Claire
Yoo
Uncovering C-type Lectin Mediated Host-Microbe Interactions
Abstract profile. Full document pending author claim.
Authors:
Claire Yoo, Deepsing Syangtan, Marco Jost
Date Created:
2025-01-01
Course Title:
Professor:
Not specified
About Paper:
C-typelectinreceptors(CLRs)arealargefamilyoftransmembrane rRNA gene sequencing to determine their taxonomic orientation. carbohydrate binding proteins that are primarily expressed on the Simultaneously, I run a parallel assay with multiple strains of surface of immune cells such as dendritic cells, macrophages, and the genus Bacteroides, a common member of the gut microbial monocytes. These receptors bind in a calcium-dependent manner. community. Initial results show that DC-SIGN binds to multiple CLRs initiate a range of intracellular signaling cascades with strains of the genus Bacteroides, with particularly strong binding significant health implications upon ligand enagement, including observed for Bacteroides thetaiotaomicron (B. theta, 36:67%), cytokine production as well as antigen presentation. Thus, Bacteroides uniformis (B. uniformis, 54:57%), Bacteroides identifying microbe partners for these CLRs would enable the ovatus (B. ovatus, 63:63%), and Bacteroides xylanisolvens (B. elucidationofmechanismsthroughwhichgutmicrobiotainfluence xylanisolvens, 73:54% ). the host immune system. In preliminary work, the Jost Group has To investigate the molecular basis of this interaction, we tested found that Dectin-2 and DC-SIGN, two members of CLRs, bind a panel of B. theta with mutations affecting their capsular microbes in human fecal samples. polysaccharides (CPS). Binding assays were conducted in the Building on this, to identify these microbe-CLR interactions, presence and absence of calcium ions and the mannose polymer I use a combination of flow cytometry and high-throughput mannan, a known DC-SIGN ligand, to assess specificity and DNA sequencing. A plasmid with the proper DC-SIGN domain competitive inhibition. and carbenicillin expression vector is created through a gibson assembly. It is then inserted into Ecoli, selected for using Flow cytometry analysis revealed that mutant strains CPS 7 and ΔCPS exhibited the highest levels of DC-SIGN binding, 95:85%, carbenicillin, and used to trasfect HEK293 cells which express and and 93:23% respectively. Both of these mutants possess thin secrete the DC-SIGN domain. The proteins go through column capsular layers, suggesting these polysaccharides may expose purification and are incubated in microbial cellsisolated from fecal samples by our collaborators at Kiessling Lab for further analysis.a DC-SIGN-binding epitop but are not the binding epitope Binding events are then detected using fluorescently labeled anti- themselves. Currently, I am testing lipopolysaccharide (LPS) mutants as well as engineering other CLRs Mannose Receptor Fc antibodies and analyzed by flow cytometry. Sorted CLR- C-type 1 (MRC1), Mannose Receptor C-type 2 (MRC2), CD207 bound microbial populations are subsequently subjected to 16S (Langerin), and CD62L (L-selectin).
Abstract:
C-typelectinreceptors(CLRs)arealargefamilyoftransmembrane rRNA gene sequencing to determine their taxonomic orientation. carbohydrate binding proteins that are primarily expressed on the Simultaneously, I run a parallel assay with multiple strains of surface of immune cells such as dendritic cells, macrophages, and the genus Bacteroides, a common member of the gut microbial monocytes. These receptors bind in a calcium-dependent manner. community. Initial results show that DC-SIGN binds to multiple CLRs initiate a range of intracellular signaling cascades with strains of the genus Bacteroides, with particularly strong binding significant health implications upon ligand enagement, including observed for Bacteroides thetaiotaomicron (B. theta, 36:67%), cytokine production as well as antigen presentation. Thus, Bacteroides uniformis (B. uniformis, 54:57%), Bacteroides identifying microbe partners for these CLRs would enable the ovatus (B. ovatus, 63:63%), and Bacteroides xylanisolvens (B. elucidationofmechanismsthroughwhichgutmicrobiotainfluence xylanisolvens, 73:54% ). the host immune system. In preliminary work, the Jost Group has To investigate the molecular basis of this interaction, we tested found that Dectin-2 and DC-SIGN, two members of CLRs, bind a panel of B. theta with mutations affecting their capsular microbes in human fecal samples. polysaccharides (CPS). Binding assays were conducted in the Building on this, to identify these microbe-CLR interactions, presence and absence of calcium ions and the mannose polymer I use a combination of flow cytometry and high-throughput mannan, a known DC-SIGN ligand, to assess specificity and DNA sequencing. A plasmid with the proper DC-SIGN domain competitive inhibition. and carbenicillin expression vector is created through a gibson assembly. It is then inserted into Ecoli, selected for using Flow cytometry analysis revealed that mutant strains CPS 7 and ΔCPS exhibited the highest levels of DC-SIGN binding, 95:85%, carbenicillin, and used to trasfect HEK293 cells which express and and 93:23% respectively. Both of these mutants possess thin secrete the DC-SIGN domain. The proteins go through column capsular layers, suggesting these polysaccharides may expose purification and are incubated in microbial cellsisolated from fecal samples by our collaborators at Kiessling Lab for further analysis.a DC-SIGN-binding epitop but are not the binding epitope Binding events are then detected using fluorescently labeled anti- themselves. Currently, I am testing lipopolysaccharide (LPS) mutants as well as engineering other CLRs Mannose Receptor Fc antibodies and analyzed by flow cytometry. Sorted CLR- C-type 1 (MRC1), Mannose Receptor C-type 2 (MRC2), CD207 bound microbial populations are subsequently subjected to 16S (Langerin), and CD62L (L-selectin).
Source:
Harvard / Zhixiao Yip, Mateus Lopes, Thao Nyugen, Andrew D. Luster / 2025
Topics:
binding, bacteroide, clrs, interaction, strain, cell, microbial, receptor, bind, microbe, cps, mannose
