Green fluorescent protein-expressing Escherichia coli as a selective probe for HOCl generation within neutrophils

Amy M Palazzolo; Christine Suquet; Michael E Konkel; James K Hurst

doi:10.1021/bi047342s

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Green fluorescent protein-expressing Escherichia coli as a selective probe for HOCl generation within neutrophils

Journal article

Peer reviewed

Green fluorescent protein-expressing Escherichia coli as a selective probe for HOCl generation within neutrophils

Amy M Palazzolo, Christine Suquet, Michael E Konkel and James K Hurst

Biochemistry (Easton), Vol.44(18), pp.6910-6919

05/10/2005

DOI: https://doi.org/10.1021/bi047342s

Handle:

https://hdl.handle.net/2376/114802

PMID: 15865436

Abstract

Oxidants - pharmacology

Leukocyte Elastase - pharmacology

Humans

Serine Endopeptidases - pharmacology

Green Fluorescent Proteins - genetics

Cattle

Green Fluorescent Proteins - antagonists & inhibitors

Escherichia coli - metabolism

Escherichia coli - growth & development

Neutrophils - microbiology

Neutrophils - metabolism

Green Fluorescent Proteins - metabolism

Neutrophils - enzymology

Cathepsin G

Phagocytosis - physiology

Oxidants - physiology

Hypochlorous Acid - pharmacology

Blood Bactericidal Activity - physiology

Animals

Microscopy, Fluorescence - methods

Escherichia coli - genetics

Genetic Vectors

Cathepsins - pharmacology

Hydrogen-Ion Concentration

Hypochlorous Acid - metabolism

Peroxidase - metabolism

Escherichia coli were transformed by electroporation to introduce a plasmid harboring a GFP gene-containing vector. The fluorescence of the purified GFP isolated from the transformant was quenched by myeloperoxidase (MPO)-generated HOCl, by peroxynitrous acid (ONOOH) and by enzymatically or radiolytically generated NO(2)(.) but not by other putative neutrophil-generated oxidants. Fluorescence from the bacterium was effectively quenched by HOCl but not peroxynitrite, oxidizing radicals derived from its O-O bond homolysis, or the other oxidants under study. Exposure of serum-opsonized bacteria to human neutrophils resulted in extensive loss of GFP fluorescence; fluorescence microscopy revealed that phagocytosed bacteria were completely quenched but that bacteria remaining in the external media were unquenched. Addition of sodium azide to the medium to inhibit MPO prevented neutrophil-mediated fluorescence quenching. Because the amount of HOCl required to inhibit bacterial fluorescence was an order of magnitude greater than required to inhibit colonial growth, these results imply that sufficient HOCl was formed within the neutrophil phagosome to kill the microbe.

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Title: Green fluorescent protein-expressing Escherichia coli as a selective probe for HOCl generation within neutrophils
Creators: Amy M Palazzolo - Department of Chemistry, Washington State University, Pullman, Washington 99164-4630, USA
Christine Suquet
Michael E Konkel
James K Hurst
Publication Details: Biochemistry (Easton), Vol.44(18), pp.6910-6919
Academic Unit: Chemistry, Department of; Molecular Biosciences, School of
Publisher: United States
Grant note: AI-15834 / NIAID NIH HHS
Identifiers: 99900547989301842
Language: English
Resource Type: Journal article

Green fluorescent protein-expressing Escherichia coli as a selective probe for HOCl generation within neutrophils

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