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.
Highlights
Urinary tract infections (UTIs) are common bacterial infections estimated to cause disease in 150 million patients globally per year [1]
Our work shows strong support for a mechanism called the formation of neutrophil extracellular traps (NETs), previously described for other infections and autoimmune conditions, which are involved in killing pathogens that have invaded the urinary tract
To examine whether any of these mechanisms are relevant in the context of UTIs, we examined 21 urinary pellet (UP) samples with large sediments, high leukocyte counts, and high neutrophil protein contents
Summary
Urinary tract infections (UTIs) are common bacterial infections estimated to cause disease in 150 million patients globally per year [1]. They are classified as uncomplicated when they affect the lower urinary tract, are not linked to structural and functional urinary tract abnormalities or catheterization, and occur in immunocompetent hosts [2]. Pathogens have adapted to form biofilms on urethral catheters. Many, such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis and Enterococcus species, are resistant to killing by normal sanitary measures and cause UTIs in the nosocomial environment [2,3]. The high rates of antibiotic prescription to treat UTIs and widespread acquisition of antibiotic resistance genes by the aforementioned pathogens have added to the concern of emerging bacterial strains that are no longer susceptible to any major class of antibiotic drugs [2]
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