title: Optical tracking of phagocytosis with an activatable profluorophore metabolically incorporated into bacterial peptidoglycan.
abstract: Phagocytosis is critical for immunity against pathogens. Prior imaging using dye-labeled synthetic beads or green fluorescent protein-expressing bacteria is limited by "always-on" signals which compromise discerning phagocytosed particles from adherent particles. Targeting cellular internalization of pathogens into acidic phagolysosomes, we herein report "turn-on" fluorescence imaging of phagocytosis with viable bacteria featuring peptidoglycans covalently modified with rhodamine-lactam responsive to acidic pH. Culturing of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) with d-lysine conjugated rhodamine-lactam and fluorescein isocyanate (FITC) leads to efficient metabolic incorporation of FITC and rhodamine-lactam into bacterial peptidoglycan. E. coli and S. aureus become red-emissive upon phagocytosis into Raw 264.7 macrophages. With FITC as the reference signal, the mono- and dual-color emission allow efficient in situ distinction of ingested bacteria from extracellular bacteria. Given the ease of optical peptidoglycan labeling, the prevalence of microbial peptidoglycan and preservation of microbial surface landscape, this approach would be of use for investigation on microbial pathogenesis and high-throughput screening of immunomodulators of phagocytosis.
authors.forename: Yunpeng
authors.initials: Y
authors.lastname: Tian
authors.forename: Mingzu
authors.initials: M
authors.lastname: Yu
authors.forename: Zhu
authors.initials: Z
authors.lastname: Li
authors.forename: Jiahuai
authors.initials: J
authors.lastname: Han
authors.forename: Liu
authors.initials: L
authors.lastname: Yang
authors.forename: Shoufa
authors.initials: S
authors.lastname: Han
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D007769:Lactams / Q000737:chemistry; D008264:Macrophages / Q000378:metabolism / Q000382:microbiology; D015394:Molecular Structure; D055095:Optics and Photonics; D009943:Organophosphorus Compounds / Q000737:chemistry; D010457:Peptidoglycan / Q000737:chemistry* / Q000378:metabolism; D010587:Phagocytosis; D012235:Rhodamines / Q000737:chemistry; D013211:Staphylococcus aureus / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D007769:Lactams; D009943:Organophosphorus Compounds; D010457:Peptidoglycan; D012235:Rhodamines; C062572:3-(bromopropyl)triphenylphosphonium