A New Tool to Quantify Receptor Recruitment to Cell Contact Sites during Host-Pathogen Interaction

Abstract

To understand the process of innate immune fungal recognition, we developed computational tools for the rigorous quantification and comparison of receptor recruitment and distribution at cell-cell contact sites. We used these tools to quantify pattern recognition receptor spatiotemporal distributions in contacts between primary human dendritic cells and the fungal pathogens C. albicans, C. parapsilosis and the environmental yeast S. cerevisiae, imaged using 3D multichannel laser scanning confocal microscopy. The detailed quantitative analysis of contact sites shows that, despite considerable biochemical similarity in the composition and structure of these species' cell walls, the receptor spatiotemporal distribution in host-microbe contact sites varies significantly between these yeasts. Our findings suggest a model where innate immune cells discriminate fungal microorganisms based on differential mobilization and coordination of receptor networks. Our analysis methods are also broadly applicable to a range of cell-cell interactions central to many biological problems.

Mannan Molecular Substructures Control Nanoscale Glucan Exposure in Candida

“…Glucan exposure has been linked to phagocytosis in other studies (Graus et al, 2014; Hardison and Brown, 2012), suggesting that differences in the amount of glucan exposed could relate to the changes in phagocytic efficiencies. Although the C. albicans cell wall contains 10% more b-glucan and 17% less mannose than C. glabrata (de Groot et al, 2008) by dry mass, flow cytometry determined that β-glucan exposure was 1.5-fold greater on C. glabrata than on C. albicans (Figures 1B and 1C).…”

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

“…Our previous studies allow us to estimate multivalently-engaging glucan exposure site density and area (per exposure site) as follows: SC5314—1 µm -2 density, 6.61×10 -4 µm 2 area; TRL035—4 µm -2 density, 9.62×10 -4 µm 2 area [65]. We previously measured the total area of contact sites between C. albicans and human immature dendritic cells as ∼10 µm 2 [74]. Finally, we have estimated (see above) that one Dectin-1 CRD occupies a footprint of ∼25 nm 2 .…”

Super-resolution imaging of C-type lectin spatial rearrangement within the dendritic cell plasma membrane at fungal microbe contact sites

“…3D confocal stacks of resting dendritic cells (0 h) and DC conjugates with zymosan at 1 and 4 h of interaction were imaged and analyzed by a custom procedure designed to accurately extract ssreceptor intensity from the curved contact site membranes [24]. Total contact site DC-SIGN signal increased by a factor of 67-fold ( p < 0.0005) at 1 h (relative to resting cell receptor density) and was not significantly different between 1 and 4 h (Figure 1A).…”

“…Note that while binarization was used to determine which positions were positive or negative for a given receptor, the original intensities at these positions were retained for use in intensity-based readouts. The Matlab code used for these analyses is available at the UNM STMC website software page (http://stmc.health.unm.edu/tools-and-data/index.html) and is described fully elsewhere [24]. …”