BALB/c and C57BL/6 Mice Cytokine Responses to Trypanosoma cruzi Infection Are Independent of Parasite Strain Infectivity

Trypanosoma cruzi is the etiologic agent of Chagas' disease, which affects 6-7 million people worldwide. Different strains of T. cruzi present specific genotypic and phenotypic characteristics that affect the host-pathogen interactions, and thus, the parasite has been classified into six groups (TcI to TcVI). T. cruzi infection presents two clinical phases, acute and chronic, both with distinct characteristics and important participation by the immune system. However, the specific contributions of parasite and host factors in the disease phases are not yet fully understood. The murine model for Chagas' disease is well-established and reproduces important features of the human infection, providing an experimental basis for the study of host lineages and parasite strains. Thus, we evaluated acute and chronic infection by the G (TcI) and CL (TcVI) strains of T. cruzi, which have distinct tropisms and infectivity, in two inbred mice lineages (C57BL/6 and BALB/c) that display variable degrees of susceptibility to different T. cruzi strains. Analysis of the parasite loads in host tissues by qPCR showed that CL strain established an infection faster than the G strainat the same time, the response in BALB/c mice, although diverse in terms of cytokine secretion, was initiated earlier than that in C57BL/6 mice. At the parasitemia peak in the acute phase, we observed, either by confocal microscopy or by qPCR, that the infection was disseminated in all groups analyzed, with some differences concerning parasite tropismat this point, all animals responded to infection by increasing the serum concentrations of cytokines. However, BALB/c mice seemed to better regulate the immune response than C57BL/6 mice. Indeed, in the chronic phase, C57BL/6 mice still presented exacerbated cytokine and chemokine responses. In summary, our results indicate that in these experimental models, the deregulation of immune response that is typical of chronic Chagas' disease may be due to control loss over pro-and anti-inflammatory cytokines early in the acute phase of the disease, depending primarily on the host background rather than the parasite strain.FAPESPCAPESCNPqUniv Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, Sao Paulo, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Bioquim, Sao Paulo, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, Sao Paulo, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Bioquim, Sao Paulo, BrazilFAPESP: 2011/51475-3FAPESP: 2014/21338-2CNPq: 302068/2016-3Web of Scienc

Proteomic study revealed cellular assembly and lipid metabolism dysregulation in sepsis secondary to community-acquired pneumonia

Sepsis is a life-threatening disorder characterized by organ dysfunction and a major cause of mortality worldwide. The major challenge in studying sepsis is its diversity in such factors as age, source of infection and etiology. Recently, genomic and proteomic approaches have improved our understanding of its complex pathogenesis. In the present study, we use quantitative proteomics to evaluate the host proteome response in septic patients secondary to community-acquired pneumonia (CAP). Samples obtained at admission and after 7 days of follow-up were analyzed according to the outcomes of septic patients. The patients' proteome profiles were compared with age-and gender-matched healthy volunteers. Bioinformatic analyses of differentially expressed proteins showed alteration in the cytoskeleton, cellular assembly, movement, lipid metabolism and immune responses in septic patients. Actin and gelsolin changes were assessed in mononuclear cells using immunofluorescence, and a higher expression of gelsolin and depletion of actin were observed in survivor patients. Regarding lipid metabolism, changes in cholesterol, HDL and apolipoproteins were confirmed using enzymatic colorimetric methods in plasma. Transcriptomic studies revealed a massive change in gene expression in sepsis. Our proteomic results stressed important changes in cellular structure and metabolism, which are possible targets for future interventions of sepsis.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, CNPqFAPESPUniv Fed Sao Paulo, Hosp Sao Paulo, Div Infect Dis, Escola Paulista Med, BR-04039032 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biochem, Escola Paulista Med, BR-04023900 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Escola Paulista Med, BR-04023062 Sao Paulo, BrazilUniv Fed Sao Paulo, Intens Care Unit, Hosp Sao Paulo, Escola Paulista Med, BR-04024002 Sao Paulo, BrazilHosp Israelita Albert Einstein, Intens Care Unit, BR-05652900 Sao Paulo, BrazilHosp Sirio Libanes, Intens Care Unit, BR-01409001 Sao Paulo, BrazilUniv Fed Sao Paulo, Hosp Sao Paulo, Div Infect Dis, Escola Paulista Med, BR-04039032 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biochem, Escola Paulista Med, BR-04023900 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Escola Paulista Med, BR-04023062 Sao Paulo, BrazilUniv Fed Sao Paulo, Intens Care Unit, Hosp Sao Paulo, Escola Paulista Med, BR-04024002 Sao Paulo, BrazilFAPESP: 2011/20401-4FAPESP: 2013/15636-8CNPq: 305685/2011-2Web of Scienc

Characterization of the mevalonate kinase enzyme: a bifunctional protein in Trypanosoma cruzi

Mevalonato quinase (MVK) e uma enzima essencial na via do mevalonato. Essa via e responsavel pela biossintese de isoprenoides convertendo mevalonato em esterois como colesterol em humanos e ergosterol em tripanosomatideos. MVK e conservada desde bacterias ate mamiferos e localiza-se nos glicossomos de tripanosomatideos. Ferramentas de bioInformática demonstraram que T. cruzi tem duas isoformas de MVK (TcMVK1 e TcMVK2). TcMVK1 tem um peptideo sinal bem definido localizado na porcao N-terminal, sugerindo a secrecao da proteina. Para avaliar o papel da secrecao de TcMVK na interacao com a celula hospedeira, foi produzida TcMVK recombinante (rTcMVK) assim como anticorpos -TcMVK. A forma secretada de TcMVK liga-se de maneira dose dependente a celulas HeLa, modula positivamente a internalizacao de amastigotas extracelulares de T. cruzi (EAs) mas inibe a invasao por tripomastigotas metaciclicos (MTs). TcMVK foi encontrada no sobrenadante de MTs e EAs por tecnica de western blotting pelo uso do anticorpo -TcMVK. Imagens de imunofluorescencia de MTs, adquiridas por microscopia confocal, mostraram a colocalizacao parcial de TcMVK e BiP, uma proteina constitutiva de reticulo endoplasmatico. Experimentos de sinalizacao de celulas HeLa mostraram que rTcMVK induziu a fosforilacao de componentes da via MAPK e de proteinas relacionadas com mudancas no citoesqueleto de actina. Os resultados sugerem entao TcMVK como uma proteina moonlighting, visto o seu comportamento atipico. Enzimas ou proteinas moonlighting sao proteinas que podem realizar mais de uma funcao, sendo a segunda bastante diferente da funcao original. Muitas enzimas moonlighting sao classicamente envolvidas em vias metabolicas como a via glicolitica. Adicionalmente ao seu papel tipico na biossintese de isoprenoides nos glicossomos, TcMVK e secretada e pode modular a sinalizacao celular requerida para a invasao de T. cruzi. Essa seria uma caracteristica unica para uma enzima de T. cruziBV UNIFESP: Teses e dissertaçõe

Molecular Characterization of a Novel Family of <i>Trypanosoma cruzi</i> Surface Membrane Proteins (TcSMP) Involved in Mammalian Host Cell Invasion

<div><p>Background</p><p>The surface coat of <i>Trypanosoma cruzi</i> is predominantly composed of glycosylphosphatidylinositol-anchored proteins, which have been extensively characterized. However, very little is known about less abundant surface proteins and their role in host-parasite interactions.</p><p>Methodology/ Principal Findings</p><p>Here, we described a novel family of <i>T</i>. <i>cruzi</i> surface membrane proteins (TcSMP), which are conserved among different <i>T</i>. <i>cruzi</i> lineages and have orthologs in other <i>Trypanosoma</i> species. TcSMP genes are densely clustered within the genome, suggesting that they could have originated by tandem gene duplication. Several lines of evidence indicate that TcSMP is a membrane-spanning protein located at the cellular surface and is released into the extracellular milieu. TcSMP exhibited the key elements typical of surface proteins (N-terminal signal peptide or signal anchor) and a C-terminal hydrophobic sequence predicted to be a trans-membrane domain. Immunofluorescence of live parasites showed that anti-TcSMP antibodies clearly labeled the surface of all <i>T</i>. <i>cruzi</i> developmental forms. TcSMP peptides previously found in a membrane-enriched fraction were identified by proteomic analysis in membrane vesicles as well as in soluble forms in the <i>T</i>. <i>cruzi</i> secretome. TcSMP proteins were also located intracellularly likely associated with membrane-bound structures. We demonstrated that TcSMP proteins were capable of inhibiting metacyclic trypomastigote entry into host cells. TcSMP bound to mammalian cells and triggered Ca²⁺ signaling and lysosome exocytosis, events that are required for parasitophorous vacuole biogenesis. The effects of TcSMP were of lower magnitude compared to gp82, the major adhesion protein of metacyclic trypomastigotes, suggesting that TcSMP may play an auxiliary role in host cell invasion.</p><p>Conclusion/Significance</p><p>We hypothesized that the productive interaction of <i>T</i>. <i>cruzi</i> with host cells that effectively results in internalization may depend on diverse adhesion molecules. In the metacyclic forms, the signaling induced by TcSMP may be additive to that triggered by the major surface molecule gp82, further increasing the host cell responses required for infection.</p></div

Synteny of TcSMP genes among <i>T</i>. <i>brucei</i>, T. cruzi and <i>T</i>. <i>grayi</i>.

<p>Genomic regions around the <i>T</i>. <i>cruzi</i> (CLB) TcSMP paralogs and <i>T</i>. <i>brucei</i> and <i>T</i>. <i>grayi</i> orthologs are shown. Analyses were conducted via TBLASTN using the Artemis Comparison Tool (ACT) [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004216#pntd.0004216.ref037" target="_blank">37</a>] with an E value of 500. Comparison among TcChr37 (‘‘P” chromosome assigned to the non-Esmeraldo haplotype and ‘‘S” to the Esmeraldo haplotype), <i>T</i>. <i>brucei</i> chromosome Tb10 and the contig Tgr_12_V1 of <i>T</i>. <i>grayi</i>. Homologous genes are connected by colored lines. The matches and reverse matches are represented in red and in blue, respectively. Grey blocks represent each chromosome. Chromosome markers are drawn in the sense (+) and antisense (-) strands. The numbers indicate the location on chromosomes: <i>T</i>. <i>brucei</i> TREU927 (CHR10—position: 2729733–2761348 nt), CLB <i>T</i>. <i>cruzi</i> (TcChr37-S—positions: 848335–873376 nt and TcChr37-P 848287–882361 nt) and contig of <i>T</i>. <i>grayi</i> (Tgr_12_V1 –position: 1–64810 nt). The locations of the TcSMP paralogs and <i>T</i>. <i>brucei</i> and <i>T</i>. <i>grayi</i> orthologs are depicted in chromosomes by red blocks. Abbreviations: TcSMP (TcSMP—<i>T</i>. <i>cruzi</i>/ PSSA-2—<i>T</i>. <i>brucei</i>); HIRA (HIRA-interacting protein 5, putative); ubiqui (ubiquitin-like modifier-activating enzyme ATG7, putative); RNA-bp (RNA-binding protein, putative); ubiqui E1 (ubiquitin activating E1 enzyme, putative); surf glyco (surface glycoprotein, putative); PRC (paraflagellar rod component, putative); HP (hypothetical protein, conserved); S-methyl (S-methyl-5thioribose kinase); POT (proton-dependent oligopeptide transporter, POT family); tetratrico (tetratricopeptide domain 4); 60S (putative 60S ribosomal protein L6); sterol C (sterol C-24 reductase); IFT 57 (predicted: intraflagellar transporter protein 57 homolog); tRNA-s lig (tRNA-splicing ligase RtcB); PP 2A (protein phosphatase 2A regulatory subunit).</p

Alignment of TcSMP amino acid sequences of <i>T</i>. <i>cruzi—</i>clone CL Brener (CLB).

<p>Sequences TcCLB.510129.20, TcCLB.510129.30, TcCLB.509639.10, TcCLB.507711.90, extended TcCLB.507711.100, extended TcCLB.507711.110 and TcCLB.508173.120 were deposited in the GenBank database by the <i>T</i>. <i>cruzi</i> Genome Project. Clones 23B, 24B and 24C (GenBank KJ682657, KJ682658 and KJ682659) were isolated in this work from genomic DNA (CLB) by PCR. Sequences were aligned by the MegAlign program (DNASTAR). Black, gray and light gray blocks indicate residues with 100%, 80% and 60% identity, respectively. Colored lines above the sequences denote the predicted signal peptide in green, transmembrane domains in orange and uncleaved anchor signal in blue. Red arrows indicate the putative initiator methionine. Yellow boxes correspond to peptides found in the proteomic analysis of <i>T</i>. <i>cruzi</i> membrane protein-enriched fractions [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004216#pntd.0004216.ref027" target="_blank">27</a>]. Red boxes correspond to peptides identified in the <i>T</i>. <i>cruzi</i> secretome [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004216#pntd.0004216.ref043" target="_blank">43</a>]. Blue boxes correspond to the YGQ motif present in Procyclic Surface Specific Antigen-2 (PSSA-2) of <i>T</i>. <i>brucei</i>.</p

Western blotting analysis of TcSMP proteins in the different developmental stages of <i>T</i>. <i>cruzi</i> and in the procyclic form of <i>T</i>. <i>brucei rhodesiense</i>.

<p>A) Samples of <i>T</i>. <i>cruzi</i> (CL strain) (1 x 10⁷ cells/slot) and <i>T</i>. <i>brucei</i> (5 x 10⁶ cells/slot) were lysed in Laemmli sample buffer. Proteins were separated by electrophoresis on SDS- polyacrylamide gel (10%), transferred to a nitrocellulose membrane and reacted against anti-TcSMP polyclonal antibodies. Antibody against a constitutively expressed protein (tubulin) was used as a loading control. <i>T</i>. <i>cruzi</i> developmental forms: epimastigotes (E), metacyclic trypomastigotes (M), extracellular amastigotes (A) and tissue culture trypomastigotes (T). <i>T</i>. <i>brucei rhodesiense</i> procyclic forms. B) Two μg of the recombinant TcSMP-GST purified protein was separated by SDS-PAGE and reacted against anti-TcSMP and anti-GST antibodies. The recombinant protein is coded by a 311-bp fragment of TcSMP in phase with GST (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004216#sec005" target="_blank">methods</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004216#pntd.0004216.s004" target="_blank">S4 Fig</a>). Molecular mass markers in kilodaltons (kDa) are indicated on the left and reactive proteins molecular mass on the right.</p

Phylogenetic tree of TcSMP sequences built by the Maximum-Likelihood method.

<p>The phylogram was constructed from the alignment of amino acid sequences of TcSMP proteins and their homologs in other trypanosomes. The ML tree was constructed using Phylip. Red and blue lines on the right delimit the American and African trypanosome clusters, respectively. Genes located on chromosome platforms TcChr37-P, TcChr37-S and TcChr27-P are highlighted in green, violet, and pink, respectively. Sequences were identified by their entries in TriTrypDB, except those from <i>T</i>. <i>rangeli</i> (AGN32959, ESL09843 and ESL05362) and <i>T</i>. <i>grayi</i> (KEG05859), which were identified by their GenBank accession numbers. TriTrypDB entries are prefixed by species and strain followed by the number of the sequence. <i>T</i>. <i>cruzi</i>: TcCLB., clone CL Brener; TCDM_, clone Dm28c; TCSylvio_, clone Sylvio X10/1; <i>T</i>. <i>cruzi marinkellei</i>: Tc_MARK_; <i>T</i>. <i>brucei brucei</i>: Tb927., strain TREU; Tb427., strain Lister; <i>T</i>. <i>vivax</i>: TvY486_; <i>T</i>. <i>congolense</i>: TcIL3000. Clones 23B, 24B and 24D (GenBank KJ682657, KJ682658 and KJ682659) were isolated in this work by PCR amplification using TcSMP specific primers. A trans-sialidase from <i>T</i>. <i>cruzi</i> CLB (XP_816001) was used as the outgroup.</p