Labeled Trichoderma reesei cellulase as a marker for Acanthamoeba cyst wall cellulose in infected tissues

M. Derda (Corresponding Author), J. Winiecka-Krusnell, Markus Linder, E. Linder

Research output: Contribution to journalArticleScientificpeer-review

5 Citations (Scopus)

Abstract

Some protozoans are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent. Acanthamoeba is an exception since its cyst wall contains cellulose. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible due to the similarity of the constituent β-1,4-linked hexose backbones of these molecules. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. The identification of Acanthamoeba spp., which is based primarily on morphological and biochemical features, is labor-intensive and requires cloning and axenization. We describe a novel immunocytochemical method for identification of Acanthamoeba spp. based on selective binding of Trichoderma reesei cellulase to protozoan cyst wall cellulose. A recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from T. reesei cellulases was coupled to the fluorescent dyes Alexa Fluor 350 and Alexa Fluor 568 or was labeled with biotin using EZ-Link sulfo-NHS-biotin. No staining reaction was observed with chitin-containing preparations of fungi. Thus, the recombinant CBDs can be used as a marker to distinguish between cellulose and chitin. This allows rapid identification of Acanthamoeba cyst wall cellulose in paraffin or frozen sections of infected tissues.
Original languageEnglish
Pages (from-to)6827-6830
JournalApplied and Environmental Microbiology
Volume75
Issue number21
DOIs
Publication statusPublished - 2009
MoE publication typeA1 Journal article-refereed

Fingerprint

Acanthamoeba
Trichoderma reesei
Trichoderma
Cellulase
cyst
endo-1,4-beta-glucanase
Cellulose
cellulose
Cysts
Chitin
chitin
biotin
Fluorescent Dyes
Protozoa
tissues
tissue
marker
Cellulases
Hexoses
cellulases

Keywords

  • parasites
  • infected tissues
  • Trichoderma reesei
  • cellulase
  • fungal cellulase
  • Acanthamoeba
  • cyst wall cellulose
  • cellulose binding
  • cellulose-binding domain

Cite this

Derda, M. ; Winiecka-Krusnell, J. ; Linder, Markus ; Linder, E. / Labeled Trichoderma reesei cellulase as a marker for Acanthamoeba cyst wall cellulose in infected tissues. In: Applied and Environmental Microbiology. 2009 ; Vol. 75, No. 21. pp. 6827-6830.
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abstract = "Some protozoans are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent. Acanthamoeba is an exception since its cyst wall contains cellulose. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible due to the similarity of the constituent β-1,4-linked hexose backbones of these molecules. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. The identification of Acanthamoeba spp., which is based primarily on morphological and biochemical features, is labor-intensive and requires cloning and axenization. We describe a novel immunocytochemical method for identification of Acanthamoeba spp. based on selective binding of Trichoderma reesei cellulase to protozoan cyst wall cellulose. A recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from T. reesei cellulases was coupled to the fluorescent dyes Alexa Fluor 350 and Alexa Fluor 568 or was labeled with biotin using EZ-Link sulfo-NHS-biotin. No staining reaction was observed with chitin-containing preparations of fungi. Thus, the recombinant CBDs can be used as a marker to distinguish between cellulose and chitin. This allows rapid identification of Acanthamoeba cyst wall cellulose in paraffin or frozen sections of infected tissues.",
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Labeled Trichoderma reesei cellulase as a marker for Acanthamoeba cyst wall cellulose in infected tissues. / Derda, M. (Corresponding Author); Winiecka-Krusnell, J.; Linder, Markus; Linder, E.

In: Applied and Environmental Microbiology, Vol. 75, No. 21, 2009, p. 6827-6830.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Labeled Trichoderma reesei cellulase as a marker for Acanthamoeba cyst wall cellulose in infected tissues

AU - Derda, M.

AU - Winiecka-Krusnell, J.

AU - Linder, Markus

AU - Linder, E.

PY - 2009

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N2 - Some protozoans are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent. Acanthamoeba is an exception since its cyst wall contains cellulose. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible due to the similarity of the constituent β-1,4-linked hexose backbones of these molecules. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. The identification of Acanthamoeba spp., which is based primarily on morphological and biochemical features, is labor-intensive and requires cloning and axenization. We describe a novel immunocytochemical method for identification of Acanthamoeba spp. based on selective binding of Trichoderma reesei cellulase to protozoan cyst wall cellulose. A recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from T. reesei cellulases was coupled to the fluorescent dyes Alexa Fluor 350 and Alexa Fluor 568 or was labeled with biotin using EZ-Link sulfo-NHS-biotin. No staining reaction was observed with chitin-containing preparations of fungi. Thus, the recombinant CBDs can be used as a marker to distinguish between cellulose and chitin. This allows rapid identification of Acanthamoeba cyst wall cellulose in paraffin or frozen sections of infected tissues.

AB - Some protozoans are able to encyst as a protective response to a harmful environment. The cyst wall usually contains chitin as its main structural constituent. Acanthamoeba is an exception since its cyst wall contains cellulose. Specific cytochemical differentiation between cellulose and chitin by microscopy has not been possible due to the similarity of the constituent β-1,4-linked hexose backbones of these molecules. Thus, various fluorescent brightening agents and lectins bind to both cellulose and chitin. The identification of Acanthamoeba spp., which is based primarily on morphological and biochemical features, is labor-intensive and requires cloning and axenization. We describe a novel immunocytochemical method for identification of Acanthamoeba spp. based on selective binding of Trichoderma reesei cellulase to protozoan cyst wall cellulose. A recombinant cellulose-binding protein consisting of two cellulose-binding domains (CBDs) from T. reesei cellulases was coupled to the fluorescent dyes Alexa Fluor 350 and Alexa Fluor 568 or was labeled with biotin using EZ-Link sulfo-NHS-biotin. No staining reaction was observed with chitin-containing preparations of fungi. Thus, the recombinant CBDs can be used as a marker to distinguish between cellulose and chitin. This allows rapid identification of Acanthamoeba cyst wall cellulose in paraffin or frozen sections of infected tissues.

KW - parasites

KW - infected tissues

KW - Trichoderma reesei

KW - cellulase

KW - fungal cellulase

KW - Acanthamoeba

KW - cyst wall cellulose

KW - cellulose binding

KW - cellulose-binding domain

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DO - 10.1128/AEM.01555-09

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JO - Applied and Environmental Microbiology

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SN - 0099-2240

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ER -