Synthesis of the New Cyanine-Labeled Bacterial Lipooligosaccharides for Intracellular Imaging and in Vitro Microscopy Studies

Tung Cheng Wang, Florent Cochet, Fabio Alessandro Facchini, Lenny Zaffaroni, Christelle Serba, Simon Pascal, Chantal Andraud, Andrea Sala, Flaviana Di Lorenzo, Olivier Maury, Thomas R Huser, Francesco Peri

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Endotoxin (lipooligosaccharide, LOS, and lipopolysaccharide, LPS) is the major molecular component of Gram-negative bacteria outer membrane, and very potent pro-inflammatory substance. Visualizing and tracking the distribution of the circulating endotoxin is one of the fundamental approaches to understand the molecular aspects of infection with subsequent inflammatory and immune responses, LPS also being a key player in the molecular dialogue between microbiota and host. While fluorescently labeled LPS has previously been used to track its subcellular localization and colocalization with TLR4 receptor and downstream effectors, our knowledge on lipopolysaccharide (LOS) localization and cellular activity remains almost unexplored. In this study, LOS was labeled with a novel fluorophore, Cy7N, featuring a large Stokes-shifted emission in the deep-red spectrum resulting in lower light scattering and better imaging contrast. The LOS-Cy7N chemical identity was determined by mass spectrometry, and immunoreactivity of the conjugate was evaluated. Interestingly, its application to microscopic imaging showed a faster cell internalization compared to LPS-Alexa488, despite that it is also CD14-dependent and undergoes the same endocytic pathway as LPS toward lysosomal detoxification. Our results suggest the use of the new infrared fluorophore Cy7N for cell imaging of labeled LOS by confocal fluorescence microscopy, and propose that LOS is imported in the cells by mechanisms different from those responsible for LPS uptake.

Original languageEnglish (US)
Pages (from-to)1649-1657
Number of pages9
JournalBioconjugate Chemistry
Volume30
Issue number6
DOIs
StatePublished - Jun 19 2019
Externally publishedYes

Fingerprint

Lipopolysaccharides
Microscopy
Microscopic examination
Fluorophores
Imaging techniques
Detoxification
Confocal microscopy
Fluorescence microscopy
Light scattering
Mass spectrometry
Bacteria
Endotoxins
Infrared radiation
Membranes
Toll-Like Receptor 4
Microbiota
lipid-linked oligosaccharides
In Vitro Techniques
Gram-Negative Bacteria
Fluorescence Microscopy

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biomedical Engineering
  • Pharmacology
  • Pharmaceutical Science
  • Organic Chemistry

Cite this

Synthesis of the New Cyanine-Labeled Bacterial Lipooligosaccharides for Intracellular Imaging and in Vitro Microscopy Studies. / Wang, Tung Cheng; Cochet, Florent; Facchini, Fabio Alessandro; Zaffaroni, Lenny; Serba, Christelle; Pascal, Simon; Andraud, Chantal; Sala, Andrea; Di Lorenzo, Flaviana; Maury, Olivier; Huser, Thomas R; Peri, Francesco.

In: Bioconjugate Chemistry, Vol. 30, No. 6, 19.06.2019, p. 1649-1657.

Research output: Contribution to journalArticle

Wang, TC, Cochet, F, Facchini, FA, Zaffaroni, L, Serba, C, Pascal, S, Andraud, C, Sala, A, Di Lorenzo, F, Maury, O, Huser, TR & Peri, F 2019, 'Synthesis of the New Cyanine-Labeled Bacterial Lipooligosaccharides for Intracellular Imaging and in Vitro Microscopy Studies', Bioconjugate Chemistry, vol. 30, no. 6, pp. 1649-1657. https://doi.org/10.1021/acs.bioconjchem.9b00044
Wang, Tung Cheng ; Cochet, Florent ; Facchini, Fabio Alessandro ; Zaffaroni, Lenny ; Serba, Christelle ; Pascal, Simon ; Andraud, Chantal ; Sala, Andrea ; Di Lorenzo, Flaviana ; Maury, Olivier ; Huser, Thomas R ; Peri, Francesco. / Synthesis of the New Cyanine-Labeled Bacterial Lipooligosaccharides for Intracellular Imaging and in Vitro Microscopy Studies. In: Bioconjugate Chemistry. 2019 ; Vol. 30, No. 6. pp. 1649-1657.
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