At a supra-physiological concentration, human sexual hormones act as quorum-sensing inhibitors

Abstract

N-acylhomoserine lactone (AHL)-mediated quorum-sensing (QS) regulates virulence functions in plant and animal pathogens such as Agrobacterium tumefaciens and Pseudomonas aeruginosa. A chemolibrary of more than 3500 compounds was screened using two bacterial AHL-biosensors to identify QS-inhibitors (QSIs). The purity and structure of 15 QSIs selected through this screening were verified using HPLC MS/MS tools and their activity tested on the A. tumefaciens and P. aeruginosa bacterial models. The IC50 value of the identified QSIs ranged from 2.5 to 90 µg/ml, values that are in the same range as those reported for the previously identified QSI 4-nitropyridine-N-oxide (IC50 24 µg/ml). Under the tested culture conditions, most of the identified QSIs did not exhibit bacteriostatic or bactericidal activities. One third of the tested QSIs, including the plant compound hordenine and the human sexual hormone estrone, decreased the frequency of the QS-regulated horizontal transfer of the tumor-inducing (Ti) plasmid in A. tumefaciens. Hordenine, estrone as well as its structural relatives estriol and estradiol, also decreased AHL accumulation and the expression of six QS-regulated genes (lasI, lasR, lasB, rhlI, rhlR, and rhlA) in cultures of the opportunist pathogen P. aeruginosa. Moreover, the ectopic expression of the AHL-receptors RhlR and LasR of P. aeruginosa in E. coli showed that their gene-regulatory activity was affected by the QSIs. Finally, modeling of the structural interactions between the human hormones and AHL-receptors LasR of P. aeruginosa and TraR of A. tumefaciens confirmed the competitive binding capability of the human sexual hormones. This work indicates potential interferences between bacterial and eukaryotic hormonal communications.

Figure 1. Structures of the QSIs identified in the chemical library.

Figure 2. Synthesis of the 4 diastereoisomers of QSI-1248.

Figure 3. Structure of the additional QSIs and compounds used in this study.

Figure 4. In vitro Ti plasmid transfer frequency in Agrobacterium.

The Ti plasmid transfer frequencies were measured in the presence of QSI (A) and the four 1248-diastereoisomeres (B) at 0.1 mg/ml. Histograms represent the cell density of transconjugants (CFU/ml), while black diamonds and squares, those of the donor and recipient strains, respectively. Measurements were performed in quadruplicate and the experiment was repeated twice. The cell densities of transconjugants in the presence of QSI were compared to that of the control in the presence of DMSO with a Mann and Whitney test (α = 0.05). Statistically different values are noted by asterisks.

Figure 5. QSIs modulated QS-signal accumulation in P. aeruginosa.

Growth kinetics (n = 6) of P. aeruginosa was measured (OD₆₀₀) in the presence of the QSIs (estradiol, estrone, estriol, hordenine and 4-NPO) at 0.5 mg/ml using DMSO as a negative control. Cell counts were assessed (CFU/ml) at 8- and 18-hour, and C4-HSL and OC12-HSL concentration (µM) were determined in the bacterial cultures at 18-hour. Statistically different values (Student's t test with α = 0.01) are noted by asterisks.

Figure 6. QSIs modulated QS-regulated genes in P. aeruginosa.

The β-galactosidase (b-Gal) activity in Miller unit (MU) of the transcriptional fusions lasI-lacZ (A), lasR-lacZ (B), lasB-lacZ (C), rhlI-lacZ (D), rhlR-lacZ (E), rhlA-lacZ (F), and aecA-lacZ(G) were measured in the presence of estradiol, estrone, estriol, hordenine at 0.5 mg/ml, and naringenin at 1 mg/ml as a positive control and DMSO as a negative control. The statistical significance of each test (n = 5 and three biological replicates) was evaluated by Student's t test (i.e. each test was compared with the DMSO condition). Asterisks indicate statistically different data (p value of ≤0.01).

Figure 7. QSIs modulated activity of the AHL-sensors in P. aeruginosa.

Luminescence of the reporting operon lux, expressed in relative light units (RLU/OD₆₀₀), was measured in E. coli bioindicator strains harboring the LasR (A) and RhlR (B) AHL-sensing systems of P. aeruginosa in the presence of C4-HSL and OC12-HSL, respectively. The QSIs estradiol, estrone, estriol and hordenine were added at 0.1 mg/ml, while naringenin and naringin, added at 0.5 mg/ml, were used as QSI-reference and non-QSI reference, respectively. Each test (n = 6) was compared with the DMSO-condition using Student's t test. Asterisks indicate statistically different data (p value of ≤0.01).

Figure 8. Modeling of interactions between AHL-sensors and human hormones.

Superimposed modeling of the overall binding modes of estradiol (cyan), estriol (magenta), estrone (yellow), or AHLs (OC12-HSL or OC8-HSL in gray) within the binding site of LasR (A) and of TraR (B).Superimposed modeling of the simplified binding modes showing interactions between estradiol (selected as an example in cyan) or natural ligands (OC12-HSL or OC8-HSL in green) and binding sites residues of LasR (C) and TraR (D).