Eukaryotic Cell doi:10.1128/EC.00316-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
GLYCOGEN PHOSPHORYLASE IN ACANTHAMOEBA SPP.: DETERMINING THE ROLE OF THE ENZYME DURING THE ENCYSTMENT PROCESS USING RNA INTERFERENCE (RNAi)
Jacob Lorenzo-Morales,
Jarmila Kliescikova,
Enrique Martinez-Carretero,
Luis Miguel De Pablos,
Bronislava Profotova,
Eva Nohynkova,
Antonio Osuna,
and
Basilio Valladares*
University Institute of Tropical Diseases and Public Health of the Canary Islands, University of La Laguna, Tenerife, Canary Islands, Spain; Department of Tropical Medicine, 1st Faculty of Medicine, Charles University in Prague, Czech Republic; Institute of Biotechnology, Department of Parasitology, University of Granada, Campus de Fuentenueva 18071, Granada, Spain
* To whom correspondence should be addressed. Email:
bvallada{at}ull.es.
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Abstract |
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Acanthamoeba infections are difficult to treat due to often-late diagnosis and lack of effective and specific therapeutical agents. The most important reason for unsuccessful therapy seems to be the existence of a double-wall cyst stage, which is highly resistant to the available treatments causing reinfections. The major components of the Acanthamoeba cyst wall are acid-resistant proteins and cellulose. The last one has been reported to be the major component of the inner cyst wall. It has been demonstrated 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 (i.e. Dictyostelium discoideum), glycogen phosphorylase has been reported as 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 process involved in the Acanthamoeba encystment could be similar to the ones reported in other lower eukaryotes. The catalytic domain of the glycogen phosphorylase was silenced using interference RNA (RNAi) methods and the effect of this phenomenon was analysed by light and electron microscopy, calcofluor staining, expression zymograms and northern and western blot analyses, both in siRNA 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 the cyst wall assembly, mainly for the formation of the cell wall inner layer.
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