Labeled Trichoderma reesei Cellulase as a Marker for Acanthamoeba Cyst Wall Cellulose in Infected Tissues

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 ␤-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.

Influence of temperature, oxygen and bacterial strain identity on the association of Campylobacter jejuni with Acanthamoeba castellanii

“…2). Campylobacters inside Acanthamoeba cysts could not be detected, as the cysts yielded a bright autofluorescent signal in both detection channels (data not shown), probably caused by the cellulose present in the cyst wall (Derda et al ., 2009). The same morphological changes as described for co‐culture experiments were observed during LCI.…”

Cellulose fibrils formation and organisation of cytoskeleton during encystment are essential for Acanthamoeba cyst wall architecture

“…This way we succeeded to demonstrate for the first time the cellulose fibrils in the ectocyst that were previously not visible by any other method. Immunocytochemical methods based on selective binding of recombinant cellulose-binding protein localised the cellulose in the endocyst of Acanthamoeba cysts and led to conclusion that cellulose is included only in the inner layer of the cyst wall 53 , 61 . However, it was proved previously that cellulose in Acanthamoeba cyst wall is masked by non-cellulose material (mainly proteins) and without insuring the removal of masking agent by alkali treatment, the absence of cellulose cannot be demonstrated by cytochemical methods 15 .…”

Stimulation of Acanthamoeba castellanii excystment by enzyme treatment and consequences on trophozoite growth

“…However, at the same incubation time, at 27°C and 37°C, excystment started only at 7.5 U/mL, with 24–46% of trophozoite forms observed, and became considerable, with more than 82% of trophozoites, at higher concentrations ( Figure 1B ). Interestingly, no effect was observed on A. castellanii excystment with treatments in the same conditions of temperatures and incubation times for chitinase, a glycoside hydrolase acting on chitin, a polymer of N-acetyl-β-D-glucosamine distinct from the chains of D-glucose constituting cellulose ( Supplementary Figure S1 ; Linder et al, 2002 ; Derda et al, 2009 ).…”

“…However, only small chitinase concentrations, ranging from 0.1 to 2 U/mL, were possible to be used in our conditions, indicating a potential absence of significant activity in our study. The presence of chitin in Acanthamoeba cyst wall is currently controversial ( Linder et al, 2002 ; Derda et al, 2009 ; Magistrado-Coxen et al, 2019 ). Nevertheless, several lectins used in the present study, i.e., WGA, SWGA and LEL, displayed a labeling of both ectocyst and endocyst layers in untreated cysts while they were previously described to have a better affinity for N-acetyl-β-D-glucosamine, the main component of chitin ( Elloway et al, 2004 ).…”