RESUMEN
The encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions, such as those associated with starvation, low temperatures, and biocides. Furthermore, cysteine proteases have been implicated in the massive turnover of intracellular components required for encystation. Thus, strict modulation of the activities of cysteine proteases is required to protect Acanthamoeba from intracellular damage. However, mechanisms underlying the control of protease activity during encystation have not been established in Acanthamoeba. In the present study, we identified and characterized Acanthamoeba cysteine protease inhibitor (AcStefin), which was found to be highly expressed during encystation and to be associated with lysosomes by fluorescence microscopy. Recombinant AcStefin inhibited various cysteine proteases, including human cathepsin B, human cathepsin L, and papain. Transfection with small interfering RNA against AcStefin increased cysteine protease activity during encystation and resulted in incomplete cyst formation, reduced excystation efficiency, and a significant reduction in cytoplasmic area. Taken together, these results indicate that AcStefin is involved in the modulation of cysteine proteases and that it plays an essential role during the encystation of Acanthamoeba.
Asunto(s)
Acanthamoeba castellanii/metabolismo , Cistatinas/metabolismo , Proteasas de Cisteína/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Estadios del Ciclo de Vida/genética , Proteínas Protozoarias/metabolismo , Acanthamoeba castellanii/genética , Secuencia de Aminoácidos , Animales , Catepsinas/antagonistas & inhibidores , Catepsinas/genética , Cistatinas/genética , Cistatinas/farmacología , Proteasas de Cisteína/genética , Inhibidores de Cisteína Proteinasa/genética , Inhibidores de Cisteína Proteinasa/farmacología , Regulación de la Expresión Génica , Silenciador del Gen , Humanos , Lisosomas/metabolismo , Datos de Secuencia Molecular , Proteínas Protozoarias/genética , ARN Interferente Pequeño/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Alineación de SecuenciaRESUMEN
Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner.
Asunto(s)
Acanthamoeba/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Ribosómico 18S/genética , Animales , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Sensibilidad y EspecificidadRESUMEN
Autophagy, an evolutionarily conserved protein degradation pathway in eukaryotes, plays essential roles during starvation and cellular differentiation by eliminating unwanted and/or unnecessary cell material including organelles. Autophagy protein 16 (Atg16) is an essential component of the autophagic machinery. The present study identified and characterized an Atg16 homologue (AcAtg16) in Acanthamoeba, an opportunistic pathogen responsible for several distinct diseases in humans. AcAtg16 was highly expressed during encystation and was found to be associated with small or large vesicular structures that partially colocalized with autophagolysosomes. Small interfering RNA against AcAtg16 inhibited autophagosome formation and reduced the encystation efficiency of Acanthamoeba. Moreover, most mitochondria remained undigested in these knockdown cells. Taken together, these results indicate that AcAtg16 is involved in autophagosome formation and plays an essential role in the encystation of Acanthamoeba.