Pyoverdine-Dependent Virulence of Pseudomonas aeruginosa Isolates From Cystic Fibrosis Patients

Abstract

The development of therapies that modulate or prevent pathogen virulence may be a key strategy for circumventing antimicrobial resistance. Toward that end, we examined the production of pyoverdine, a key virulence determinant, in ∼70 Pseudomonas aeruginosa isolates from pediatric cystic fibrosis patients. Pyoverdine production was heterogeneous and showed a clear correlation with pathogenicity in Caenorhabditis elegans and an acute murine pneumonia model. Examination showed pyoverdine accumulation in host tissues, including extrapharyngeal tissues of C. elegans and lung tissues of mice, where accumulation correlated with host death. Many of the isolates tested were resistant to multiple antimicrobials, so we assayed the ability of pyoverdine inhibitors to mitigate virulence and rescue pyoverdine-mediated host pathology. Representatives from three different classes of pyoverdine inhibitors (gallium, fluoropyrimidines, and LK11) significantly improved survival. Our findings highlight the utility of targeting virulence factors in general, and pyoverdine in particular, as a promising method to control bacterial pathogenesis as the utility of antimicrobials continues to diminish.

Repurposing bioactive compounds for treating multidrug-resistant pathogens

“…Liquid Killing by P. aeruginosa partially relies upon secretion of the siderophore pyoverdine, which causes worm death by scavenging iron from host mitochondria [36][37][38]. We have previously shown that limiting the production or the activity of pyoverdine extends survival of C. elegans in Liquid Killing, including upon exposure to multidrugresistant P. aeruginosa isolates [22,23,37,39]. To determine if any of our molecules may function by inhibiting pyoverdine, we cultured P. aeruginosa in the presence of two-fold serial dilutions of our bioactives and measured production of the fluorescent siderophore (Figs 4 and S4).…”

Identification and validation of a novel anti-virulent that binds to pyoverdine and inhibits its function

“…Isotopically labeled ( 15 N or 13 C and 15 N) pyoverdine was purified as above, except that P. aeruginosa PAO1 was inoculated in iron-poor M9 media supplemented with 1.0 g/L 15 NH 4 Cl as the sole nitrogen and 2.0 g/L unlabeled or uniformly labeled 13 C glucose as the carbon source for 20-22 h at 37°C. Labeled pyoverdine was then purified as described above.…”

“…To assess the effect of pyoverdine inhibitors PQ3, PQ3c, and 5-FC on the expression of virulence factors whose transcription depends on PvdS, which is dependent upon ferripyoverdine binding FpvA, bacteria were grown for 12 h in SK media supplemented with either DMSO or 100 µM inhibitor. Then cells were collected, pelleted, and RNA was extracted and purified using 13 C HSQC, CCH-TOCSY, HCCH-TOCSY, and HCCH-COSY. The assignments of aromatic chromophore were made by 1 H-13 C HSQC and 13 C-edited NOESY-HSQC.…”

“…Upon binding the compound, pyoverdine undergoes a conformational change that can be detected using heteronuclear single quantum correlation (HSQC) spectroscopy. In brief, this 2D NMR technique relies on the transfer of the magnetization from the proton directly attached to a heteronuclear atom, such as 15 N or 13 C, and back to proton [55]. The HSQC spectrum displays NMR resonances (peaks) or correlations that reflect the equivalent of a "frequency fingerprint" of the local chemical environment of these nuclei in the molecule.…”

“…Pyoverdine is a key virulence determinant in hosts ranging from cultured cells to invertebrates to mammals [12][13][14][15][16][17][18]. It directly removes iron from hosts and disrupts their mitochondrial function [14,15,19] and also regulates the transcription of several important virulence factors, including the translational inhibitor exotoxin A, the protease PrpL, and pyoverdine itself [20][21][22][23].…”