RESUMO
Toxoplasma protein disulfide isomerase (PDI) is a 52 KDa thioredoxin of interest because have a great immunogenicity for humans. We cloned and produced a recombinant protein (recTgPDI) used to test its effect during infection to different human cell lines (epithelial and retinal). We also determine if there were differences in gen expression during in vitro infection. Expression of the gen was lower after entry into the host cells. PDI's inhibitors bacitracin and nitroblue tetrazolium reduced the percent of infected cells and small amounts of recTgPDI proteins interfered with the invasion step. All these results support a role of Toxoplasma PDI during the first steps of infection (adhesion and invasion). Toxoplasma PDI is a protein linked to early steps of invasion, it would be of importance to identify the host proteins substrates during invasion steps.
Assuntos
Isomerases de Dissulfetos de Proteínas/metabolismo , Tiorredoxinas/metabolismo , Toxoplasma/enzimologia , Toxoplasma/fisiologia , Linhagem Celular , Clonagem Molecular , Células Ependimogliais/parasitologia , Fibroblastos/parasitologia , Regulação Enzimológica da Expressão Gênica , Células HeLa/parasitologia , Humanos , Modelos Estruturais , Conformação Proteica , Isomerases de Dissulfetos de Proteínas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Análise de Sequência de DNA , Toxoplasma/genéticaRESUMO
Apicomplexa are an extremely diverse group of unicellular organisms that infect humans and other animals. Despite the great advances in combating infectious diseases over the past century, these parasites still have a tremendous social and economic burden on human societies, particularly in tropical and subtropical regions of the world. Proteases from apicomplexa have been characterized at the molecular and cellular levels, and central roles have been proposed for proteases in diverse processes. In this work, 16 new genes encoding for trypsin proteases are identified in 8 apicomplexan genomes by a genome-wide survey. Phylogenetic analysis suggests that these genes were gained through both intracellular gene transfer and vertical gene transfer. Identification, characterization and understanding of the evolutionary origin of protease-mediated processes are crucial to increase the knowledge and improve the strategies for the development of novel chemotherapeutic agents and vaccines.
Assuntos
Apicomplexa/enzimologia , Apicomplexa/genética , Evolução Molecular , Genoma de Protozoário , Tripsina/genética , Sequência de Aminoácidos , DNA de Protozoário , Transferência Genética Horizontal , Genes de Protozoários , Dados de Sequência Molecular , Filogenia , Alinhamento de SequênciaRESUMO
The histone-like proteins (HU) belong to a family of DNA architectural proteins that stabilize nucleoprotein complexes. We found a putative HU protein (TgGlmHMM_3045) in Toxoplasma gondii genome that was homologous to the bacterial HU protein. This putative sequence was located in the scaffold TGG_995361 of the chromosome 10. The sequence included the prokaryotic bacterial histone-like domain, KFGSLGlRRRGERVARNPRT (ID number PS00045). HU protein sequences were also found in Plasmodium falciparum, Neospora caninum, Theileria parva and Theileria annulata. We found that the homology of the putative HU protein in Apicomplexa was greater with bacterial histone-like proteins than with eukaryotic histone proteins. The phylogenetic tree indicated that the putative HU protein genes were acquired in Apicomplexa by means of a secondary endosymbiotic event from red algae and later they were transferred from the apicoplast organelle to the nuclear genome.
Assuntos
Genoma de Protozoário , Histonas/genética , Proteínas de Protozoários/genética , Toxoplasma/genética , Sequência de Aminoácidos , Animais , Biologia Computacional , DNA de Protozoário/genética , Histonas/metabolismo , Dados de Sequência Molecular , Proteínas de Protozoários/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Toxoplasma gondii has a nitrite production and a putative nitric oxide synthase (NOS) motif genomic sequence. In order to demonstrate that this sequence is functional and could be involved in the metabolism of l-arginine derivatives, we constructed a baculovirus carrying the previously identified Toxoplasma NOS-like DNA sequence. The recombinant protein was expressed into insect Sf9 cells and his activity was tested in serial microplate colorimetric assays. The protein produced 21 nmol/min/ml nitrites per microgram of protein and followed Michaelis-Menten kinetics, with a K(m) for L-arginine of 2.3mM. Furthermore, the optimal pH, temperature and incubation time for the recombinant Toxoplasma NOS-like protein were established. Toxoplasma NOS runs as a band of 11.6 kDa on tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results indicate that the recombinant protein derived from the putative genomic sequence, at the chromosome 1b of T. gondii, is able to produce nitrites from L-arginine as substrate.
Assuntos
Óxido Nítrico Sintase/metabolismo , Toxoplasma/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Colorimetria , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Camundongos , Camundongos Endogâmicos ICR , Dados de Sequência Molecular , Óxido Nítrico Sintase/química , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/isolamento & purificação , Nucleopoliedrovírus/genética , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Spodoptera , Toxoplasma/genéticaRESUMO
The serine-rich (SR) protein family is involved in the pre-mRNA splicing process and the DNA sequences of the corresponding genes are highly conserved in the metazoan organisms. The mammalian SR proteins consist of one or two characteristic RNA binding domains (RBD), containing the signature sequences RDAEDA and SWQDLKD and a RS (arginine/serine-rich) domain. We used the amino acid and nucleotide sequences deposited in GenBank and Swiss-Prot databases to perform a phylogenetic analysis using bioinformatics tools. The results of the phylogenetic trees suggest that this family has evolved by several gene duplication events as a result of a positive selection mechanism.