An In Vitro Cell Culture Model for Pyoverdine-Mediated Virulence

Abstract

Pseudomonas aeruginosa is a multidrug-resistant, opportunistic pathogen that utilizes a wide-range of virulence factors to cause acute, life-threatening infections in immunocompromised patients, especially those in intensive care units. It also causes debilitating chronic infections that shorten lives and worsen the quality of life for cystic fibrosis patients. One of the key virulence factors in P. aeruginosa is the siderophore pyoverdine, which provides the pathogen with iron during infection, regulates the production of secreted toxins, and disrupts host iron and mitochondrial homeostasis. These roles have been characterized in model organisms such as Caenorhabditis elegans and mice. However, an intermediary system, using cell culture to investigate the activity of this siderophore has been absent. In this report, we describe such a system, using murine macrophages treated with pyoverdine. We demonstrate that pyoverdine-rich filtrates from P. aeruginosa exhibit substantial cytotoxicity, and that the inhibition of pyoverdine production (genetic or chemical) is sufficient to mitigate virulence. Furthermore, consistent with previous observations made in C. elegans, pyoverdine translocates into cells and disrupts host mitochondrial homeostasis. Most importantly, we observe a strong correlation between pyoverdine production and virulence in P. aeruginosa clinical isolates, confirming pyoverdine's value as a promising target for therapeutic intervention. This in vitro cell culture model will allow rapid validation of pyoverdine antivirulents in a simple but physiologically relevant manner.

Keywords: Pseudomonas aeruginosa; macrophages; pyoverdine; virulence.

Figure 1

Pyoverdine production is important for virulence in murine macrophages. (A,B) Bacterial growth (A) and pyoverdine production (B) of wild-type P. aeruginosa PAO1 or the pyoverdine biosynthetic mutant PAO1ΔpvdF after 16 h incubation in Eagle’s Minimal Essential Medium. (C) RAW264.7 murine macrophage viability after 1.5 h exposure to filtrates from wild-type PAO1 or PAO1ΔpvdF. Cell viability was measured using an alamarBlue assay. (D) Visualization of macrophage cell death following filtrate exposure using counterstaining with Sytox Orange, a cell-impermeant nucleic acid stain (red). Cells were prelabeled with Hoechst 33342 (blue) then treated with filtrate in the presence of Sytox Orange for 1 h. (E) Macrophage viability after 1.5 h exposure to filtrates from bacteria grown in the presence of 50 µM 5-fluorocytosine (5-FC) or 10 µM 5-fluorouridine (5-FUR). (F) Macrophage viability after 2.5 h exposure to bacterial filtrates from wild-type PAO1 or PAO1ΔpvdF before and after the removal of large biomolecules by centrifugal filtration (<5 kDa flowthrough) or after pre-saturating the pyoverdine in the flowthrough with 250 µM gallium (III) nitrate. Error bars represent SEM from at least three biological replicates. * corresponds to p < 0.01, # corresponds to p < 0.05, and NS corresponds to p > 0.05 based on Student’s t-test.

Figure 2

Pyoverdine translocates into macrophages. (A) Pyoverdine fluorescence in macrophage cell lysates after 2 h exposure to low-molecular weight flowthrough from bacterial filtrates. Lysate fluorescence was quenched by exogenous ferric iron. (B) Pyoverdine fluorescence within macrophages visualized via confocal laser-scanning microscopy after 1.5 h exposure to flowthrough pretreated with iron (III) chloride, gallium (III) nitrate, or solvent control. (C) Quantification of pyoverdine fluorescence within 50 individual cells. (D) Pyoverdine–gallium fluorescence within cells labeled with Syto 9 green fluorescent nucleic acid stain and CellMask deep red plasma membrane stain. The bottom row shows an enlarged micrograph of one representative cell. (E) Pyoverdine and dextran fluorescence within macrophages were visualized after 1.5 h exposure to wild-type flowthrough supplemented with dextran-Texas Red (10,000 MW). The bottom row shows an enlarged micrograph of one representative cell. Error bars in (A) represent SEM from three biological replicates. * corresponds to p < 0.01 based on Student’s t-test. Cells labeled with Syto 9, CellMask, or dextran in the absence of pyoverdine are shown in Figure S7.

Figure 3

Pyoverdine production disrupts host mitochondrial networks. (A) Visualization of mitochondrial morphology in murine macrophages via MitoTracker Green FM staining after 2.5 h exposure to 50 µM CCCP or DMSO solvent control. (B) MitoTracker Green FM staining in macrophages after 1 h exposure to flowthrough. Bottom row shows enlarged micrograph of a representative cell.

Figure 4

In vitro macrophage pathogenesis model recapitulates results from C. elegans. (A) Pyoverdine production by P. aeruginosa cystic fibrosis isolates after growth in Liquid Killing media in the presence of C. elegans. (B) Pyoverdine production by P. aeruginosa cystic fibrosis isolates in Eagle’s Minimum Essential Medium with or without 50 µM 5-fluorocytosine (5-FC). (C) C. elegans survival after exposure to P. aeruginosa under Liquid Killing conditions. (D) Macrophage survival after exposure to bacterial filtrates from P. aeruginosa. Error bars represent SEM from three biological replicates. * corresponds to p < 0.01 and # corresponds to p < 0.05 based on Student’s t-test.

Figure 5

Pyoverdine production correlates with pathogen virulence in P. aeruginosa clinical and environmental isolates. (A) Pyoverdine production by a panel of 23 P. aerugionosa isolates after 16 h incubation in serum-free Eagle’s Minimum Essential Medium. (B–D) Correlation between pyoverdine production (B), bacterial growth (C), or pyoverdine normalized to growth (D) and filtrate toxicity against murine macrophages. Error bars represent SEM from two biological replicates. Each point represents the average of two biological replicates.