Dectin-1 Molecular Aggregation and Signaling is Sensitive to β-Glucan Structure and Glucan Exposure on Candida albicans Cell Walls

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. Comparison of receptor modeling data with FRET measurements 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 a few 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.

Dectin-1A is a C-type Lectin innate immunoreceptor that recognizes β-(1,3;1,6)-glucan, 23 a structural component of Candida species cell walls. The higher order structure of β-24 glucans ranges from random coil to insoluble fiber due to varying degrees of tertiary 25 (helical) and quaternary structure. Model Saccharomyces cerevisiae β-glucans of 26 medium and high molecular weight (MMW and HMW, respectively) are highly 27 structured. In contrast, low MW glucan (LMW) is much less structured. Despite similar 28 affinity for Dectin-1A, the ability of glucans to induce Dectin-1A mediated calcium influx 29 and Syk phosphorylation positively correlates with their degree of higher order structure. 30 Chemical denaturation and renaturation of MMW glucan showed that glucan structure 31 determines agonistic potential, but not binding affinity, for Dectin-1A. We explored the 32 role of glucan structure on Dectin-1A oligomerization, which is thought to be required for relevant virulence factor, playing roles in adhesion, colonization and immune recognition 56 [8,9]. 57 Due to the abundant amount of mannan in the outer cell wall, β-glucan exhibits a very 58 limited, punctate pattern of nanoscale surface exposure. The extent of this glucan 59 masking is influenced by environmental conditions such as intestinal pH or lactate levels 60 [10,11]. In addition, interactions with neutrophils have been shown to "unmask" the 61 mannose layer through a neutrophil extracellular trap-mediated mechanism [12]. 62 Furthermore, our lab and others have determined that anti-fungal drugs "unmask" the 63 fungal cell wall, which leads to increases in nanoscale regions of glucan exposure and 64 correlates with enhanced host defense [13][14][15]. Therefore, fungal species use masking 65 as a way to evade immune recognition of β-glucan by the host's immune system [16].
Interestingly, the response to MMW glucan was uniform at the single cell level. 158 However, cells stimulated with HMW glucan exhibited a more heterogeneous response, 159 with some cells achieving comparable maximum amplitudes as with MMW and others 160 exhibiting little change from basal calcium levels (Fig. 1A, B). We expect that HMW 161 glucan is present as larger particles than MMW, so at equal mass/volume 162 concentrations, the HMW solution will have a lower concentration of particles. Non-163 responder cells in HMW experiments may have stochastically encountered too few 164 glucan particles to achieve a detectable signaling response. When we stimulated cells 165 with MMW or HMW at equimolar concentrations, which should have similar glucan 166 particulate concentrations, we observed a minor difference in peak amplitude, but we 167 saw that the integrated Ca 2+ flux over time was the same for MMW and HMW glucans 168 (Supplemental Fig. 1). Furthermore, single cell data demonstrated a similarly uniform 169 response of Dectin-1 to MMW and HMW glucans under these conditions. These results 170 indicate that Dectin-1A drives differential Ca 2+ flux to glucan ligands that vary in size 171 and structure. 172 To determine how these differently-structured, soluble glucans impacted cellular 173 patterns of Syk phosphorylation, we stimulated HEK-293 cells expressing Dectin1A-174 mEmerald with H 2 O (vehicle), LMW, MMW, or HMW. Whole cell lysate was collected 175 and Syk phosphorylation was determined by western blot analysis. Likewise, our results 176 show an increase in Syk phosphorylation in the larger, highly structured glucan MMW 177 and HMW compared to unstimulated and LMW stimulated cells ( Fig. 1 D,E).
with MMW glucan (Fig. 1F). These results indicate that glucans with higher order 180 structure are better able to activate Dectin-1A-mediated Ca 2+ signaling and that this is a 181 Syk dependent process.   To determine if the glucan structure affects signaling outcomes, we denatured MMW 208 (highly stimulatory glucan) using DMSO, a chaotrope that promotes a reduction in 209 glucan tertiary structure, thus shifting MMW's triple helix structure to a more single glucan, we did not observe calcium signaling in cells expressing Dectin-1A ( Fig. 2A, B). 212 However, when we renatured the glucans by removing DMSO via dialysis we observe 213 partial recovery of calcium signaling. We found that renatured glucans induce a 214 significant increase in peak amplitude [Ca 2+ ] i response compared to DMSO denatured 215 MMW and renatured MMW stimulated untransfected HEK-293 cells (Fig. 2 C). In 216 addition, we confirmed the loss of helical structure via a Congo Red assay. Congo Red 217 specifically binds to β-(1,3)-glucans with a triple helix conformation as their tertiary 218 structure. This binding is detected by bathochromic shift in absorbance maximum from 219 488 to 516 nm [52]. Our results indicated a loss in glucan structure after denaturation in 220 a DMSO solution that was regained upon renaturation ( Fig. 2 D). Moreover, we 221 repeated these experiments by stimulating cells with glucan denatured with NaOH or 222 neutralized renatured glucan [17]. Similarly, our results show that cells lose the ability to 223 activate Dectin-1A calcium signaling when stimulated with denatured MMW glucan but 224 regain the ability to stimulate Dectin-1A activation when the glucan is renatured (Fig. 2  225 E,F). We found that renatured glucans induce a significant increase in peak amplitude 226 [Ca 2+ ] i response compared to NaOH denatured MMW and renatured MMW stimulated 227 untransfected HEK-293 cells (Fig. 2 G). In addition, we confirmed that glucan structure 228 was lost when NaOH was added and regained when neutralized (Fig. 2 H). These 229 results suggest that glucan structure is an important factor in activating a Dectin-1A 230 response. 231  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29 3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29 3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  21  22  23  24  25  26  27  28  29  suggesting that the size of the glucans is correlated with their higher-order structure. 280 Together, these results indicate that downstream signaling of the receptor is determined 281 by the structure of the glucan rather than affinity alone. 282  receptor diffusion coefficient decreased, potentially due to increasing hydrodynamic 298 radius as an initially monomeric receptor formed larger clusters/oligomers. We obtained 299 average diffusion coefficients and spatial number density of our receptor using Raster 300 Image Correlation Spectroscopy (RICS). RICS allowed us to survey multiple areas of 301 the cell for molecular parameters such as diffusion coefficient and receptor density. 302 This section pertains to receptor diffusion measurement by RICS, while receptor density 303 is treated in a separate section below. Furthermore, because fluorescence was probed 304 within a large cell area, RICS analyses suffered much less from photobleaching and 305 location specific artifacts than analogous single-point measurements. Previous research 306 has described RICS in more detail [53,54]. Briefly, we generated a volume of excitation 307 using focused laser illumination and calibrated a confocal observation volume using  the receptors (configuration to be used for experimental determination of receptor 374 aggregation by FRET) (Fig. 5 B). The observed decay curves were analyzed by 375 performing a mono-exponential and bi-exponential fit. For donor only and our negative 376 control we observed a negative amplitude for the second component in the bi-377 exponential fit, indicating a mono-exponential fit was superior for these conditions. This indicated that unstimulated and LMW stimulated cells contained a significantly higher 446 amount of monomer pixels compared to MMW and HMW glucan treated cells (Fig. 6D). technique accurately resolves objects from the diffraction limit (~300 nm, the resolution 472 limit of conventional fluorescence microscopy methods) or above, down to ~15 nm (a 473 typical resolution limit of dSTORM using our configuration). H-SET analysis detected 474 sites of Dectin-1 labeling as "singlet" objects or "multiple" clustered objects. Multiple 475 clustered objects are those with three or more resolvable individual Dectin-1 molecules. 476 Singlet objects are those that appear to contain only a single, resolvable Dectin-1 477 labeling event, though it is possible that multiple Dectin-1 molecules in very close 478 proximity (<15 nm separation) would be unresolvable and appear as a singlet object. 479 We detected no significant change in the density of singlet objects or multiple object 480 clusters before vs after MMW glucan stimulation (Fig. 7A,B; Supplemental Fig. 2).  value significantly below that experimentally observed, we would propose that a minor fraction of Dectin-1 molecules may participate in oligomeric aggregates on cell 547 membranes, even in the absence of glucan. Using simulations with maximum radial 548 values of 5 and 6 nm, our results indicate that the experimentally observed amount of 549 Dectin-1 receptors dimerizing (Donor-Acceptor population) prior to stimulation match 550 closely to our simulated results (Fig. 8D). This indicated that the FRET signal we 551 observe prior to stimulation was attributable to random "collisional" interactions of  images show TRL035 forming a phagocytic cup more efficiently than SC5314 (Fig. 9A). 577 Our results show that HEK-293 cells co-transfected with Dectin-1A-mEmerald and from approximately 15% in non-contact site membrane to about 25% in contact sites of 580 high glucan exposing yeast TRL035 and C. albicans derived glucan particles. 581 Interestingly, we did not measure a significant increase in receptor aggregation between 582 non-contact membranes and contact sites with low glucan exposing SC5314 (Fig. 9  583 B,C), which may indicate that the amount of aggregated Dectin-1 at SC5314 contacts 584 was quite small and below the detection limit. Furthermore, we observed no significant 585 difference in FRET efficiency between any conditions tested (Fig. 9B), similarly to our 586 observations with soluble glucan. The significance and interpretation of these findings is 587 further discussed below. These results suggest that the larger cell wall glucan exposure 588 results in an increase of Dectin-1 in molecular aggregates with associated signaling, 589 resulting in a more efficient recognition of yeast by the Dectin-1A receptor. 590   We showed that glucans with higher order structure are better able to activate Dectin-607 1A signaling. Upon activation by stimulatory soluble glucan, Dectin-1A enters 608 aggregated states that contain dimers and higher order oligomers, but these appear to 609 remain as small nanoscale domains containing relatively small numbers of receptors. suggests that the biological response to glucans with strong helical structure (e.g., 640 HMW glucan) seems bi-stable in nature, with response/non-response being correlated 641 with glucan structure but the amplitude of calcium signal being similar in single cells, 642 once successfully triggered. our reported Dectin-1 density (Fig. 4), the contact sites we measured would contain 706 ~46000 total Dectin-1 proteins. So, the Dectin-1 system is able to drive signaling 707 responses when, at most, only a few hundred receptors, corresponding to less than 1% 708 of the total contact site resident Dectin-1 proteins, are aggregated in the contact. These 709 results and estimates suggest that fungal recognition requires the Dectin-1 system to 710 engage in a search for rare sites of multivalent interaction with glucan. Signal initiation 711 must be sensitive to activation of relatively small numbers of Dectin-1 proteins. In the 712 future, it will be important to achieve a better understanding of Dectin-1's collaboration 713 with other anti-fungal receptors (e.g., DC-SIGN and CD206). Such receptors may be 714 important for building and stabilizing a fungal contact that can effectively promote 715 Dectin-1's ability to search for and find its rare sites of glucan exposure.
undergo Syk-dependent signaling. Here we provide evidence in support of a model in 718 which highly structured glucans induce stable dimerization and/or oligomerization of the 719 receptor. This allows their (hem)ITAM domains to become close enough for a sufficient 720 period of time to allow for the activation of Syk, leading to further signaling cascades. 721 Greater understanding of receptor activation is required to better understand the role of 722 Dectin-1A and its agonists as a potential way forward for adjuvant and immunotherapy Cells were grown in an incubator at 37°C at 5% CO 2 and saturating humidity. Cells were 733 maintained at 37°C, 5% CO 2 , and 75% relative humidity during imaging. 734

Soluble Glucan Linkage Analysis 778
Desalted and lyophilized samples of the fractions were dissolved in dimethylsulfoxide 779 (DMSO) and treated with NaOH and methyl iodide to methylate all free hydroxyl groups 780 [75]. The methylated material was purified by extraction with dichloromethane and 781 washing with water. The purified material was then hydrolyzed with trifluoroacetic acid, 782 the reducing ends of the resulting sugars were reduced with NaBD 4 , and then the 783 resulting free hydroxyl groups were acetylated with acetic anhydride. The mixture of derivative corresponding to a particular linkage has been identified by a characteristic 786 retention time and mass spectrum using a mass detector. The relative amount of each 787 derivative was measured by gas chromatography with flame ionization detection. The 788 areas obtained for each observed peak were used to calculate the relative amounts of 789 each type of linkage found in the sample ( Table 1). The 3,6-linked residues represent 790 branch points. 791  Fig. 3). 803 Table 2.  taken immediately after stimulated with β-glucans at a concentration of 10 µg/ml. 913 Analysis was conducted on minute time points (1-5 minutes) by averaging ten frames cells were resuspended in 1 ml of PBS. 100 µl of the solution was added to HEK-293 916 cells in 35 mm dishes 15 minutes prior to imaging. 23 frames were collected per cell. 917 Images were collected at a maximum of 45 minutes after the addition of yeast per plate. 918 Analysis was conducted on the plasma membrane by masking out internal cellular 919 compartments on the images. For our fungal contact site studies, analysis was 920 conducted on the plasma membrane that was in contact with the fungus and a separate 921 masking for plasma membrane that was not in contact with any yeast. A bi-exponential 922 fit was performed to the decay curve. For donor only as well as donor and acceptor on 923 opposite sides of the plasma membrane (negative control), the decay curve indicated a 924 negative amplitude for one of the components, thus indicating a mono-exponential 925 decay. Therefore, decay curves from these samples were analyzed using a mono-926 exponential fit. For cells with donor-acceptor on the cytosolic tail, data was fit to a bi- Images collected were also used for our Numbers and Brightness analysis; we used 955 192 nM EGFP in solution and purified mEmerald-Dectin-1 protein to set the average 956 brightness of our monomeric protein (Supplemental Fig. 4). Furthermore, the S-factor 957 was calculated using the background image. We divided each brightness distribution by (minPts), that is, the minimum number of objects composing a multi-cluster, and the 980 maximum distance between the objects within a multi-cluster (epsilon). We optimized 981 these parameters, defining them as 3 and 27 nm, respectively, according to optimization 982 procedures previously described [64]. fluorophore molecules on the membrane in a specific membrane area on the real cell. 986 As the experiments have shown that the ratio of donors to acceptors is roughly 1:1, in 987 our model 50% of the particles were donors while the rest were acceptors. The absolute 988 number of donor and acceptor molecules was based on the experimentally determined 989 Dectin-1A membrane density presented in Fig. 8. 990 The initial location of each particle in the simulation space was defined by drawing 991 random numbers from a uniform distribution. Throughout the simulation, particle 992 movement was modeled by using a random walk process. The distance each particle 993 moved at each time point was determined by the diffusion coefficient that was 994 experimentally determined and reported in Fig. 4. 995 The simulation space included monomer molecules as donors and acceptors. The size 996 of this simulation space corresponded to an experimental area equivalent to 0.16 µm 2 of 997 the cell membrane. The total duration of the simulation was equivalent to the total 998 amount of time required to acquire data from 5 pixels of an experimental FLIM FRET 999 observation (equivalent to 24 μs of total data acquisition time). 1000 To simulate a TCSPC FLIM experiment, each simulation run included 2000 sequential 1001 excitation pulses, followed by a window of simulated fluorescence decay observation 1002 with a length of 12 ns (0.1ns time resolution). At the start of each pulse, 30% of the 1003 donors were selected to act as excited particles, which corresponded with fractional 1004 donor excitation observed under our FLIM experimental conditions (Supplemental Fig.  1005 5). Note that we consider that this value represents an upper bound on % donors 1006 excited under experimental conditions due to non-linearity in response at high laser where R 0 is the Förster distance, and R i is the distance between donor and acceptor. 1027 The FRET efficiency calculation is more complicated when a donor transfers its energy 1028 to more than a single acceptor (see "FRET efficiency" below). In both cases, the donor 1029  25 1.56 1.87 2.18 2.49 2.81 3.12 3.43 3.74 4.05