At host-pathogen contact sites with Candida albicans, Dectin-1 activates pro-inflammatory signaling, while DC-SIGN promotes adhesion to the fungal surface. We observed that Dectin-1 and DC-SIGN collaborate to enhance capture/retention of C. albicans under fluid shear culture conditions. Therefore, we devised a cellular model system wherein we could investigate the interaction between these two receptors during the earliest stages of host-pathogen interaction. In cells expressing both receptors, DC-SIGN was quickly recruited to contact sites (103.15% increase) but Dectin-1 did not similarly accumulate. Once inside the contact site, FRAP studies revealed a strong reduction in lateral mobility of DC-SIGN (but not Dectin-1), consistent with DC-SIGN engaging in multivalent adhesive binding interactions with cell wall mannoprotein ligands. Interestingly, in the absence of Dectin-1 co-expression, DC-SIGN recruitment to the contact was much poorer--only 35.04%. These data suggested that Dectin-1 promotes the active recruitment of DC-SIGN to the contact site. We proposed that Dectin-1 signaling activates the RHOA pathway, leading to actomyosin contractility that promotes DC-SIGN recruitment, perhaps via the formation of a centripetal ActoMyosin Flow (AMF) directed into the contact site. Indeed, RHOA pathway inhibitors significantly reduced Dectin-1 associated DC-SIGN recruitment to the contact site. We used agent based modeling to predict DC-SIGN transport kinetics with ("Directed+Brownian") and without ("Brownian") the hypothesized actomyosin flow-mediated transport. The Directed+Brownian transport model predicted a DC-SIGN contact site recruitment (108.72%), similar to that we observed experimentally under receptor co-expression. Brownian diffusive transport alone predicted contact site DC-SIGN recruitment of only 54.02%. However, this value was similar to experimentally observed recruitment in cells without Dectin-1 or treated with RHOA inhibitor, suggesting that it accurately predicted DC-SIGN recruitment when a contact site AMF would not be generated. TIRF microscopy of nascent cell contacts on glucan-coated glass revealed Dectin-1 dependent DC-SIGN and F-actin (LifeAct) recruitment kinetics to early-stage contact site membranes. DC-SIGN entry followed F-actin with a temporal lag of 8.35 {+/-} 4.57 seconds, but this correlation was disrupted by treatment with RHOA inhibitor. Thus, computational and experimental evidence provides support for the existence of a Dectin-1/RHOA-dependent AMF that produces a force to drive DC-SIGN recruitment to pathogen contact sites, resulting in improved pathogen capture and retention by immunocytes. These data suggest that the rapid collaborative response of Dectin-1 and DC-SIGN in early contact sties might be important for the efficient acquisition of yeast under flow conditions, such as those that prevail in circulation or mucocutaneous sites of infection.

https://biorxiv.org/content/10.1101/2020.12.15.422964v1.full.pdf

Dectin-1 mediated DC-SIGN Recruitment to Candida albicans Contact Sites

Glucans

Dectin-1A is a C-type Lectin innate immunoreceptor that recognizes β-(1,3:1,6)-glucan, a structural component of Candidaspecies 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 cerevisiaeβ-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. Forster 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 (

https://www.biorxiv.org/content/biorxiv/early/2020/05/25/824995.full.pdf

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 {beta}-(1,3:1,6)-glucan, a structural component of Candida species cell walls. The higher order structure of {beta}-glucans ranges from random coil to insoluble fiber due to varying degrees of tertiary (helical) and quaternary structure. Model Saccharomyces cerevisiae {beta}-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. Forster 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 {beta}-glucans through their varying ability to drive receptor dimer/oligomer formation and activation of membrane proximal signaling events.nnAuthor SummaryCandidemia 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 {beta}-glucan, a highly immunogenic component of the fungal cell wall. During innate immune recognition of Candida, the organization of cell wall {beta}-glucan is an important determinant of a successful immune activation. However, there have been many reports showing conflicting biological activities of {beta}-glucans with different size, branching and structure. Here, using quantitative fluorescence imaging techniques, we investigate how differential size and structure of {beta}-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 {beta}-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 {beta}-glucan exposure.

/pdf/innate-antifungal-immune-receptor-dectin-1-undergoes-ligand-1y3zheqll3.pdf

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

Mike Danielson

Papers

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

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