A monoclonal antibody that specifically binds chitosan in vitro and in situ on fungal cell walls

Schubert, Max

doi:10.4014/jmb.1001.02023

Cited by 9 publications

(11 citation statements)

References 30 publications

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“…This conversion was often deduced from a concomitant induction of a chitin deacetylase rather than by direct histochemical proof of the presence of chitosan due to lack of a commercially available specific stain for chitosan (15). Polyclonal chitosan-specific antisera have been produced repeatedly, and recently, the generation of a monoclonal antibody specifically directed against chitosan has been described (12,18,20,23,30,31,40), but they have rarely been used in histochemical staining due to their typically low affinity toward the antigen (12,38). Lectins with specificity to chitosan have been described (10,25) but are not commercially available, and our attempts at reproducing the findings were not successful.…”

Section: Discussionmentioning

confidence: 99%

Fusion of a Novel Genetically Engineered Chitosan Affinity Protein and Green Fluorescent Protein for Specific Detection of Chitosan In Vitro and In Situ

Nampally

Moerschbacher

Kolkenbrock

2012

Appl Environ Microbiol

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ABSTRACTChitin 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 His6tags for purification and detection. The recombinant chitosan affinity protein (CAP) expressed inEscherichia coliwas 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-His5antibodies. 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 forin situchitosan staining.

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Section: Discussionmentioning

confidence: 99%

Fusion of a Novel Genetically Engineered Chitosan Affinity Protein and Green Fluorescent Protein for Specific Detection of Chitosan In Vitro and In Situ

Nampally

Moerschbacher

Kolkenbrock

2012

Appl Environ Microbiol

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“…First, mycelium resulting from growth in liquid complete medium (CM) was stained with the anti-chitosan antibody mAbG7 (Schubert et al, 2010). However, this revealed only weak staining of the hyphae ( Figure S1), which nevertheless suggested the presence of chitosan in the cell walls of vegetative hyphae.…”

Section: Chitosan Is a Component Of The Cell Wall In Vegetative Hyphaementioning

confidence: 99%

“…OGA488 staining was performed essentially as described in Geoghegan and Gurr (2016). Samples (mycelial pellets and coverslips that were overgrown with hyphae or had previously been inoculated Staining with the monoclonal anti-chitosan antibody mAbG7, a generous gift from Stefan Schillberg (Schubert et al, 2010), was performed as described previously (Geoghegan & Gurr, 2016).…”

Section: Confocal Imagingmentioning

confidence: 99%

“…Δcsn strains were first stained for the presence of chitosan, to determine whether deletion of CSN had resulted in alterations in either the amount or distribution of chitosan. However, staining of vegetative hyphae with OGA488, germlings with the anti-chitosan antibody mAbG7 (Schubert et al, 2010), or conidia with eosin Y did not reveal any differences in the patterns or intensity of staining in Δcsn, compared with the WT strain (Figures 5b and S13). The morphology of hyphae, germlings, and conidia was also unaffected in the Δcsn strain.…”

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confidence: 95%

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Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae

Geoghegan

Gurr

2017

Cellular Microbiology

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SummaryChitin deacetylation results in the formation of chitosan, a polymer of β1,4‐linked glucosamine. Chitosan is known to have important functions in the cell walls of a number of fungal species, but its role during hyphal growth has not yet been investigated. In this study, we have characterized the role of chitin deacetylation during vegetative hyphal growth in the filamentous phytopathogen Magnaporthe oryzae. We found that chitosan localizes to the septa and lateral cell walls of vegetative hyphae and identified 2 chitin deacetylases expressed during vegetative growth—CDA1 and CDA4. Deletion strains and fluorescent protein fusions demonstrated that CDA1 is necessary for chitin deacetylation in the septa and lateral cell walls of mature hyphae in colony interiors, whereas CDA4 deacetylates chitin in the hyphae at colony margins. However, although the Δcda1 strain was more resistant to cell wall hydrolysis, growth and pathogenic development were otherwise unaffected in the deletion strains. The role of chitosan hydrolysis was also investigated. A single gene encoding a putative chitosanase (CSN) was discovered in M. oryzae and found to be expressed during vegetative growth. However, chitosan localization, vegetative growth, and pathogenic development were unaffected in a CSN deletion strain, rendering the role of this enzyme unclear.

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“…flavus and A. parasiticus conidia were harvested from an overgrown PDA plate as described previously (Schubert et al 2010) and the retained conidia were washed three times with sterile phosphate-buffered saline (PBS) by centrifugation (40009g, 10 min, room temperature). Aliquots of each conidial suspension (80 ll, 4 9 10 4 conidia/ml) were incubated with 0.5-50 lM of each chemically-synthesized peptide (thanatin, metchnikowin, gallerimycin, D4E1, magainin or MSI-99) at 37°C in the dark (Table 1).…”

Section: In Vitro Bioassay For Antifungal Activitymentioning

confidence: 99%

Thanatin confers partial resistance against aflatoxigenic fungi in maize (Zea mays)

et al. 2015

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Aflatoxin-producing fungi can contaminate plants and plant-derived products with carcinogenic secondary metabolites that present a risk to human and animal health. In this study, we investigated the effect of antimicrobial peptides on the major aflatoxigenic fungi Aspergillus flavus and A. parasiticus. In vitro assays with different chemically-synthesized peptides demonstrated that the broad-spectrum peptide thanatin from the spined soldier bug (Podisus maculiventris) had the greatest potential to eliminate aflatoxigenic fungi. The minimal inhibitory concentrations of thanatin against A. flavus and A. parasiticus were 3.13 and 12.5 µM, respectively. A thanatin cDNA was subsequently cloned in a plant expression vector under the control of the ubiquitin-1 promoter allowing the recombinant peptide to be directed to the apoplast in transgenic maize plants. Successful integration of the thanatin expression cassette was confirmed by PCR and expression was demonstrated by semi-quantitative RT-PCR in transgenic maize kernels. Infection assays with maize kernels from T1 transgenic plants showed up to three-fold greater resistance against Aspergillus spp. infections compared to non-transgenic kernels. We demonstrated for the first time that heterologous expression of the antimicrobial peptide thanatin inhibits the growth of Aspergillus spp. in transgenic maize plants offering a solution to protect crops from aflatoxin-producing fungi and the resulting aflatoxin contamination in the field and under storage conditions.

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A monoclonal antibody that specifically binds chitosan in vitro and in situ on fungal cell walls

Cited by 9 publications

References 30 publications

Fusion of a Novel Genetically Engineered Chitosan Affinity Protein and Green Fluorescent Protein for Specific Detection of Chitosan In Vitro and In Situ

Fusion of a Novel Genetically Engineered Chitosan Affinity Protein and Green Fluorescent Protein for Specific Detection of Chitosan In Vitro and In Situ

Investigating chitin deacetylation and chitosan hydrolysis during vegetative growth in Magnaporthe oryzae

Thanatin confers partial resistance against aflatoxigenic fungi in maize (Zea mays)

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