RESUMO
Amoebic keratitis (AK) is a sight-threatening infection characterized by a severe inflammation of the cornea, caused by the free-living protozoan of the genus Acanthamoeba. Identification of amoebic proteins involved in AK pathogenesis may help to elucidate molecular mechanisms of infection and contribute to indicate diagnostic and therapeutic targets. In this study, we evaluated changes in the expression profile of Acanthamoeba proteins triggered by the invasive process, using an approach involving two-dimensional polyacrylamide gel electrophoresis (2DE PAGE), followed by mass spectrometry identification (ESI-IT-TOF LC-MSn). AK was induced by intrastromal inoculation in Wistar rats, using trophozoites from a T4 genotype, human case-derived A. castellanii strain under prolonged axenic culture. Cultures re-isolated from the lesions after two successive passages in the animals were used as biological triplicate for proteomic experiments. Analysis of the protein profile comparing long-term and re-isolated cultures indicated 62 significant spots, from which 27 proteins could be identified in the Acanthamoeba proteome database. Five of them (Serpin, Carboxypeptidase A1, Hypothetical protein, Calponin domain-containing protein, aldo/keto reductase) were exclusively found in the re-isolated trophozoites. Our analysis also revealed that a concerted modulation of several biochemical pathways is triggered when A. castellanii switches from a free-living style to a parasitic mode, including energetic metabolism, proteolytic activity, control of gene expression, protein degradation and methylation of DNA, which may be also involved in gain of virulence in an animal model of AK.
Assuntos
Ceratite por Acanthamoeba/metabolismo , Acanthamoeba castellanii/metabolismo , Proteínas de Protozoários/biossíntese , Ceratite por Acanthamoeba/parasitologia , Análise de Variância , Animais , Modelos Animais de Doenças , Humanos , Masculino , Proteômica , Proteínas de Protozoários/genética , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização por Electrospray , Eletroforese em Gel Diferencial BidimensionalRESUMO
Amoebic keratitis (AK) is a sight-threatening infection characterized by a severe inflammation of the cornea, caused by the free-living protozoan of the genus Acanthamoeba. Identification of amoebic proteins involved in AK pathogenesis may help to elucidate molecular mechanisms of infection and contribute to indicate diagnostic and therapeutic targets. In this study, we evaluated changes in the expression profile of Acanthamoeba proteins triggered by the invasive process, using an approach involving two-dimensional polyacrylamide gel electrophoresis (2DE PAGE), followed by mass spectrometry identification (ESI-IT-TOF LC-MSn). AK was induced by intrastromal inoculation in Wistar rats, using trophozoites from a T4 genotype, human case-derived A. castellanii strain under prolonged axenic culture. Cultures re-isolated from the lesions after two successive passages in the animals were used as biological triplicate for proteomic experiments. Analysis of the protein profile comparing long-term and re-isolated cultures indicated 62 significant spots, from which 27 proteins could be identified in the Acanthamoeba proteome database. Five of them (Serpin, Carboxypeptidase A1, Hypothetical protein, Calponin domain-containing protein, aldo/keto reductase) were exclusively found in the re-isolated trophozoites. Our analysis also revealed that a concerted modulation of several biochemical pathways is triggered when A. castellanii switches from a free-living style to a parasitic mode, including energetic metabolism, proteolytic activity, control of gene expression, protein degradation and methylation of DNA, which may be also involved in gain of virulence in an animal model of AK.
RESUMO
Free-living amoebae of the genus Acanthamoeba are causative agents of Acanthamoeba keratitis and amoebic encephalitis in humans, both of which are serious infections. The ability to produce proteases is one of the factors involved in the pathogenesis of Acanthamoeba infections. The aim of this study was to evaluate the secreted proteases of six Acanthamoeba strains from distinct genotypes (T1, T2, T4 and T11) maintained in prolonged axenic culture and following three successive passages in Madin-Darby Canine Kidney (MDCK) cells. Conditioned medium was obtained from cultures before and after interaction with the MDCK monolayers, resolved in SDS-PAGE containing gelatine, then subjected to quantitative azocasein assays. Zymography profiles varied between the strains, with the predominant proteases found to be serine-type proteases from 49 to 128 kDa. A T1 genotype strain isolated from dust showed quantitatively higher protease secretion compared to the other strains. No changes were detected in the zymography profiles of MDCK-interacted cultures compared to long-term axenic cultures. Two strains presented lower proteolytic activity post-MDCK interaction, while the remaining strains presented similar values before and after MDCK passages. In conclusion, this study confirms the predominance of serine-type protease secretion by Acanthamoeba, with distinct profiles presented by the different strains and genotypes studied. Also, interaction of trophozoites with MDCK cells did not alter the zymography pattern.
Assuntos
Acanthamoeba/enzimologia , Acanthamoeba/metabolismo , Serina Proteases/metabolismo , Acanthamoeba/genética , Ceratite por Acanthamoeba/parasitologia , Animais , Cultura Axênica , Caseínas/análise , Linhagem Celular , Cães , Genótipo , Humanos , Células Madin Darby de Rim Canino , Trofozoítos/metabolismoRESUMO
Acanthamoeba keratitis (AK) is a progressive corneal infection that demands rapid and sensitive techniques for diagnosis to avoid risk of visual impairment. We evaluated two DNA extraction techniques and a semi-nested-PCR (snPCR) targeting the 18S rRNA gene to detect Acanthamoeba cysts and trophozoites. The most effective protocol was evaluated in samples of corneal scrapings and biopsies from an AK rat model and applied to diagnosis of human cases of AK. DNA extraction performed with a commercial kit based on DNA binding to magnetic beads was more efficient than a method based on alkaline lysis, allowing the detection of one trophozoite and one cyst of Acanthamoeba in samples prepared from cultures. This technique and sn-PCR were applied in corneal scrapings of rats experimentally infected with Acanthamoeba (n = 6), resulting in 100% of positivity, against 16.7% (n = 6) of positive identification in culture method using non-nutrient agar (NNA) with Escherichia coli. Corneal biopsies from rats were also tested (n = 6) and resulted in positivity in all samples in both molecular and culture methods. Eight out of ten presumptive human cases of Acanthamoeba keratitis were also confirmed by sn-PCR of scrapping samples, while the culture method was positive in only four cases. We discuss that animal model of AK can be an efficient tool to validate diagnostic methods and conclude that DNA extraction with the kit and snPCR protocol described here is an effective alternative for diagnosis of AK.