylation reactions that are part of the GPI biosynthesis. This pathway was based on the structure of the <i>T. cruzi</i> GPI and sequence homology of <i>T. cruzi</i> genes with genes known to encode components of this pathway in <i>Saccharomyces cerevisiae</i>, <i>Homo sapiens</i>, <i>Trypanosoma brucei</i> and <i>Plasmodium falciparum</i>. Not shown in the figure, free glycoinositolphospholipids (GIPLs), also present in the <i>T. cruzi</i> membrane, are likely to be by-products of the same GPI biosynthetic pathway.</p

<i>T. cruzi</i> genes encoding enzymes of the GPI biosynthetic pathway.

<p>(*) Gene ID numbers refer to the non-Esmeraldo-like haplotype, except for <i>TcGPI16</i> and <i>TcGPI19</i>, for which only the Esmeraldo-like alleles were identified.</p><p>(**) Names for the yeast and human orthologs are shown in parentheses.</p><p>ni: not identified.</p

Ocular toxoplasmosis with positive polymerase chain reaction in peripheral blood: report of two cases, São Paulo State, Brazil = Toxoplasmose ocular com reação em cadeia da polimerase positiva em sangue periférico: relato de dois casos, estado de São Paulo, Brasil

Objetivos: Descrever o uso da reação em cadeia da polimerase (PCR) no sangue periférico e demonstrar sua importância no acompanhamento clínico de pacientes com toxoplasmose ocular. Descrição dos casos: Dois pacientes imunocompetentes foram clinicamente diagnosticados com toxoplasmose ocular aguda. Rotineiramente, a avaliação clínica foi feita por fundoscopia com o uso de oftalmoscópio binocular indireto, retinografia colorida, angiografia fluorescente e tomografia de coerência óptica espectral. A sorologia foi realizada por ensaio imunoenzimático (ELISA) e confirmada por ensaio imunoenzimático fluorescente ELFA (IgG, IgM). O diagnóstico molecular foi realizado por PCR em sangue periférico usando o gene B1 de Toxoplasma gondii como marcador. O paciente mais jovem era do sexo masculino, apresentava lesão prévia no olho direito, queixa de baixa acuidade visual no olho esquerdo e estava sob tratamento. O paciente mais velho era do sexo masculino, apresentava descolamento de retina e súbita diminuição de visão no olho direito. A fundoscopia revelou cicatriz coriorretiniana no olho esquerdo. Ambos os pacientes tinham IgG reagente, IgM não reagente e PCR positivo em sangue periférico. Novas amostras de sangue foram coletadas para monitoramento sorológico e molecular e a PCR permaneceu positiva em ambos os casos. Seis semanas após o início do tratamento com sulfadiazina e pirimetamina oral, os resultados do PCR tornaram-se negativos. Conclusões: Os resultados mostram que antígenos de T. gondii podem ser encontrados em sangue periférico durante as reativações oculares e que a PCR parece ser uma boa ferramenta para o acompanhamento de pacientes com toxoplasmose ocula

Cell membrane mucins in <i>T. cruzi GPI8</i> mutants.

<p>Immunoblot of total (T), cytoplasmic (C) and membrane (M) fractions of WT epimastigotes, <i>TcGPI8</i> single allele knockout, neomycin resistant (+/−N2) and double resistant <i>TcGPI8</i> (N/H2) mutant cell lines. Equivalent amounts of protein from each fraction, as showed by the Coomassie blue stained bands (bottom panel), were transferred to nitrocellulose membranes and incubated with anti-mucin antibodies and revealed with horseradish peroxidase conjugated secondary antibodies.</p

Cellular localization of <i>T. cruzi</i> enzymes of the GPI biosynthetic pathway.

<p>Epimastigotes were transiently transfected with the plasmids pTREX-TcDPM1-GFP (<b>A</b>), pTREX-TcGPI3-GFP (<b>B</b>), pTREX-TcGPI12-GFP (<b>C</b>) or pTREXnGFP as a control plasmid (<b>D</b>) and (<b>E</b>). Transfected parasites were fixed with 4% paraformaldehyde, incubated with the ER marker anti-BiP (1∶1000) and the secondary antibody conjugated to Alexa 555 (1∶1000). Cells were also stained with DAPI showing the nuclear and kinetoplast DNA. In panel <b>E</b>, parasites that were not incubated with the primary, anti-BiP antibody are shown as negative controls. Images were captured with the Nikon Eclipse Ti fluorescence microscope. Scale bars: 5 µm.</p