Glycogen Phosphorylase in Acanthamoeba spp.: Determining the Role of the Enzyme during the Encystment Process Using RNA Interference

Abstract

Acanthamoeba infections are difficult to treat due to often late diagnosis and the lack of effective and specific therapeutic agents. The most important reason for unsuccessful therapy seems to be the existence of a double-wall cyst stage that is highly resistant to the available treatments, causing reinfections. The major components of the Acanthamoeba cyst wall are acid-resistant proteins and cellulose. The latter has been reported to be the major component of the inner cyst wall. It has been demonstrated previously that glycogen is the main source of free glucose for the synthesis of cellulose in Acanthamoeba, partly as glycogen levels fall during the encystment process. In other lower eukaryotes (e.g., Dictyostelium discoideum), glycogen phosphorylase has been reported to be the main tool used for glycogen breakdown in order to maintain the free glucose levels during the encystment process. Therefore, it was hypothesized that the regulation of the key processes involved in the Acanthamoeba encystment may be similar to the previously reported regulation mechanisms in other lower eukaryotes. The catalytic domain of the glycogen phosphorylase was silenced using RNA interference methods, and the effect of this phenomenon was assessed by light and electron microscopy analyses, calcofluor staining, expression zymogram assays, and Northern and Western blot analyses of both small interfering RNA-treated and control cells. The present report establishes the role of glycogen phosphorylase during the encystment process of Acanthamoeba. Moreover, the obtained results demonstrate that the enzyme is required for cyst wall assembly, mainly for the formation of the cell wall inner layer.

Resistance of Acanthamoeba Cysts to Disinfection Treatments Used in Health Care Settings

“…Neff's encystment medium was chosen since it allows recovery of high numbers of cysts and it may better represent the conditions encountered by amoebae in water networks than other conditions used to produce cysts (25). SDS (0.5%) was used after encystment to kill immature cysts and dissociate aggregates (35), although these aggregates may need to be further considered, as they FIG. 2.…”

Detection of Acanthamoeba on the ocular surface in a Spanish population using the Schirmer strip test: pathogenic potential, molecular classification and evaluation of the sensitivity to chlorhexidine and voriconazole of the isolated Acanthamoeba strains

“…Regarding AK, an early diagnosis is required to achieve a successful therapeutic outcome (Khan, 2006;Lorenzo-Morales et al, 2013). Nevertheless, effective therapies need to be developed against Acanthamoeba infections, owing mainly to the existence of a highly resistant cyst stage in the life cycle of these amoebae and also to the emergence of strains that are resistant to the currently used anti-amoebic (Khan, 2006;Lorenzo-Morales et al, 2008Siddiqui & Khan, 2012).…”

Therapeutic Potential of a Combination of Two Gene-Specific Small Interfering RNAs against Clinical Strains of Acanthamoeba

“…RNAi appears promising for silencing gene expression in parasitic pathogens, such as protozoans and helminths, as well as disease vectors, by specific target mRNA interference (9,10,18,20). Analysis of gene functions in pathogens of infectious diseases and their vectors is important for research in drug development, and the silencing effects may be directly employed to control parasite transmission and development.…”

“…Since it was first applied in the genus Acanthamoeba (9), a few genes involved in Acanthamoeba pathogenesis and the formation of mature cysts have been silenced using siRNA in order to identify them as possible targets for chemotherapy and gene therapy or as key items in cellular functions (4,9,10,13,14). In previous studies, extracellular serine proteases were silenced using siRNAs, resulting in the Acanthamoeba organisms not being able to degrade human corneal cells (9).…”

“…More recently, it was established by using siRNAs that serine proteases also play an important role in trophozoite differentiation and reemergence during excystment (4). Furthermore, the role of glycogen phosphorylase in the formation of mature cysts in Acanthamoeba was also established recently by using siRNA (10). Thus, blocking the entry and exit paths of these amoebae during their pathogenesis process could be achieved by using these siRNA molecules.…”