Identification and Functional Analysis of Trypanosoma cruzi Genes That Encode Proteins of the Glycosylphosphatidylinositol Biosynthetic Pathway

Abstract

Background Trypanosoma cruzi 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, T. cruzi GPI anchors have immunostimulatory activities, including the ability to stimulate the synthesis of cytokines by innate immune cells. Therefore, T. cruzi genes related to GPI anchor biosynthesis constitute potential new targets for the development of better therapies against Chagas disease.Methodology/Principal Findings In silico analysis of the T. cruzi 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 T. cruzi 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. T. cruzi genes TcDPM1, TcGPI10 and TcGPI12 complement conditional yeast mutants in GPI biosynthesis. Attempts to generate T. cruzi knockouts for three genes were unsuccessful, suggesting that GPI may be an essential component of the parasite. Regarding TcGPI8, 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.Conclusions/SignificanceAnalyses of T. cruzi 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 T. cruzi genes resulted in yeast cell lines that can now be employed in high throughput screenings of drugs against this parasite.

A Riboswitch‐based Inducible Gene Expression System for Trypanosoma brucei

“…) have shown that disruption of a single allele results in a 40% (Cardoso et al. ) to 50% (Campos et al. ) reduction in the levels of the target mRNA in T. cruzi and up to three‐ to fivefold reduction in the target mRNA in L. donovani (Dumas et al.…”

“…Interestingly, C-terminal tagging of the proteins encoded by only one allele of several genes resulted in downregulation of gene expression at the mRNA level demonstrating that both alleles were transcribed. In this regard, when targeting genes for deletion, several studies in T. cruzi (Campos et al 2011;Cardoso et al 2013;Perez Brandan et al 2011) and Leishmania donovani (Dumas et al 1997) have shown that disruption of a single allele results in a 40% (Cardoso et al 2013) to 50% (Campos et al 2011) reduction in the levels of the target mRNA in T. cruzi and up to three-to fivefold reduction in the target mRNA in L. donovani (Dumas et al 1997) allowing the assessment of the phenotype of cells with this reduced expression. One possible reason for these dramatic effects could be the differential expression of different alleles.…”

Estudos da dinâmica do núcleo da célula hospedeira durante a infecção por Trypanosoma cruzi

“…Outra glicoproteína envolvida nesse processo invasivo é a Gp82, que juntamente com fosfolipase C e IP3, fazem parte de uma cascata de sinalização que induz a mobilização de cálcio necessário para a invasão (De Souza et al, 2010). Vale a pena ressaltar que as âncoras de GPI de T. cruzi possuem atividade imunoestimulatória, como a habilidade de estimular citocinas em células da imunidade inata (Previato et al, 2003;Cardoso et al, 2013).…”

“…A entrada do parasito na célula hospedeira também é associada à presença de mucinas, que representam a maior parte das glicoproteínas de superfície em Trypanosoma cruzi, sendo os seus resíduos de carboidratos que interagem com a membrana da célula hospedeira de mamífero (Souza et al, 2010;Schenkman et al, 1991;Villalta et al, 1984;Yoshida et al, 1989). Muitos outros compostos, como o ácido siálico, são incorporados à trans-silalidases principalmente em mucinas presentes nas membranas dos tripomastigotas, a fim de burlar o sistema complemento, evitando a resposta imune (Souza et al, 2010;Schenkman et al, 1991;Tomlinson et al, 1994, Cardoso et al, 2013.…”

Síntese de análogos de âncora de GPI: uma contribuição para a descoberta de novos alvos moleculares de Trypanosoma cruzi

“…(Table 1, Line 2). The lipid linked to the PI moiety varies along the parasite's developmental state: Epimastigotes GPIs contain mainly 1-O-(C16:0)-alkyl-2-O-(C16:0)-acylglycerol-3-PI; metacyclic trypomastigotes present GPI anchors predominantly composed by different types of phosphoceramide-inositol, containing dihydrosphingosine (C18:0), lignoceric (C24:0) or palmitic acid (C16:0) ( Figure 1) (CARDOSO et al, 2013;SERRANO et al, 1995; Herein, what is acknowledged about the biosynthesis of T. cruzi is described with the assistance of Figure 4, and some examples of divergent biosynthesis from other eukaryotes will be discussed. routes, that require GlcNAc to occur inside ER (SMITH et al, 2007).…”