Carnosol attenuates Acinetobacter baumannii virulence by interfering with indole-mediated quorum sensing

Abstract

Many bacterial pathogens utilize quorum sensing (QS) signals to modulate the biological functions in a cell density-dependent manner. Acinetobacter baumannii is a harmful pathogen and a major cause of hospital-acquired infections due to its severe drug-resistance and pathogenic nature. Therefore, the development of innovative antibacterial strategies for A. baumannii infections is important. Earlier studies have proved that A. baumannii utilizes N-acyl-L-homoserine lactones (AHLs) and indole signalling systems to modulate its biological functions and virulence. Here, we report that carnosol, which is isolated from Salvia officinalis L. (S. officinalis), inhibited the pathogenicity of A. baumannii by interfering with indole-mediated quorum sensing. Phenotypic and virulence experiments have revealed that carnosol decreased the formation of biofilms, motility, and cytotoxicity of A. baumannii, without affecting its growth rate. Genetic and biochemical analysis results showed that carnosol reduced the regulatory effect of AbiR on the transcription of target genes by inhibiting the binding of AbiR to the promoters of these genes. Our results suggest that the indole QS system of A. baumannii could be used as a new target and that carnosol might be developed as a new anti-virulence agent against A. baumannii infections.

Keywords: Acinetobacter baumannii; carnosol; indole; quorum sensing; virulence.

Figure 1.

Influence of S. officinalis extract on the phenotypes and virulence of A. baumannii. Effect of 1% S. officinalis extract (0.1 mg/mL) on the motility (a), biofilm formation (b) and cytotoxicity (c) of A. baumannii. The data are presented as the means ± standard deviations of three independent experiments. The significance was determined by a two-sided Student’s t-test, (***p < 0.001).

Figure 2.

Effects of carnosol on the biological functions of A. baumannii. (a) Chromatogram of bioactive fractions of S. officinalis extract. (b) The chromatographic peaks of the active substance are pointed by the arrow. ESI-MS spectra and structure of carnosol. Effects of the addition of different concentrations of carnosol on biofilm formation (c), motility (d) and cytotoxicity (e). The expression of the abaI, abaR, abiS and abiR in A. baumannii ATCC 17978 treated with 100 μM carnosol was evaluated by RT-qPCR (f). The effects of carnosol on the gene expression levels of abaI, abaR, abiS and abiR were evaluated by assessing the β-galactosidase activity of promoter-lacZ in the wild-type strain (OD₆₀₀ = 3.0) with or without the addition of 100 μM carnosol (g). The compound was dissolved in DMSO, and the same volume of DMSO was used as the solvent for the compounds as a control. The data of c-g are presented as the means ± standard deviations of three independent experiments. The significance was determined by one-way ANOVA, (ns = no significant; *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 3.

Differential gene expression profiles of the A. baumannii ATCC17978 cultured in the presence or absence of carnosol as measured by RNA-seq (Log₂ fold-change ≥1.0). (a) The number of genes that were upregulated and downregulated with the addition of 100 μM carnosol in the A. baumannii ATCC 17978 strain . (b) Cluster of orthologous groups (COG) term enrichment analysis of differentially expressed genes between the A. baumannii ATCC 17978 strain treated with and without carnosol treatment . The differentially regulated genes are not depicted in these diagrams but can be found in supplementary table 3. (c) RT–qPCR analysis of the genes whose expression differed among the A. baumannii ATCC 17978 strain treated with carnosol, the abiR strain, and the wild-type strain.

Figure 4.

Analysis of the binding of carnosol to AbiR and the influence of carnosol on the regulatory activity of AbiR. (a) SDS–PAGE of the purified AbiR protein (35.82 kDa). M: Marker. (b)MST analysis indicated that carnosol binds to AbiR with a K_D of 0.3 ± 1.3 μM. The expression of the csuA/B and pilW in A. baumannii ATCC 17978 treated with the carnosol was evaluated by RT-qPCR (c). The effects of carnosol on the gene expression levels of csuA/BABCDE and pilW were evaluated by assessing the β-galactosidase activity from PcsuA/BABCDE-lacZ (d) and PpilW-lacZ (e) in the A. baumannii ATCC 17978. EMSA detection of the in vitro binding of AbiR to the promoters of csuA/BABCDE and pilW in the presence of different amounts of carnosol (f-g). The potential residues of AbiR involved in carnosol binding were analyzed by AutoDock Vina software (h). MST analysis of the binding of carnosol to AbiR^Y209A (i), AbiR^H266A (j), AbiR^I236A (k) and AbiR^W267A (l). EMSA analysis of the effect of carnosol on the binding of AbiR^Y209A to the promoter of csuA/BABCDE in vitro (m). The data of b-e and i-l are presented as the means ± standard deviations (SD) from three independent experiments. The error bars indicate the SDs. The significance was determined by one-way ANOVA, (ns = no significant; *p < 0.05; **p < 0.01; ***p < 0.001). The experiments described in f, g and m were performed three times, and representative images from one experiment are shown.

Figure 5.

The effect of carnosol on the pathogenicity of A. baumannii ATCC 17978 in a mouse infection model. (a) All mice were allowed free access to food and water. After 7 days of adaptive feeding, forty BALB/c mice between the ages of 6 and 8 weeks were randomly divided into four groups (n = 10). Mortality was determined after BALB/c mice were infected with the A. baumannii ATCC 17978 strain in the absence or presence of carnosol for 7 days. Effects of carnosol on the production of inflammatory factors. RAW264.7 cells were infected with A. baumannii ATCC 17978 in the presence or absence of carnosol for 8 h. RT – qPCR analysis of the expression levels of caspase-1 (b), ASC (c), NLRP3 (d), IL-1β (e), IL-6 (f) and TNF-α (g). The data of b-g are presented as the means ± standard deviations of three independent experiments. The significance of the results was determined by one-way ANOVA (***p < 0.001).

Figure 6.

Analysis of synergistic effects between carnosol and antibacterial drugs. Effects of carnosol (20 µM) and its synergistic effects with different concentration of amikacin (16, 32 and 64 µg/mL) on the biofilm formation (a) and virulence (b) of the clinical drug-resistant A. baumannii strain. The data are presented as the means ± standard deviations of three independent experiments. The significance of the results was determined by one-way ANOVA (***p < 0.001).