Sex Steroids Induce Membrane Stress Responses and Virulence Properties in Pseudomonas aeruginosa

Abstract

Estrogen, a major female sex steroid hormone, has been shown to promote the selection of mucoid Pseudomonas aeruginosa in the airways of patients with chronic respiratory diseases, including cystic fibrosis. This results in long-term persistence, poorer clinical outcomes, and limited therapeutic options. In this study, we demonstrate that at physiological concentrations, sex steroids, including testosterone and estriol, induce membrane stress responses in P. aeruginosa This is characterized by increased virulence and consequent inflammation and release of proinflammatory outer membrane vesicles promoting in vivo persistence of the bacteria. The steroid-induced P. aeruginosa response correlates with the molecular polarity of the hormones and membrane fluidic properties of the bacteria. This novel mechanism of interaction between sex steroids and P. aeruginosa explicates the reported increased disease severity observed in females with cystic fibrosis and provides evidence for the therapeutic potential of the modulation of sex steroids to achieve better clinical outcomes in patients with hormone-responsive strains.IMPORTANCE Molecular mechanisms by which sex steroids interact with P. aeruginosa to modulate its virulence have yet to be reported. Our work provides the first characterization of a steroid-induced membrane stress mechanism promoting P. aeruginosa virulence, which includes the release of proinflammatory outer membrane vesicles, resulting in inflammation, host tissue damage, and reduced bacterial clearance. We further demonstrate that at nanomolar (physiological) concentrations, male and female sex steroids promote virulence in clinical strains of P. aeruginosa based on their dynamic membrane fluidic properties. This work provides, for the first-time, mechanistic insight to better understand and predict the P. aeruginosa related response to sex steroids and explain the interindividual patient variability observed in respiratory diseases such as cystic fibrosis that are complicated by gender differences and chronic P. aeruginosa infection.

Keywords: Pseudomonas aeruginosa; gender; hormones; membrane stress; steroids.

FIG 1

Sex steroids promote Pseudomonas aeruginosa virulence in vitro and reduce lung clearance in vivo. (A) Chemical structures of the various sex steroid hormones with associated logP values. (B) P. aeruginosa PAO1 was cultured for 16 to 24 h in tryptic soy broth (TSB) medium supplemented with ethanol (vehicle control) or the respective sex steroid hormones at concentrations varying from 250 pM to 250 μM, and alginate production was assessed. Sex steroids significantly promote P. aeruginosa in vitro production of alginates in a concentration-dependent manner with an inverse relationship with molecular polarity. (C to I) P. aeruginosa PAO1 was cultured for 16 to 24 h in TSB medium supplemented with ethanol (vehicle control) and the respective sex steroid hormones (at optimal concentrations varying from 25 nM to 250 μM). Changes in virulence were assessed by (C) increased swarming motility, (D) increased rhamnolipid production, (E) elevated elastase levels, and (F) altered biofilm architecture with (G) reduced roughness coefficient and increased biovolumes, (H) unchanged microbial cell burden, and (I) reduced diffusivity for the neutral molecule of dextran (150 kDa) in the matrix. Each experiment was performed in triplicate, and results are presented as the geometric mean and standard error (B, G to I) or box and whisker plots with median (horizontal bar), interquartile range (boxes), and minimum and maximum (whiskers) (C to E). Statistical significance was determined by one-way ANOVA corrected using Dunnett’s test for multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (J and K) P. aeruginosa PAO1 was cultured for 24 h in Luria-Bertani medium supplemented with ethanol (vehicle control), testosterone (2.5 μM), or estriol (25 nM). Then, 10⁶ CFU were used to infect the lungs of BALB/c female mice (6 to 10 weeks old) intranasally. Bacterial cell burden measured at 6 h postinoculation in bronchoalveolar lavage fluid (BALF) revealed a significantly reduced P. aeruginosa lung clearance with (J) testosterone (2.5 μM) and a trend toward reduced clearance with (K) estriol (25 nM). Each experiment was performed in duplicate, and results are presented as box and whisker plots with median (horizontal bar), interquartile range (boxes), and minimum and maximum (whiskers). Statistical significance was determined by one-way ANOVA corrected using Tukey’s test for multiple comparisons. *, P < 0.05; ***, P < 0.001. EE2, ethinylestradiol; T, testosterone; E3, estriol; TSDG.HCl, 17β-N,N-dimethylglycinate testosterone hydrochloride; AU, arbitrary unit; RFU, relative fluorescence unit.

FIG 2

The sex steroids testosterone and estriol induce Pseudomonas aeruginosa virulence through molecular signaling pathways controlled by surface sensing and membrane stress. (A) P. aeruginosa PAO1 was cultured for 16 to 24 h in tryptic soy broth (TSB) medium supplemented with ethanol (vehicle control) or testosterone (2.5 μM) and subjected to RNA sequencing. Significant increases in key virulence genes (marked in blue) involved in surface sensing (c-di-GMP regulation, pili), membrane stress (polysaccharides, alginates, quorum sensing, membrane proteins), and motility (flagella) illustrate the global regulation driven by testosterone. Three genes involved in flagellar motility, rhamnolipids, and polysaccharide regulation were found to be significantly downregulated (marked in red). All the genes reported with a significant change in expression (defined as a >0.5-fold increase or decrease compared to the control) are represented. (B and C) P. aeruginosa PAO1 was cultured for 24 to 72 h in TSB medium supplemented with ethanol (vehicle control), testosterone (2.5 μM), estriol (25 nM), or colistin (0.4 μM, 1/4 MIC) as a positive control for the membrane stress response. Northern blot gels (B) show increased mucA and pilA mRNA expression following testosterone, estriol, and colistin exposure, further illustrated by their (C) corresponding normalized band intensities. T, testosterone; E3, estriol.

FIG 3

The sex steroid hormones testosterone and estriol induce enhanced Pseudomonas aeruginosa motility, rhamnolipid, elastase, and alginate production through the muc operon. (A) P. aeruginosa PAO1 and mutants lacking genes from the muc operon (algR, mucA, and mucB) were cultured for 24 h in tryptic soy broth medium supplemented with ethanol (vehicle control), testosterone (2.5 μM), estriol (25 nM), or colistin (0.4 μM, 1/4 MIC), and virulence was assessed. Testosterone and estriol induced increases in (A) swarming motility, (B) rhamnolipids, (C) elastase, and (D) alginate production in PAO1 but not in the mucA, mucB, or algR mutants, and the phenotype was restored following mucB complementation. Each experiment was performed in triplicate, and results are presented as the geometric mean and standard error. Statistical significance was determined by one-way ANOVA corrected using Dunnett’s test for multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. T, testosterone; E3, estriol; AU, arbitrary unit; RFU, relative fluorescence unit.

FIG 4

The sex steroid hormones testosterone and estriol induce release of Pseudomonas aeruginosa outer membrane vesicles with diminished inflammatory capability. P. aeruginosa PAO1 was cultured for 24 h in Tryptic soy broth medium supplemented with ethanol (vehicle control), testosterone (2.5 μM), or estriol (25 nM). Outer membrane vesicles (OMVs) were extracted from the resulting supernatant and further purified as described in Materials and Methods. (A) Representative electron photomicrographs of extracted OMVs from cultures exposed to ethanol (vehicle control), testosterone (2.5 μM), or estriol (25 nM) (arrows indicate OMVs with their respective vesicle size in nm). (B) OMV amount, but not size, differs under sex steroid stress (range, 80 to 400 nm). (C) OMV extracts subjected to proteomic analysis (Table S2B) reveal significant differences in protein composition and diversity and consist of predominantly outer membrane (OM) proteins. (D and E) Extracted OMVs (10 ng total protein) were administered to the lungs of female C57BL/6 mice (n = 5, 6 to 8 weeks old) using the intratracheal route. After 24 h, bronchoalveolar lavage fluid was obtained, and inflammatory (D) cytokines and (E) chemokines were measured, which illustrate a significantly diminished inflammatory response from OMVs secreted under sex steroid stress. Each experiment was performed in triplicate, and results are presented as the geometric mean and standard error. Statistical significance was determined by one-way ANOVA corrected using Dunnett’s test for multiple comparisons. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. T, testosterone; E3, estriol; OM, outer membrane; EC, extracellular; P, periplasm; C, cytoplasm; U, unknown.

FIG 5

Variations in membrane fluidity among Pseudomonas aeruginosa clinical isolates explains sex hormone responsiveness. (A and B) Membrane fluidity (assessed using fluorescence recovery after photobleaching [FRAP]) of nine clinical P. aeruginosa isolates from nine patients was compared to PAO1. Four of the nine isolates (P. aeruginosa from patients 1 to 4) demonstrate lower membrane fluidity, while the other five (P. aeruginosa from patients 5 to 9) show higher membrane fluidity compared to PAO1. (B) Clinical P. aeruginosa isolates were cultured for 24 h in tryptic soy broth (TSB) medium supplemented with ethanol (vehicle control) or testosterone at two concentrations (physiological concentration, 25 nM; supraphysiological but optimal concentration for PAO1, 2.5 μM). The motility response (swarming) was assessed as an indicator of the sex steroid virulence response. The four isolates with the lowest membrane fluidity compared to PAO1 (P. aeruginosa from patients 1 to 4) did not demonstrate any motility changes following testosterone exposure (termed “nonresponsive”). In contrast, four of the five isolates (P. aeruginosa from patients 5 to 7 and patient 9) demonstrated significantly increased motility (and higher membrane fluidity) compared to PAO1 following testosterone exposure (termed “hormone-responsive”). (C to E) Two hormone-responsive clinical isolates (patients 7 and 9) were selected and assessed for further virulence studies. Strains were cultured for 24 h in TSB medium supplemented with ethanol (vehicle control), testosterone, or estriol, each at two separate concentrations (25 nM and 2.5 μM as described above). Both hormone-responsive strains exhibited significant upregulation of virulence markers upon hormone exposure as evidenced by elevated (C) rhamnolipids, (D) elastase, and (E) alginate production. Each experiment was performed in triplicate, and results are presented as the geometric mean and standard error. Statistical significance was determined by one-way ANOVA corrected using Dunnett’s test for multiple comparisons. *, P < 0.05; **, P < 0.01; *** P < 0.001; ****, P < 0.0001. T, testosterone; E3, estriol; AU, arbitrary unit; RFU, relative fluorescence unit.

FIG 6

Proposed mechanistic model for sex steroid-driven regulation of Pseudomonas aeruginosa virulence and persistence. (1) The polarity of the various sex steroid hormones influences their ability to diffuse across the P. aeruginosa (lipid bilayer) membrane with their degree of impact on membrane dynamics dependent on the diffusion rate. More polar molecules such as estriol (with lower hydrophobicity) diffuse more slowly and therefore are more likely to interact with lipopolysaccharides and proteins in the bacterial cell envelope compared to less polar molecules such as testosterone or ethinylestradiol (with higher hydrophobicity), which diffuse more rapidly through the bacterial membrane. (2) Interactions between polar sex steroids, membrane proteins, and lipopolysaccharides are sensed by surface signaling systems, including the muc operon (mucA/B), leading to (3) activation of the alginate biosynthetic pathway through the muc operon, (4) upregulation of downstream genes involved in surface sensing (pili, c-di-GMP) and quorum sensing (pqs, las, rhl, vfr), and (5) biofilm formation (polysaccharides, alginates). (6) The increased secretion of quorum sensing autoinducer (AI) molecules coupled to increased membrane protein production alters membrane composition and fluidity and promotes “lipid raft” formation and membrane stress-inducing (7) OMV release that dampens host inflammatory responses and further promotes bacterial persistence and tissue damage. (8) In chronic P. aeruginosa infection with “hormone-responsive” strains, the constant pressure exerted by sex steroids, particularly on the bacterial envelope, promotes adaptive change, which aims to protect the bacterium by creating defensive barriers such as alginates that surround bacterial cells, leading to mucoid conversion with bacterial persistence and poorer clinical outcomes. EE2, ethinylestradiol; T, testosterone; E3, estriol; TSDG.HCl, 17β-N,N-dimethylglycinate testosterone hydrochloride; OMV, outer membrane vesicle; LPS, lipopolysaccharides.