Ajoene, a sulfur-rich molecule from garlic, inhibits genes controlled by quorum sensing

Abstract

In relation to emerging multiresistant bacteria, development of antimicrobials and new treatment strategies of infections should be expected to become a high-priority research area. Quorum sensing (QS), a communication system used by pathogenic bacteria like Pseudomonas aeruginosa to synchronize the expression of specific genes involved in pathogenicity, is a possible drug target. Previous in vitro and in vivo studies revealed a significant inhibition of P. aeruginosa QS by crude garlic extract. By bioassay-guided fractionation of garlic extracts, we determined the primary QS inhibitor present in garlic to be ajoene, a sulfur-containing compound with potential as an antipathogenic drug. By comprehensive in vitro and in vivo studies, the effect of synthetic ajoene toward P. aeruginosa was elucidated. DNA microarray studies of ajoene-treated P. aeruginosa cultures revealed a concentration-dependent attenuation of a few but central QS-controlled virulence factors, including rhamnolipid. Furthermore, ajoene treatment of in vitro biofilms demonstrated a clear synergistic, antimicrobial effect with tobramycin on biofilm killing and a cease in lytic necrosis of polymorphonuclear leukocytes. Furthermore, in a mouse model of pulmonary infection, a significant clearing of infecting P. aeruginosa was detected in ajoene-treated mice compared to a nontreated control group. This study adds to the list of examples demonstrating the potential of QS-interfering compounds in the treatment of bacterial infections.

Fig 1

Ajoene and derivatives. Compound 1, ajoene, present in two isomers, E and Z; compounds 2 to 5, ajoene derivatives.

Fig 2

Expression of QS-controlled specific fluorescence (GFP expression/cell density). The QS bioassays used were with P. aeruginosa harboring either the rhlA-gfp or the lasB-gfp fusion and E. coli harboring the luxI-gfp fusion incubated with synthesized ajoene.

Fig 3

Fold change in gene expression of rhlA and lasB measured by RT-PCR (dark gray bars) and DNA microarray (light gray bars). Data represent the average of three individual experiments. *, P < 0.05, Student's t test. Error bars are means ± SDs.

Fig 4

Total rhamnolipid concentration in untreated (no added ajoene [no add]) and ajoene-treated planktonic P. aeruginosa. The cultures were grown in medium supplemented with 10 μg/ml, 20 μg/ml, 40 μg/ml, and 80 μg/ml of ajoene (rhamnolipid is below the detection level for the 40-μg/ml and 80-μg/ml ajoene treatments at an OD of 1.5). Samples were retrieved at an OD₆₀₀ of 1.5 (light gray bars) and at an OD₆₀₀ of 2.0 (dark gray bars). Data represent the average of three individual experiments. Error bars are means ± SDs.

Fig 5

Concentrations of C₄-HSL and 3-oxo-C₁₂-HSL in untreated (no added ajoene [no add]) and ajoene-treated planktonic cultures of P. aeruginosa. The cultures were grown in medium supplemented with 10 μg/ml, 20 μg/ml, 40 μg/ml, and 80 μg/ml of ajoene. Samples were retrieved at an OD₆₀₀ of 1.5 (dark gray bars), at an OD₆₀₀ of 1.8 (light gray bars), and at an OD₆₀₀ of 2.0 (medium gray bars).

Fig 6

Combined fluorescence and light microscopic investigations at day 4 of biofilms of P. aeruginosa exposed to PMNs (arrow) for 180 min at 37°C and then subsequently stained with the DNA stain PI. (A) Biofilm grown without ajoene in the medium; (B) biofilm grown in the presence of 100 μg/ml ajoene in the medium. Red fluorescence indicates lysed PMNs, and green fluorescence indicates the P. aeruginosa biofilm.

Fig 7

Biofilms of P. aeruginosa PAO1 (green) (A) and clinical P. aeruginosa isolate CF438 (green; stained with Syto 9) (B) at day 4 after no treatment, treatment with 100 μg/ml ajoene for 4 days, treatment with 10 μg/ml tobramycin for the final 24 h, or treatment with a combination of tobramycin-ajoene. Dead cells are stained with the DNA stain PI (red). The yellow color reflects a mixture of live and dead cells. The biofilms were visualized with a confocal scanning laser microscope.

Fig 8

Combined results of three separate experiments of ajoene treatment versus no treatment (placebo) using the mouse model of pulmonary infection. The BALB/c mice were intratracheally challenged (at day 0) with alginate beads containing 1.5 × 10⁸ CFU/ml P. aeruginosa. The two groups of mice were either untreated (placebo) or treated with ajoene at 25 μg/g BW once a day. The mice were given 2 days of prophylactic treatment or placebo. Mice were sacrificed on day 1 or day 3 postinfection, and the contents of bacteria in the lungs were determined. The median values are indicated with filled black squares. The statistical significance of the difference in clearance was tested by a Mann-Whitney U test (analysis of nonparametric data), and P values for the difference at day 1 and day 3 were 0.9 and 0.002, respectively.