Development of Negative Controls for Fc-C-Type Lectin Receptor Probes

ABSTRACT 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 β-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 β-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 β-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 β-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.

1. This study performed with two major fungal pathogens, Candida albicans and Aspergillus fumigatus. With C. albicans, there was no non-specific binding, while A. fumigatus shows high non-specific binding. Can the authors discuss a bit more on this subject? 2. In the Material and Methods section, it is described that after fixing with 1% PFA overnight, the fungal samples were washed with PBS before taking them for stimulating study. As the authors may be aware, PFA crosslinks two proteins and some PFA may be having only one binding partner having other arm of the PFA with free reactive group, which may have an impact on immunostimulation. Therefore, after PFA fixation, introducing a quenching step will be important. Did the authors try quenching PFA fixed fungal samples before taking them for BWZ-reporter cell assay?
The G. Brown lab's generation of Fc-Dectin-1 chimeric proteins many years ago was a seminal step in the ability to detect exposed beta-glucans using immuno-detection methods. However, because the Fc-Dectin-1 molecules are not true antibodies, non-immune serum could not be used as a negative control to show binding specificity. To remedy this, Hatinguias et al. constructed mutant forms of the Fc-Dectin-1 that are unable to bind to beta-glucans (either lacking the binding domain or mutating key residues required for beta-glucan binding). The results are verified with multiple fungal particles providing scientific rigor. Intriguingly, the authors demonstrate that the negative control Fc-Dectin-1 molecules can bind to Aspergillus conidia in a beta-glucan independent manner suggesting some inherent "stickiness" of conidia. This however is unexplored. The results will be useful for the fungal field.

Suggestions
The D1mut construct is particularly useful as a negative control for the Fc-Dectin-1 probe, but should not be extrapolated to use as the negative control for other Fc-CTLR constructs (e.g., those binding mannan). The authors should acknowledge this in the discussion The authors should at least speculate on the nature of the binding of the D1mut to Aspergillus resting conidia. Since the Fc-only construct showed a similar level of binding to conidia, the binding of Fc-D1mut appears to be independent of the lectin region. Do antibodies also display a low level of non-specific binding to conidia? Would the inclusion of low levels of detergent reduce dispersion force interactions sufficiently to reduce it? line 76 add "carbohydrate-recognizing" extracellular domain of CTRLs to provide better context for those unfamiliar with the Ctype lectin receptor structure line 147 beta-glucan exposure is relatively low and should be qualified as such line 190 typo Fig. 12 should be Fig. 2 line 267, 302 define "depleted zymosan"

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Dear reviewers,
Thank for taking the time to review our manuscript. You will find in this letter a point-by-point response to your comments.

This study performed with two major fungal pathogens, Candida albicans and Aspergillus fumigatus. With C. albicans, there was no non-specific binding, while A. fumigatus shows high non-specific binding. Can the authors discuss a bit more on this subject?
It is a good point that we should have discussed indeed, especially as both reviewers have asked about it. We have now commented on this observation in the Discussion section of the manuscript.

In the Material and Methods section, it is described that after fixing with 1% PFA overnight,
the fungal samples were washed with PBS before taking them for stimulating study. As the authors may be aware, PFA crosslinks two proteins and some PFA may be having only one binding partner having other arm of the PFA with free reactive group, which may have an impact on immunostimulation. Therefore, after PFA fixation, introducing a quenching step will be important. Did the authors try quenching PFA fixed fungal samples before taking them for BWZ-reporter cell assay?
Yes, fixation quenching (with glycine for instance) would have be a good step to include in our protocol. We did not quench the PFA in either the staining protocol or the stimulation, which might be a concern although the fixative is extensively washed. Prior to the stimulation, samples are prepared in batch in RPMI 10% FCS medium that contain a number of proteins and amino acids, including glycine, any of them might be react with potential free arms of the PFA, and therefore would to prevent the fixation of the PFA to any receptor at the surface of the cells. These proteins/amino acid could also affect the stimulation of the cells. However, we have also observed activation of the BWZ Dectin-1 by heat-inactivated conidia and also ΔpyrG conidia (background A1160) that are unable to germinate in the absence of uracil and uridine, without the need to inactivate the spores, which is always likely to affect the outcome of such assay. Therefore, we do not think that omitting the quenching of the fixation is likely to have a strong biological effect in this context.

Reviewer #2
The D1mut construct is particularly useful as a negative control for the Fc-Dectin-1 probe, but should not be extrapolated to use as the negative control for other Fc-CTLR constructs (e.g., those binding mannan)

. The authors should acknowledge this in the discussion
We have now made this explicit. Although we believe that using either the Fc-control or the Fc-D1mut as negative control is better than having none, we agree that ideally specific negative controls, with point-mutation(s) in the carbohydrate-binding site would constitute ideal controls for Fc-CTLR assays.
The authors should at least speculate on the nature of the binding of the D1mut to Aspergillus resting conidia. Since the Fc-only construct showed a similar level of binding to conidia, the binding of Fc-D1mut appears to be independent of the lectin region. Do antibodies also display a low level of non-specific binding to conidia? Would the inclusion of low levels of detergent reduce dispersion force interactions sufficiently to reduce it?
We have now added a paragraph in the discussion to address the difference in non-specific binding of Fc-CTLRs by C. albicans and A. fumigatus. We believe this difference lies in potential differences in the production of fungal lectins by the two fungi, which could lead to different carbohydrate-binding specificity (Fc-CTLRs are glycosylated). We have not checked whether antibodies generally bind to the spores, although from these experiments we only see a marginal non-specific binding of the fluorescent anti-human Fc (as estimated by the low gMFI compared with unstained conidia). We have not tried to add detergent at step of the staining protocol.
line 76 add "carbohydrate-recognizing" extracellular domain of CTRLs to provide better context for those unfamiliar with the C-type lectin receptor structure We added "ligand binding" to take into account non-carbohydrate ligands.
line 147 beta-glucan exposure is relatively low and should be qualified as such

Modified.
line 190 typo Fig. 12 should be Fig. 2 Corrected, thanks for the careful reading.
line 267, 302 define "depleted zymosan" The term is now defined line 101 when zymosan is mentioned first in the results section.
We thank again the reviewers for their time and hope that we addressed their comments appropriately.
The authors.