EFFECT OF UPEC BIOFILM BACTERIA AND THEIR SUPERNATANTS ON THE SECRETION OF MYELOPEROXIDASE AND CATHEPSIN G BY NEUTROPHILS AND MONONUCLEAR CELLS IN HUMAN PERIPHERAL BLOOD

Abstract

The ability of uropathogenic Escherichia coli (UPEC) to form biofilms is one of the factors causing recurrent urinary tract infections. Myeloperoxidase and cathepsin G of phagocytes form antimicrobial protection during inflammation. The existence of cross-reactions between UPEC bacteria, the extracellular matrix of biofilms and effector cells of the immune system cannot be excluded. The aim of the study was to evaluate the secretion of myeloperoxidase and cathepsin G by neutrophils and mononuclear cells of human peripheral blood upon interaction with biofilm cells and their supernatants of reference and clinical UPEC. Neutrophils and mononuclear cells of peripheral blood of healthy men (n = 6) isolated on a double Ficoll-Urografin gradient (1.077 g/ml and 1.112 g/ml) were used in the work. The reference strain E. coli DL82 (fimH, papC, papGII, sfa, hlyA, usp, fyuA, iucD, iroCD, iroN) and the clinical isolate E. coli R44 (fimH) (10⁶ cell/ml) were grown in 96-well polystyrene plates on LB medium for 24 h. The biofilm supernatant was sterilized by filtration (0.22 μm). Bacterial cells were released from the biofilm using ultrasound. Neutrophils were cultured with biofilm bacteria or their supernatants for 1 h, then centrifuged at 400 g, the supernatant was collected and frozen at -20 °C. MPO and cathepsin G activity was assessed using O-phenylenediamine dihydrochloride and N-benzoyl-L-tyrosine ethyl ester, respectively, by optical density in neutrophil and mononuclear cell supernatants. Statistical processing was performed using Excel. It was shown that MPO secretion by neutrophils increased upon interaction with E. coli DL82 biofilm supernatants, in contrast to E. coli R44 supernatants. E. coli DL82 and E. coli R44 cells of biofilm did not affect MPO secretion. MPO secretion by human peripheral blood mononuclear cells remained at the control level when exposed to UPEC cells and supernatants. Contact of neutrophils with bacteria and supernatants of the E. coli R44 strain led to a decrease in the concentration of cathepsin G in the medium compared to the control. Thus, exometabolites of UPEC bacterial biofilms likely have a more significant impact on neutrophil secretory activity than the bacteria themselves.