566. Inactivation of a Bacterial Quorum-Sensing Signal by Expression of Bacterial Autoinducer Inhibitor, aiiA, in Mammalian Cells

Abstract

Many bacteria regulate production of extracellular virulence factors and biofilm formation through the cell-cell communication system, quorum-sensing. Quorum-sensing molecules frequently take the form of acylated homoserine lactones (HSLs). A gene recently isolated from Bacillus sp. 240B1 encodes an enzyme, AiiA, that inactivates HSLs through cleavage of the lactone ring. We asked whether expression of aiiA in mammalian cells would be sufficient to inactivate bacterial quorum-sensing signals in the cells' environment. The aiiA gene was cloned into a mammalian expression vector under the control of the CMV promoter. This construct was transfected into HeLa cells and allowed to express for 18 hours. Full-length transcripts of the gene were detected by RT-PCR in HeLa cells transfected with the aiiA vector, but were not detected in cells transfected with a GFP control vector. Recombinant AiiA activity was found in HeLa cell lysates and cell medium by means of a homoserine lactone bioassay. This bioassay employs a strain of E. coli producing β-galactosidase in response to HSL concentration. The level of HSL in the medium correlates linearly to β-galactosidase expression as assessed by the Galacton assay. We found that cell medium from aiiA-transfected HeLa cells incubated for five hours with 1μM butyryl-HSL (C4-HSL) showed a 20% reduction in HSL levels when compared to medium from control transfected cells and a 30% reduction in HSL levels when compared to medium from untransfected cells. Similarly, aiiA-transfected HeLa cell lysates were found to decrease HSL levels by 20% when compared to both control-transfected and untransfected HeLa cell lysates. Our findings represent the first report of aiiA transcription and function in mammalian cells. Based on these results, recombinant expression of aiiA in vivo may be a novel therapeutic intervention to prevent bacterial biofilm infections in the lungs of cystic fibrosis patients, in the bone in cases of osteomyelitis, or other tissues.