TY - JOUR
T1 - Development of Negative Controls for Fc-C-Type Lectin Receptor Probes
AU - Hatinguais, Rémi
AU - Kay, Madalaine
AU - Salazar, Fabián
AU - Conn, Daniel P
AU - Williams, David L
AU - Cook, Peter C
AU - Willment, Janet A
AU - Brown, Gordon D
N1 - Funding Information: This work was also supported, in part, by RO1GM119197 from the National Institutes of Health. For the purpose of open access, the author has applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. Funding Information: We acknowledge funding from the Wellcome Trust (102705, 217163), the MRC Centre for Medical Mycology at the University of Exeter (MR/N006364/1; MR/N006364/2) and the NIHR Exeter Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. Publisher Copyright: Copyright © 2023 Hatinguais et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
PY - 2023/5
Y1 - 2023/5
N2 - Fc-C-type lectin receptor (Fc-CTLRs) probes are soluble chimeric proteins constituted of the extracellular domain of a CTLR fused with the constant fraction (Fc) of the human IgG. These probes are useful tools to study the interaction of CTLRs with their ligands, with applications similar to those of antibodies, often in combination with widely available fluorescent antibodies targeting the Fc fragment (anti-hFc). In particular, Fc-Dectin-1 has been extensively used to study the accessibility of b-glucans at the surface of pathogenic fungi. However, there is no universal negative control for Fc-CTLRs, making the distinction of specific versus nonspecific binding difficult. We describe here 2 negative controls for Fc-CTLRs: a Fc-control constituting of only the Fc portion, and a Fc-Dectin-1 mutant predicted to be unable to bind b-glucans. Using these new probes, we found that while Fc-CTLRs exhibit virtually no nonspecific binding to Candida albicans yeasts, Aspergillus fumigatus resting spores strongly bind Fc-CTLRs in a nonspecific manner. Nevertheless, using the controls we describe here, we were able to demonstrate that A. fumigatus spores expose a low amount of b-glucan. Our data highlight the necessity of appropriate negative controls for experiments involving Fc-CTLRs probes. IMPORTANCE While Fc-CTLRs probes are useful tools to study the interaction of CTLRs with ligands, their use is limited by the lack of appropriate negative controls in assays involving fungi and potentially other pathogens. We have developed and characterized 2 negative controls for Fc-CTLRs assays: Fc-control and a Fc-Dectin-1 mutant. In this manuscript, we characterize the use of these negative controls with zymosan, a b-glucan containing particle, and 2 human pathogenic fungi, Candida albicans yeasts and Aspergillus fumigatus conidia. We show that A. fumigatus conidia nonspecifically bind Fc-CTLRs probes, demonstrating the need for appropriate negative controls in such assays.
AB - Fc-C-type lectin receptor (Fc-CTLRs) probes are soluble chimeric proteins constituted of the extracellular domain of a CTLR fused with the constant fraction (Fc) of the human IgG. These probes are useful tools to study the interaction of CTLRs with their ligands, with applications similar to those of antibodies, often in combination with widely available fluorescent antibodies targeting the Fc fragment (anti-hFc). In particular, Fc-Dectin-1 has been extensively used to study the accessibility of b-glucans at the surface of pathogenic fungi. However, there is no universal negative control for Fc-CTLRs, making the distinction of specific versus nonspecific binding difficult. We describe here 2 negative controls for Fc-CTLRs: a Fc-control constituting of only the Fc portion, and a Fc-Dectin-1 mutant predicted to be unable to bind b-glucans. Using these new probes, we found that while Fc-CTLRs exhibit virtually no nonspecific binding to Candida albicans yeasts, Aspergillus fumigatus resting spores strongly bind Fc-CTLRs in a nonspecific manner. Nevertheless, using the controls we describe here, we were able to demonstrate that A. fumigatus spores expose a low amount of b-glucan. Our data highlight the necessity of appropriate negative controls for experiments involving Fc-CTLRs probes. IMPORTANCE While Fc-CTLRs probes are useful tools to study the interaction of CTLRs with ligands, their use is limited by the lack of appropriate negative controls in assays involving fungi and potentially other pathogens. We have developed and characterized 2 negative controls for Fc-CTLRs assays: Fc-control and a Fc-Dectin-1 mutant. In this manuscript, we characterize the use of these negative controls with zymosan, a b-glucan containing particle, and 2 human pathogenic fungi, Candida albicans yeasts and Aspergillus fumigatus conidia. We show that A. fumigatus conidia nonspecifically bind Fc-CTLRs probes, demonstrating the need for appropriate negative controls in such assays.
KW - C-type lectin receptors
KW - host-pathogen interaction
KW - in vitro assays
KW - pathogen-associated molecular patterns (PAMPs)
KW - pattern-recognition receptor (PRRs)
UR - http://www.scopus.com/inward/record.url?scp=85163914160&partnerID=8YFLogxK
U2 - https://doi.org/10.1128/spectrum.01135-23
DO - https://doi.org/10.1128/spectrum.01135-23
M3 - Article
C2 - 37158741
SN - 2165-0497
VL - 11
SP - e0113523
JO - Microbiology spectrum
JF - Microbiology spectrum
IS - 3
ER -