A Novel Use of Allopurinol as A Quorum-Sensing Inhibitor in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa can cause a variety of healthcare-associated infections by its arsenal of virulence factors. Virulence factor production is largely controlled by the cell-to-cell communication system termed quorum sensing (QS). Targeting QS may be a good approach to inhibit the production of virulence factors and attenuate pathogenicity without exerting selective stress on bacterial growth. This will greatly reduce the emergence of resistant mutants. In this work, we investigated the anti-virulence and anti-QS activities of the FDA-approved drug allopurinol against the P. aeruginosa PAO1 strain. Allopurinol at 200 µg/mL (1/10 MIC) significantly decreased the production of the QS-controlled Chromobacterium violaceum CV026 violet pigment violacein and other P. aeruginosa QS-controlled virulence factors phenotypically. Furthermore, allopurinol reduced the infiltration of P. aeruginosa and leucocytes and diminished the congestion in the liver and kidney tissues of infected mice. In silico study showed that allopurinol could compete with the autoinducers on binding to the receptors LasR and RhlR by hydrogen bonding. On the molecular level, qRT-PCR proved that allopurinol showed a significant downregulating effect on all tested QS-encoding genes that regulate virulence factor production. In summary, allopurinol is a promising QS inhibitor that may be useful in the future treatment of P. aeruginosa infection.

Keywords: Pseudomonas aeruginosa; allopurinol; quorum sensing; virulence inhibition.

Figure 1

Allopurinol effect on P. aeruginosa PAO1 growth: (A) the turbidities of overnight bacterial cultures in the absence or presence of 1/10 MIC of allopurinol were measured at OD 600 nm; (B) viable counting of allopurinol-treated and control PAO1 cultures after overnight incubation. Allopurinol showed no statistically significant inhibitory effect on bacterial growth.

Figure 2

Effect of allopurinol on (A) C. violaceum CV026 growth. The turbidities of overnight bacterial cultures in the absence or presence of 1/10 MIC of allopurinol were measured at OD 600 nm. Allopurinol had no significant effect on bacterial growth. (B) Violacein pigment production: violacein was extracted by DMSO from the bacterial cells treated with or without 1/10 MIC of allopurinol. Allopurinol significantly reduced violacein production.

Figure 3

Inhibitory effect of allopurinol against biofilm formation in P. aeruginosa PAO1. A crystal violet assay was used to stain the biofilm-forming cells in the presence and absence of allopurinol at sub-MIC. (A) Light microscopic images showing a few scattered adhered P. aeruginosa cells when treated with allopurinol at sub-MIC. (B) The absorbances of the extracted crystal violet that stained the biofilm-forming cells in the presence or absence of allopurinol at sub-MIC were measured at 590 nm. Allopurinol significantly reduced biofilm formation. The data are presented as the percentage change from the untreated P. aeruginosa control.

Figure 4

Diminishment of the (A) swimming, (B) twitching, and (C) swarming motilities of P. aeruginosa PAO1 by allopurinol. Significantly, the bacterial motilities were diminished by allopurinol at sub-MIC.

Figure 5

Allopurinol inhibitory effect on P. aeruginosa virulence. Allopurinol at sub-MIC significantly reduced the production of protease, hemolysins and elastase enzymes, pyocyanin pigment, and rhamnolipids. The supernatants of the treated and untreated allopurinol bacteria were used for the assays. Protease activity was assayed by measuring the clear zones around the wells made in skim milk agar plates. Hemolysin activity was assayed spectrophotometrically by measuring hemoglobin absorbance. Elastase activity was measured by the elastin congo red method. Pyocyanin was measured spectrophotometrically at 691 nm, and rhamnolipid was assayed using the oil displacement method by measuring the clearance zone produced by the addition of the supernatant cultures to oil. The data are presented as the percentage change from the untreated P. aeruginosa control.

Figure 6

Histopathological examination of liver and renal tissues. Five groups of three-week-old albino mice (Mus musculus), with each group including five mice, were used in the experiment. Two negative control groups were intraperitoneally injected with sterile PBS or not infected. Two positive control groups were intraperitoneally injected with P. aeruginosa PAO1 (1 × 10⁶ CFU/mL) or with dimethyl sulfoxide-treated P. aeruginosa PAO1. The test group was injected with allopurinol-treated P. aeruginosa PAO1 (1 × 10⁶ CFU/mL). The mice were observed for five days and then euthanized by cervical dislocation. Both kidneys and liver were dissected from mice, rinsed with normal saline, and fixed in neutral buffered formalin (10%) for histopathological examination. Representative photomicrographs (H&E × 200) are depicted from control (un-infected), P. aeruginosa infected, and P. aeruginosa treated with allopurinol (1/10 MIC) mice groups. (A) Photomicrograph of mice liver from the non-infected (control) group showing apparently normal tissue architecture and cellular details. (B,C) Photomicrographs of mice liver from the P. aeruginosa infected group showing diffuse severe congestion of hepatic blood vessels (arrows) with perivascular cellular infiltration and colonization of P. aeruginosa rods (arrowheads), coagulative necrosis represented by pyknosis (tailed arrows), and the focal area of leucocytic cell infiltrated hepatic parenchyma with colonization of P. aeruginosa rods (arrows). (D,E) Photomicrographs of mice liver from the allopurinol-treated P. aeruginosa group showing mild perivascular leucocytic cells infiltration (arrowheads) with a few scattered P. aeruginosa rods in addition to mild congestion. (F) Photomicrograph of mice kidney from the non-infected (control) group showing apparently normal renal cortex with normal glomeruli and renal tubules. (G,H) Photomicrographs of mice kidney from the P. aeruginosa infected group showing perivascular colonization of P. aeruginosa (arrows) with cellular infiltration (arrowhead, (G)), leucocytic cell infiltration, hyperplasia of renal epithelium (arrowhead, (H)), and degenerated renal tubules. (I,J) Photomicrographs of mice kidney from the allopurinol-treated P. aeruginosa group showing mild focal infiltration of a few P. aeruginosa rods and less leucocytic cell infiltration (arrows (I)) beside milder cystic dilation of some renal tubules (arrowheads), and focal mild degeneration of some renal tubules (arrows (J)).

Figure 7

Allopurinol reduced the expressions of QS-encoding genes in P. aeruginosa. Allopurinol significantly reduced the expression of the genes that encode the three main QS systems in P. aeruginosa, namely, rhlI, rhlR, lasI, lasR, pqsA, and pqsR. *** Statistically significant difference.

Figure 8

In silico allopurinol binding to QS receptors. (A,C) The molecular docking of allopurinol into the LasR and RhlR receptors’ active sites, respectively; 3D (Left) and 2D schematic views of the binding (Right). (B,D) The molecular docking of natural ligand into the active site of LasR and RhlR receptors, respectively; 3D (Left) and 2D schematic views of the binding (Right). Allopurinol binds efficiently to QS receptors and clearly exerts antagonist activity.