Fusion of a novel genetically engineered chitosan affinity protein and green fluorescent protein for specific detection of chitosan in vitro and in situ

Abstract

Chitin is the second most abundant polysaccharide, present, e.g., in insect and arthropod exoskeletons and fungal cell walls. In some species or under specific conditions, chitin appears to be enzymatically de-N-acetylated to chitosan-e.g., when pathogenic fungi invade their host tissues. Here, the deacetylation of chitin is assumed to represent a pathogenicity mechanism protecting the fungus from the host's chitin-driven immune response. While highly specific chitin binding lectins are well known and easily available, this is not the case for chitosan-specific probes. This is partly due to the poor antigenicity of chitosan so that producing high-affinity, specific antibodies is difficult. Also, lectins with specificity to chitosan have been described but are not commercially available, and our attempts to reproduce the findings were not successful. We have, therefore, generated a fusion protein between a chitosanase inactivated by site-directed mutagenesis, the green fluorescent protein (GFP), and StrepII, as well as His(6) tags for purification and detection. The recombinant chitosan affinity protein (CAP) expressed in Escherichia coli was shown to specifically bind to chitosan, but not to chitin, and the affinity increased with decreasing degree of acetylation. In vitro, CAP detection was possible either based on GFP fluorescence or using Strep-Tactin conjugates or anti-His(5) antibodies. CAP fluorescence microscopy revealed binding to the chitosan exposing endophytic infection structures of the wheat stem rust fungus, but not the chitin exposing ectophytic infection structures, verifying its suitability for in situ chitosan staining.

Fig 1

Schematic representation of the CAP gene. The scale above indicates the nucleotide position.

Fig 2

SDS-PAGE of the crude extract from E. coli Rosetta 2(DE3)(pET-22b-StrepII-CSN-eGFP-HIS₆ E122Q, pLysSRARE2); (60 μg, lane 1) and CAP purified using affinity chromatography (6 μg; lane 2), either stained using Coomassie brilliant blue G-250 (A) or Western blotted and detected by chemiluminescence using HRP-coupled StrepII affinity protein (B). The band at ca. 75 kDa represents CAP. M, molecular mass markers.

Fig 3

Dot activity assay to check the enzymatic activity of wild-type chitosanase and CAP. Lane 1, crude extract from E. coli Rosetta 2(DE3)(pET-22b(+), pLysSRARE2); lane 2, purified wild-type chitosanase; lane 3, purified CAP. Chitosanase activity is revealed as a dark spot in the calcofluor white-stained gel.

Fig 4

Binding specificity of CAP. Different chitosans (DAs from 10% to 56%) and glycol-chitin (DA of 100%) were spotted in dilution series (starting with 1,000 ng) onto nitrocellulose membrane, and the membrane was blocked with BSA, washed, and incubated with CAP (0.1 mg ml⁻¹ in TBS containing 5% [wt/vol] BSA) for 1 h at room temperature before excess CAP was washed off. Bound CAP was detected using StrepII affinity protein (A) or anti-His₅ antibody (B) or by the fluorescence of eGFP on a transilluminator (Dark Reader, excitation wavelength of 420 to 500 nm; MoBiTech, Germany) (C) or under a UV lamp (AlphaImager, set to 365 nm; Alpha Innotech Corp., United States) (D). Glycol-chitin (DA of 100%) was detected by fluorescence (Dark Reader) after binding to WGA-FITC conjugate to show that it was not lost during fixation (E).

Fig 5

Double staining of in vitro-induced infection structures of the wheat stem rust fungus Puccinia graminis f. sp. tritici. 1, spore; 2, germ tube; 3, appressorium; 4, substomatal vesicle; 5, infection hypha. Germ tube, appressorium, and the tip of the infection hypha were labeled by WGA conjugated to Texas Red (red fluorescence), indicating the presence of chitin, while CAP staining revealed the presence of chitosan in the substomatal vesicle and, less marked, infection hypha (green fluorescence).