Green fluorescent protein-expressing Escherichia coli as a selective probe for HOCl generation within neutrophils.
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.
Palazzolo
Amy M
AM
Department of Chemistry, Washington State University, Pullman, Washington 99164-4630, USA.
Suquet
Christine
C
Konkel
Michael E
ME
Hurst
James K
JK
D000818:Animals; D001770:Blood Bactericidal Activity / Q000502:physiology; D056649:Cathepsin G; D002403:Cathepsins / Q000494:pharmacology; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006997:Hypochlorous Acid / Q000378:metabolism* / Q000494:pharmacology; D019272:Leukocyte Elastase / Q000494:pharmacology; D008856:Microscopy, Fluorescence / Q000379:methods; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism* / Q000382:microbiology*; D016877:Oxidants / Q000494:pharmacology / Q000502:physiology; D009195:Peroxidase / Q000378:metabolism; D010587:Phagocytosis / Q000502:physiology; D012697:Serine Endopeptidases / Q000494:pharmacology
D016877:Oxidants; D049452:Green Fluorescent Proteins; D006997:Hypochlorous Acid; D009195:Peroxidase; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D019272:Leukocyte Elastase