Cytotoxic Interactions between Pseudomonas aeruginosa Virulence Factors and Metal‐Based Antimicrobials In Vitro

Abstract

Emergence of multidrug‐resistant (MDR) bacterial pathogens has triggered a search for alternatives to current antibiotic‐based treatments. Toxic metal salts are widely used as anticeptics, but gallium(III) may act systemically, by disrupting iron metabolism in MDR pathogens like Pseudomonas aeruginosa. As P. aeruginosa infections release a range of toxins on their own, we asked whether bacterial toxins may interact with metal‐based drugs against the host.Using stress‐inducible transcription factor (Nrf2, HIF‐1α, ATF‐6, and MTF‐1) reporter constructs, we have developed a cell‐based protocol for simultaneous evaluation of the cellular metabolic activity, transcriptional/translational activity, and activation of intracellular stress responses in NIH‐3T3 fibroblasts and BEAS‐2B bronchial epithelial cells. The cytotoxicities of four P. aeruginosa cytotoxic virulence factors phenazine‐1‐carboxylate, phenazine‐1‐carboxamide, pyocyanin (Pyo) and its main metabolite, 1‐hydroxyphenazine (1HP), alone and in combinations with five clinically approved antimicrobial metal salts (Ag, Zn, Ce, Bi, and Ga) were evaluated in vitro.Bacterial phenazines Pyo and 1HP displayed cytotoxicities (IC50 30–90 μM) at concentrations comparable with those detected in sites of P. aeruginosa infections. Silver nitrate was the most cytotoxic metal salt (IC50 < 10 μM), while citrates of cerium(III) and bismuth(III) were not affecting cell proliferation even at 1 mM. In combinations, Bi and Ce inhibited cytotoxicity of Pyo and 1HP, increasing the IC50 2‐fold. In contrast, zinc(II) and gallium(III) strongly synergized with the phenazines, with a decrease in the IC50 reaching over 100‐fold in some experiments. Both Pyo and 1HP strongly (200–500‐fold) activated Nrf2 in the reporter cells, suggesting oxidative stress is a significant contributor to their cytotoxicity mechanism. Both Zn and Ga induced activity of MTF‐1, at subtoxic concentrations, thus implying that interaction of these metals with cytotoxic phenazines occurs along pathways maintaining intracellular redox homeostasis.This is the first report documenting cytotoxic interactions between clinically approved antimicrobial metal salts and bacterial phenazines at physiologically relevant concentrations. It provides a rationale for further mechanistic and in vivo studies of the phenomenon.Support or Funding InformationThis research was supported by the University of Missouri Agricultural Station Chemical Laboratories, the Cystic Fibrosis Association of Missouri (CFAM), the Leda J Sears Trust Fund for Child Health Research; and National Institute of Food and Agriculture (NIFA) grant No. MO‐HABC0002.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.