Multiscale Invasion Assay for Probing Macrophage Response to Gram-Negative Bacteria

Abstract

The immune system is a complex network of various cellular components that must differentiate between pathogenic bacteria and the commensal bacteria of the human microbiome, where misrecognition is linked to inflammatory disorders. Fragments of bacterial cell wall peptidoglycan bind to pattern recognition receptors within macrophages, leading to immune activation. To study this complex process, a methodology to remodel and label the bacterial cell wall of two different species of bacteria was established using copper (I) catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC). Additionally, an approach for three-dimensional (3D) culture of human macrophages and their invasion with relevant bacteria in a well-defined hydrogel-based synthetic matrix inspired by the microenvironment of the gut was established. Workflows were developed for human monocyte encapsulation and differentiation into macrophages in 3D culture with high viability. Bacteria invaded into macrophages permitted in situ peptidoglycan labeling. Macrophages exhibited biologically-relevant cytokine release in response to bacteria. This molecularly engineered, multi-dimensional bacteria-macrophage co-culture system will prove useful in future studies to observe immunostimulatory, bacterial fragment production and localization in the cell at the carbohydrate level for insights into how the immune system properly senses bacteria.

Shedding Light on Bacterial Physiology with Click Chemistry

“…Recently, the Grimes group multiplexed NAM bioorthogonal labeling with three‐dimensional (3D) human cell cultures to simulate human gut environments [26] . Pseudomonas aeruginosa and Escherichia coli were pre‐labeled with NAM probes, and fluorescently labeled E. coli was introduced into 3D hydrogels embedded with macrophages to model more physiologically relevant immune responses and imaged in real‐time during infection.…”

Minimalist Tetrazine N-Acetyl Muramic Acid Probes for Rapid and Efficient Labeling of Commensal and Pathogenic Peptidoglycans in Living Bacterial Culture and During Macrophage Invasion

“…The copper-catalyzed azide–alkyne cycloaddition (CuAAC) has been applied extensively because of the small reaction partner sizes, but the requirement of a cytotoxic copper catalyst has limited its use in live-cell applications . To avoid the use of copper, strain-promoted azide–alkyne cycloaddition (SPAAC) has been employed to image and study glycans through metabolic engineering, albeit with modest live-cell kinetics. − …”

Investigating Peptidoglycan Recycling Pathways inTannerella forsythiawithN-Acetylmuramic Acid Bioorthogonal Probes

“…In addition, strainpromoted azide−alkyne cycloaddition (SPAAC) was attempted as an alternative methodology for fluorescent labeling that allows for live cell imaging based on established protocols for EQKU cells. 17 Utilizing SPAAC, we are also able to label DH5α-Tf-KU cells (Figure 4A). These data suggest that SPAAC can be utilized to label wild-type T. forsythia.…”

“…Previous work has utilized the recycling enzymes AmgK and MurU from the Pseudomonas family to incorporate NAM probes and perform chemistries on the PG of P. putida and P. aeruginosa for visualization of the cell wall. − This has been possible by utilizing near lethal doses of fosfomycin to block native PG biosynthesis, forcing the cell to recycle exogenous NAM and unnatural NAM substrates. In addition, through insertion of recycling enzymes AmgK and MurU from P. putida via plasmids or directly into the genome, this work has expanded to label and study PG in other species such as E. coli , Bacillus subtilis , and Helicobater pylori , which do not naturally express such recycling machinery. , Since T. forsythia is predicted to have its own homologues of AmgK (Tanf_10225) and MurU (Tanf_1030), we desired to apply our remodeling methodology and synthetic NAM probes to determine if recycling and labeling of PG was possible in T. forsythia without genetically supplying exogenous AmgK and MurU.…”

“…Analysis of the data with Xcalibur processing software confirmed the presence of the modified fragments (Figure B). In addition, strain-promoted azide–alkyne cycloaddition (SPAAC) was attempted as an alternative methodology for fluorescent labeling that allows for live cell imaging based on established protocols for EQKU cells . Utilizing SPAAC, we are also able to label DH5α-Tf-KU cells (Figure A).…”