Investigating Sulforaphane's anti-virulence and anti-quorum sensing properties against Pseudomonas aeruginosa

Abstract

Background: P. aeruginosa, a significant bacterium, can cause severe illness and resistance to antibiotics. Quorum sensing (QS) systems regulate virulence factors production. Targeting QS could reduce bacteria pathogenicity and prevent antibiotic resistance. Cruciferous vegetables contain sulforaphane, known for its anti-inflammatory, antioxidant, anticancer, and antimicrobial properties.

Aim: We aimed to examine the inhibitory influences of sulforaphane, at a sub-inhibitory concentration (¼ minimum inhibitory concentration, MIC), on virulence and QS in P. aeruginosa.

Materials and methods: The sulforaphane's anti-virulence actions at sub-inhibitory concentrations were explored in vitro and in vivo. A sub-MIC concentration of sulforaphane was combined with anti-pseudomonal drugs, and the results of this combination were assessed. The virtual affinity of sulforaphane for the receptors of QS was studied, and its effect on the expression of QS genes was quantified.

Results: Sulforaphane significantly decreased the biofilm formation, motility, ability to withstand oxidative stress, and the synthesis of virulence extracellular enzymes such as proteases, hemolysins, and elastase, as well as other virulence factors like pyocyanin. In addition, sulforaphane lessened the severity of P. aeruginosa infection in mice. Sulforaphane reduced the antipseudomonal antibiotics' MICs when used together, resulting in synergistic effects. The observed anti-virulence impacts were attributed to the ability of sulforaphane to inhibit QS via suppressing the QS genes' expression.

Conclusion: Sulforaphane shows promise as a potent anti-virulence and anti-QS agent that can be used alongside conventional antimicrobials to manage severe infections effectively. Furthermore, this study paves the way for further investigation of sulforaphane and similar structures as pharmacophores for anti-QS candidates.

Keywords: Pseudomonas aeruginosa; Sulforaphane; bacterial virulence; quorum sensing; resistance to antibiotics.

FIGURE 1

Sulforaphane, when used at a concentration below the minimum inhibitory concentration of 2.5 μg/mL, does not have a noticeable impact on the proliferation of PAO1. A one-way ANOVA test was performed to determine the statistical significance. ns, non-significant.

FIGURE 2

Sulforaphane reduced the production of biofilms in PAO1. (A) Photographs depicting the biofilms that were developed with and without the presence of sulforaphane at sub-minimum inhibitory concentration (sub-MIC). (B) The optical density of crystal violet, which stained the bacterial cells creating the biofilm, was measured, and the impact on biofilm formation was determined by calculating the percentage change compared to the untreated control. Sulforaphane, when used at a concentration below the minimum inhibitory concentration of 2.5 μg/mL, showed a highly significant (***) reduction in biofilm development.

FIGURE 3

The presence of sulforaphane at sub-MIC levels led to a considerable reduction in the synthesis of PAO1 virulence factors (A) proteases, (B) hemolysins, (C) elastases, and (D) pyocyanin. (E) Sulforaphane significantly increased the sensitivity of PAO1 to oxidative conditions. (F) Sulforaphane, when used at a concentration below the minimum inhibitory concentration (sub-MIC), dramatically reduced the motility of PAO1. The data are displayed as the percentage difference compared to the untreated control. *** indicates a highly significant difference.

FIGURE 4

3D visualization of the interactions between sulforaphane and LuxR-type quorum-sensing system transcription factors from A. tumefaciens [traR, (A)], P. aeruginosa [QscR, (B)], and C. violaceum [CviR, (C)].

FIGURE 5

Molecular dynamics simulations of the co-crystallized ligand LAE (A) and sulforaphane (B) with traR. U, potential energy (kcal/mol); K, kinetic energy (kcal/mol); t, time; ps, picosecond.

FIGURE 6

Molecular dynamics simulations of the co-crystallized ligand OHN (A) and sulforaphane (B) with QscR. U, potential energy (kcal/mol); K, kinetic energy (kcal/mol); t, time; ps, picosecond.

FIGURE 7

Molecular dynamics simulations of the co-crystallized ligand HLC (A) and sulforaphane (B) with CviR. U, potential energy (kcal/mol); K, kinetic energy (kcal/mol); t, time; ps, picosecond.

FIGURE 8

Sulforaphane, at a concentration below the minimum inhibitory concentration (sub-MIC), reduced the expression of the following quorum sensing (QS)-encoding genes in PAO1: (A) rhlI, (B) rhlR, (C) lasI, (D) lasR, (E) pqsA, and (F) pqsR. *** indicates a highly significant difference.

FIGURE 9

Sulforaphane protected mice and reduced the PAO1 pathogenesis. (A) Three fatalities were documented among the mice who received untreated PAO1 injections, but only one fatality was recorded in the group of mice injected with sulforaphane-treated PAO1. No fatalities were documented in the negative control group. The log-rank test for trend was utilized to confirm the statistical significance (p = 0.0295). Bacterial loads of liver and kidney tissues in each group are presented in (B,C), respectively. The colonized bacteria in the organ tissues isolated from sulforaphane treated PAO1 injected mice were significantly decreased as compared to tissues isolated from mice injected with untreated PAO1. (D) Histopathological organs sections were stained from untreated PAO1 and sulforaphane treated PAO1 using hematoxylin and eosin dye, (i) the liver tissue section of the untreated PAO1 group exhibited substantial and widespread dilation of the hepatic sinusoids (shown by arrows), with focal individualization of some hepatocytes (indicated by tailed arrows) and infiltration of specific areas of the liver parenchyma by leukocytes (indicated by arrowheads), (ii) liver section of infected group showing focal leucocytic cells infiltrated hepatic parenchyma represented by neutrophils (tailed arrow), lymphocytes (arrows) and macrophages (arrowhead), (iii) liver section of sulforaphane treated PAO1 showed dilation of hepatic sinusoids (arrows head), (iv) liver section of sulforaphane treated PAO1 showed perivascular nuclear hyperchromasia of some hepatocytes (arrows) with leucocytic cellular infiltration (arrowhead), (v) kidney section of untreated PAO1 showed focal nephritis of renal cortex represented in congestion of blood vessel (arrowhead)with leucocytic cells infiltration (arrow), (vi) kidney section of untreated PAO1 showed casts deposition inside some renal tubules with the renal medulla (arrowhead), (vii) kidney section of sulforaphane treated PAO1 showed perivascular edema, fibrosis (arrows) with leucocytic cells infiltration (arrowhead) in the renal cortex, (viii) the kidney tissue section of PAO1 treated with sulforaphane exhibited hyper cellularity of renal glomeruli (shown by the arrow), along with degeneration of certain renal tubules (indicated by the arrowhead). (bar = 100 µm). ** indicates a significant difference.