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Applied and Environmental Microbiology, May 2002, p. 2503-2508, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2503-2508.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Use of Recombinant Cellulose-Binding Domains of Trichoderma reesei Cellulase as a Selective Immunocytochemical Marker for Cellulose in Protozoa

Markus Linder,1 Jadwiga Winiecka-Krusnell,2 and Ewert Linder2,3*

VTT Biotechnology, FIN 02044-VTT, Espoo, Finland,1 Swedish Institute for Infectious Disease Control,2 Microbiology and Tumorbiology Centre, Karolinska Institute, S 171 82, Solna, Sweden3

Received 20 June 2001/ Accepted 29 November 2001

Some unicellular organisms are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent, but in some cases, as in Acanthamoeba, it consists of cellulose instead. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible, due to the similarity of their constituent ß-1,4-linked hexose backbones. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. We have used a recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from Trichoderma reesei cellulases linked together in combination with monoclonal anticellulase antibodies and anti-mouse immunoglobulin fluorescein conjugate to specifically stain cellulose in the cysts of Acanthamoeba strains for fluorescence microscopy imaging. Staining was observed in ruptured cysts and frozen sections of cysts but not in intact mature cysts. No staining reaction was observed with the chitin-containing cyst walls of Giardia intestinalis, Entamoeba dispar, or Pneumocystis carinii. Thus, the recombinant CBD can be used as a marker to distinguish between cellulose and chitin. Thirteen of 25 environmental or clinical isolates of amoebae reacted in the CBD binding assay. All 13 isolates were identified as Acanthamoeba spp. Five isolates of Hartmannella and seven isolates of Naegleria tested negative in the CBD binding assay. Whether cyst wall cellulose really is a unique property of Acanthamoeba spp. among free-living amoebae, as suggested by our findings, remains to be shown in more extensive studies.

* Corresponding author. Mailing address: Swedish Institute for Infectious Disease Control (SMI), 171 82 Solna, Sweden. Phone: 46 8 457 2520. Fax: 46 8 310525. E-mail: ewert.linder{at}smi.ki.se.

Applied and Environmental Microbiology, May 2002, p. 2503-2508, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2503-2508.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Derda, M., Winiecka-Krusnell, J., Linder, M. B., Linder, E. (2009). Labeled Trichoderma reesei Cellulase as a Marker for Acanthamoeba Cyst Wall Cellulose in Infected Tissues. Appl. Environ. Microbiol. 75: 6827-6830 [Abstract] [Full Text]  
  • Lorenzo-Morales, J., Kliescikova, J., Martinez-Carretero, E., De Pablos, L. M., Profotova, B., Nohynkova, E., Osuna, A., Valladares, B. (2008). Glycogen Phosphorylase in Acanthamoeba spp.: Determining the Role of the Enzyme during the Encystment Process Using RNA Interference. Eukaryot Cell 7: 509-517 [Abstract] [Full Text]  
  • DebRoy, S., Dao, J., Soderberg, M., Rossier, O., Cianciotto, N. P. (2006). Legionella pneumophila type II secretome reveals unique exoproteins and a chitinase that promotes bacterial persistence in the lung. Proc. Natl. Acad. Sci. USA 103: 19146-19151 [Abstract] [Full Text]  


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