Transient Proteotoxicity of Bacterial Virulence Factor Pyocyanin in Renal Tubular Epithelial Cells Induces ER-Related Vacuolation and Can Be Efficiently Modulated by Iron Chelators

Abstract

Persistent infections of biofilm forming bacteria, such as Pseudomonas aeruginosa, are common among human populations, due to the bacterial resistance to antibiotics and other adaptation strategies, including release of cytotoxic virulent factors such as pigment pyocyanin (PCN). Urinary tract infections harbor P. aeruginosa strains characterized by the highest PCN-producing capacity, yet no information is available on PCN cytotoxicity mechanism in kidney. We report here that renal tubular epithelial cell (RTEC) line NRK-52E responds to PCN treatments with paraptosis-like activity features. Specifically, PCN-treated cells experienced dilation of endoplasmic reticulum (ER) and an extensive development of ER-derived vacuoles after about 8 h. This process was accompanied with hyper-activation of proteotoxic stress-inducible transcription factors Nrf2, ATF6, and HSF-1. The cells could be rescued by withdrawal of PCN from the culture media before the vacuoles burst and cells die of non-programmed necrosis after about 24–30 h. The paraptosis-like activity was abrogated by co-treatment of the cells with metal-chelating antioxidants. A microscopic examination of cells co-treated with PCN and agents aiming at a variety of the cellular stress mediators and pathways have identified iron as a single most significant co-factor of the PCN cytotoxicity in the RTECs. Among biologically relevant metal ions, low micromolar Fe2+ specifically mediated anaerobic oxidation of glutathione by PCN, but catechol derivatives and other strong iron complexing agents could inhibit the reaction. Our data suggest that iron chelation could be considered as a supplementary treatment in the PCN-positive infections.

Interaction of Bacterial Phenazines with Colistimethate in Bronchial Epithelial Cells

“…1 and 4 ) that PYO and 1-HP activate the transcription factor Nrf2, which is responsive to intracellular oxidative stress ( 31 ), we next asked whether their toxicity in bronchial epithelial cells could be modulated by antioxidants. The bacterial phenazine PYO can cause a surge in the levels of intracellular reactive oxygen species (ROS) and oxidative stress due to its redox cycling ability ( 21 ), while iron may be required as a mediator of such activity ( 32 ). Potential inhibitors of phenazine-induced oxidative stress would therefore possess one or more of the following chemical properties: (i) quenching ROS, (ii) binding a metal redox catalyst, and/or (iii) reducing oxidized intracellular proteins and lipids back to the original state.…”

“…The most interesting combination is the colistimethate-pyocyanin pair, since it showed the strongest synergistic interaction against all three airway cell lines. This observation is rather unexpected, as the main suggested mechanism of pyocyanin cytotoxicity in epithelial cells is oxidative stress ( 21 , 32 ). In contrast, the cellular stress induced by CMS and other polymyxins seemingly took a different pathway, as evidenced by the lack of Nrf2 activation ( Fig.…”

“…While many research groups over the years have described PYO-induced intracellular oxidative stress ( 20 , 47 , 48 ), we recently identified iron as an essential mediator in the pyocyanin-catalyzed autoxidation of intracellular thiols in renal tubular epithelial cells ( 32 ). Indeed, strong iron chelators appeared to be the single most potent inhibitors of pyocyanin cytotoxicity in kidney epithelial ( 32 ) and lung epithelial (this study) cells. CMS cytotoxicity was effectively inhibited by N -acetyl-cysteine, a protector of cellular thiol homeostasis.…”

“…The reagent-grade phenazines 1-hydroxyphenazine (1-HP), phenazine-1-carboxylic acid (PCA), and phenazine-1-carboxamide (PCN) were obtained from ArkPharm and were used without further purification. Crystalline pyocyanin was purchased from Cayman or prepared by photolysis of a 2 mM solution of phenazine methosulfate in 20 mM HEPES buffer (pH 7.4), as reported previously ( 32 ). In-house nanopure water was generated by double distillation and used in all experiments.…”

Intramolecular 1,5-S...N σ-hole interaction in (E)-N′-(pyridin-4-ylmethylidene)thiophene-2-carbohydrazide

“…For example, hydrazone-based iron chelators have found applications as analytical reagents (Singh et al, 1982) and have been proposed for the treatment of bacterial, fungal, and protozoan infections (Narang et al, 2012;Rzhepishevska et al, 2014), as well as health disorders involving alterations in iron metabolism, such as hemochromatosis (Jansová & Š imů nek, 2019), cancer (Lovejoy & Richardson, 2003), and neurodegenerative diseases (Richardson, 2004). In addition, since iron has been identified as a critical co-factor of bacterial phenazine cytotoxicity to mammalian host cells (Mossine et al, 2016), the application of efficient iron chelators to the infection sites could not only restrict proliferation of the pathogen but also protect the infected tissue from injury caused by toxic bacterial metabolites (Mossine et al, 2018).…”