Aii20J, a wide-spectrum thermostable N-acylhomoserine lactonase from the marine bacterium Tenacibaculum sp. 20J, can quench AHL-mediated acid resistance in Escherichia coli

Abstract

Acyl homoserine lactones (AHLs) are produced by many Gram-negative bacteria to coordinate gene expression in cellular density dependent mechanisms known as quorum sensing (QS). Since the disruption of the communication systems significantly reduces virulence, the inhibition of quorumsensing processes or quorum quenching (QQ) represents an interesting anti-pathogenic strategy to control bacterial infections. Escherichia coli does not produce AHLs but possesses an orphan AHL receptor, SdiA, which is thought to be able to sense the QS signals produced by other bacteria and controls important traits as the expression of glutamate-dependent acid resistance mechanism, therefore constituting a putative target for QQ. A novel AHL-lactonase, named Aii20J, has been identified, cloned and over expressed from the marine bacterium Tenacibaculum sp. strain 20 J presenting a wide-spectrum QQ activity. The enzyme, belonging to the metallo-β-lactamase family, shares less than 31 % identity with the lactonase AiiA from Bacillus spp. Aii20J presents a much higher specific activity than the Bacillus enzyme, maintains its activity after incubation at 100 ºC for 10 minutes, is resistant to protease K and α-chymotrypsin, and is unaffected by wide ranges of pH. The addition of Aii20J (20 μg/mL) to cultures of E. coli K-12 to which OC6-HSL was added resulted in a significant reduction in cell viability in comparison with the acidresistant cultures derived from the presence of the signal. Results confirm the interaction between AHLs and SdiA in E. coli for the expression of virulence-related genes and reveal the potential use of Aii20J as anti-virulence strategy against important bacterial pathogens and in other biotechnological applications.

Inhibition ofSteptococcus mutansbiofilm formation by extracts ofTenacibaculumsp. 20J, a bacterium with wide-spectrum quorum quenching activity

“…To exclude that 20J-PCE affects bioluminescence in V. harveyi through factors nondependent on QS, the mutant V. harveyi JAF548 [31] in which bioluminescence is expressed independently of QS control was used. Moreover, the Furanone C30, which was suggested to prevent biofilm formation in S. mutans previously [22,23], and extracts obtained from the closely related species T. maritimum NCBI2154 T , a strain with no AHL-lactonase activity [36], were also tested. Results showed that after 8 h, the 20J extract completely eliminates light production by the reporters BB170 and JMH597 compared to control wells (Figure 1(a)).…”

“…A preliminary analysis, including dialysis and ultracentrifugation, indicated that the active compound in 20J extracts is a large, possibly membrane-associated molecule. Despite the bioactive molecule is not affected by heat treatment and proteinase K digestion, it cannot be disregarded that the anti-biofilm activity is enzymatic since similar treatments could not neutralize enzymatic AHL degradation in 20J extracts [36]. …”

“…20J was also treated with proteinase K (100 μg ml −1 ; Sigma-Aldrich, MO) at 30°C for 1 h [36]. Thermal stability of the bioactive compound was tested by incubating the PCE of Tenacibaculum sp.…”

“…Thermal stability of the bioactive compound was tested by incubating the PCE of Tenacibaculum sp. 20J at 100°C for 10 min [36]. The residual anti-biofilm activity was measured by using the xCELLigence® System RTCA (ACEA, Biosciences Inc., CA).…”

“…E-plates 16 or E-plate 96 (ACEA, Biosciences Inc.) were inoculated with 180 μl of a 24-h culture of S. mutans in BHI broth supplemented with 0.1% sucrose (OD 600 adjusted to 0.05) and 20 μl of extracts or inhibitors. Furanone C30 (0.02 µg ml −1 , Sigma-Aldrich) and the AHL-lactonase Aii20J (20 µg ml −1 [36]) were also tested. Twenty microlitres of PBS pH 6.5 were added to the control wells.…”