title: Characterization of Early-Phase Neutrophil Extracellular Traps in Urinary Tract Infections.
abstract: Neutrophils have an important role in the antimicrobial defense and resolution of urinary tract infections (UTIs). Our research suggests that a mechanism known as neutrophil extracellular trap (NET) formation is a defense strategy to combat pathogens that have invaded the urinary tract. A set of human urine specimens with very high neutrophil counts had microscopic evidence of cellular aggregation and lysis. Deoxyribonuclease I (DNase) treatment resulted in disaggregation of such structures, release of DNA fragments and a proteome enriched in histones and azurophilic granule effectors whose quantitative composition was similar to that of previously described in vitro-formed NETs. The effector proteins were further enriched in DNA-protein complexes isolated in native PAGE gels. Immunofluorescence microscopy revealed a flattened morphology of neutrophils associated with decondensed chromatin, remnants of granules in the cell periphery, and myeloperoxidase co-localized with extracellular DNA, features consistent with early-phase NETs. Nuclear staining revealed that a considerable fraction of bacterial cells in these structures were dead. The proteomes of two pathogens, Staphylococcus aureus and Escherichia coli, were indicative of adaptive responses to early-phase NETs, specifically the release of virulence factors and arrest of ribosomal protein synthesis. Finally, we discovered patterns of proteolysis consistent with widespread cleavage of proteins by neutrophil elastase, proteinase 3 and cathepsin G and evidence of citrullination in many nuclear proteins.
authors.affiliation: The J. Craig Venter Institute, Rockville, MD, United States of America.
authors.forename: Yanbao
authors.initials: Y
authors.lastname: Yu
authors.affiliation: The J. Craig Venter Institute, Rockville, MD, United States of America.
authors.forename: Keehwan
authors.initials: K
authors.lastname: Kwon
authors.affiliation: The J. Craig Venter Institute, Rockville, MD, United States of America.
authors.forename: Tamara
authors.initials: T
authors.lastname: Tsitrin
authors.affiliation: The J. Craig Venter Institute, Rockville, MD, United States of America.
authors.forename: Shiferaw
authors.identifier: 0000-0003-1570-3879
authors.initials: S
authors.lastname: Bekele
authors.affiliation: The J. Craig Venter Institute, Rockville, MD, United States of America.
authors.forename: Patricia
authors.initials: P
authors.lastname: Sikorski
authors.affiliation: The J. Craig Venter Institute, Rockville, MD, United States of America.
authors.forename: Karen E
authors.initials: KE
authors.lastname: Nelson
authors.affiliation: The J. Craig Venter Institute, Rockville, MD, United States of America.
authors.forename: Rembert
authors.initials: R
authors.lastname: Pieper
mesh_terms: D015153:Blotting, Western; D002853:Chromatography, Liquid; D065206:Extracellular Traps / Q000276:immunology*; D006801:Humans; D013058:Mass Spectrometry; D014552:Urinary Tract Infections / Q000276:immunology*