Innate Antifungal Immune Receptor, Dectin-1, Undergoes Ligand-Induced Oligomerization with Highly Structured β-Glucans and at Fungal Cell Contact Sites

Dectin-1A is a C-type Lectin innate immunoreceptor that recognizes β-(1,3:1,6)-glucan, a structural component of Candida species cell walls. The higher order structure of β-glucans ranges from random coil to insoluble fiber due to varying degrees of tertiary (helical) and quaternary structure. Model Saccharomyces cerevisiae β-glucans of medium and high molecular weight (MMW and HMW, respectively) are highly structured. In contrast, low MW glucan (LMW) is much less structured. Despite similar affinity for Dectin-1A, the ability of glucans to induce Dectin-1A mediated calcium influx and Syk phosphorylation positively correlates with their degree of higher order structure. Chemical denaturation and renaturation of MMW glucan showed that glucan structure determines agonistic potential, but not binding affinity, for Dectin-1A. We explored the role of glucan structure on Dectin-1A oligomerization, which is thought to be required for Dectin-1 signaling. Glucan signaling decreased Dectin-1A diffusion coefficient in inverse proportion to glucan structural content, which was consistent with Dectin-1A aggregation. Förster Resonance Energy Transfer (FRET) measurements revealed that molecular aggregation of Dectin-1 occurs in a manner dependent upon glucan higher order structure. Number and Brightness analysis specifically confirmed an increase in the Dectin-1A dimer and oligomer populations that is correlated with glucan structure content. Receptor modeling data confirms that in resting cells, Dectin-1A is predominantly in a monomeric state. Super Resolution Microscopy revealed that glucan-stimulated Dectin-1 aggregates are very small (<15 nm) collections of engaged receptors. Finally, FRET measurements confirmed increased molecular aggregation of Dectin-1A at fungal particle contact sites in a manner that positively correlated with the degree of exposed glucan on the particle surface. These results indicate that Dectin-1A senses the solution conformation of β-glucans through their varying ability to drive receptor dimer/oligomer formation and activation of membrane proximal signaling events. Author Summary Candidemia is the most common bloodstream infection in the United States. During infection, the fungal cell wall is an important virulence factor, playing roles in adhesion, immune recognition and colonization. The human innate immune system recognizes β-glucan, a highly immunogenic component of the fungal cell wall. During innate immune recognition of Candida, the organization of cell wall β-glucan is an important determinant of a successful immune activation. However, there have been many reports showing conflicting biological activities of β-glucans with different size, branching and structure. Here, using quantitative fluorescence imaging techniques, we investigate how differential size and structure of β-glucan impacts activation of the innate immune receptor, Dectin-1A. Our results indicate a positive correlation between highly structured glucans and Dectin-1A activation. Furthermore, we determined this is due to the higher ordered β-glucan causing Dectin-1A receptors to form aggregates that are below 15 nm in size. Finally, Dectin-1A receptor aggregation has also been shown to form at fungal particle contact sites with high β-glucan exposure.

in soluble form or as particulate "zymosan" are common models for stimulation of innate 159 immunocytes by fungal pathogen cell wall glucan. The above glucans have overall very 160 similar composition and structures to C. albicans yeast glucan, though relatively minor 161 differences in β-(1,6)-glucan side chain length and branching frequency have been 162 reported between these species [51,52]. 163 Using these glucans, we performed intracellular calcium ([Ca 2+ ] i ) measurement 164 experiments using HEK-293 cells transfected with Dectin-1A. We stimulated the cells 165 using either LMW, MMW, or HMW (Fig.1A). We found that large, highly ordered glucans 166 (HMW and MMW) induced more than a two-fold increase in peak amplitude of [Ca 2+ ] i . In 167 contrast, the glucans with a random coil structure (LMW) showed little induction of 168 calcium signaling compared to unstimulated cells. These results indicate that Dectin-1A 169 drives differential Ca 2+ flux to glucan ligands that vary in size and structure. 170 To determine how these differently structured soluble glucans impacted cellular patterns  (Fig. 1C). These results indicate that glucans with higher order structure are better able 178 to activate Dectin-1A-mediated Ca 2+ signaling and that this is a Syk dependent process.  this glucan is partially regained when the glucan is renatured (Fig. 2C). In addition, we 211 confirmed that glucan structure was lost when NaOH was added and regained when 212 neutralized with HCl (Fig. 2D). These results suggest that glucan structure is an 213 important factor in activating a Dectin-1A response.     and calibrated this volume using standard fluorophores of known diffusion coefficient. 279 We then observed fluorescent molecules (i.e., Dectin-1A-mEmerald) diffusing in and out  compared to LMW and unstimulated cells (Fig. 4A). This finding is consistent with an 288 increase in receptor aggregation upon stimulation, which we examine in greater detail 289 below. Moreover, we observed a significant decrease in receptor density after treatment 290 with MMW or HMW, compared to non-simulated or LMW cells (Fig. 4B). This finding      technique accurately resolves objects from the diffraction limit (~300 nm, the resolution 440 limit of conventional fluorescence microscopy methods) or above, down to ~15 nm (a 441 typical resolution limit of dSTORM using our configuration). H-SET analysis detected 442 sites of Dectin-1 labeling as "singlet" objects or "multiple" clustered objects. Multiple 443 clustered objects are those with three or more resolvable individual Dectin-1 molecules.

444
Singlet objects are those that appear to contain only a single, resolvable Dectin-1 445 labeling event, though it is possible that multiple Dectin-1 molecules in very close 446 proximity (<15 nm separation) would be unresolvable and appear as a singlet object. 447 We detected no significant change in the density of singlet objects or multiple object predictions closely at maximum radii between 5 and 6 nm (Fig. 7C). Simulations at 500 these radial parameters were then compared to experimental results with respect to the 501 donor-acceptor population percent that they predicted.

502
We used the predictions of this computational model to test the hypothesis that random   Our results demonstrate that the structure of β-glucan impacts receptor signaling by 555 determining the membrane organization and molecular aggregation state of Dectin-1A. 556 We showed that glucans with higher order structure are better able to activate Dectin-557 1A signaling. Upon activation by stimulatory soluble glucan, Dectin-1A enters 558 aggregated states that contain dimers and higher order oligomers, but these appear to 559 remain as small nanoscale domains containing relatively small numbers of receptors.  Table 3 for convenient reference. However, these ensemble-based studies concern β-glucan stimulation at the population 578 level. Therefore, we took a single cell approach to understand nanoscale glucan 579 signaling dynamics using glucans that vary in size and structure. In line with previous 580 studies, our results revealed that Dectin-1A activation is influenced by the β-glucan 581 triple helical structure. Together, these findings demonstrate that β-glucan impacts 582 receptor signaling due to structure and not merely through its affinity or size.    (Fig. 4), we would expect ~46000 total Dectin-1 proteins to be present in this 682 entire phagocytic contact membrane. However, an important caveat is that, in our model 683 system, we noted that SC5314 phagocytic synapses were much smaller than those for  Cells were maintained in an incubator at 37°C at 5% CO 2 levels. Cells were maintained 731 at 37°C, 5% CO 2 , and 75% humidity during imaging.   The samples were dissolved in DMSO-d6/D2O (6:1 by volume) at 100°C for 1 h.      To minimize the drift that occurred during data acquisition, a self-registration algorithm 966 was implemented.

967
The Dectin-1A nanodomain density was quantified by Super resolution imaging and Dectin-1A membrane density presented in Fig. 4.

982
The initial location of each particle was defined by drawing random numbers from a 983 uniform distribution. Throughout the simulation, particle movement was modeled by 984 using a random walk process. The distance each particle moved at each time point was 985 determined by the diffusion coefficient that was experimentally determined and reported 986 in Fig. 4. All particle movements are independent of their type and status. The simulation space included monomer molecules as donors and acceptors. This 988 simulation space-corresponded to total area of 0.64 µm 2 (equivalent to ~20 pixels 989 membrane area in an experimental FLIM dataset). The total duration of the simulation 990 was 97.6 μs (equivalent to the total experimental data acquisition time for 20 pixels).