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N-Acylhomoserine lactones involved in quorum sensing control the type VI secretion system, biofilm formation, protease production, and in vivo virulence in a clinical isolate of Aeromonas hydrophila

¹ Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555-1070, USA
² Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-1070, USA
³ Department of Medical and Research Technology, University of Maryland School of Medicine, Baltimore, MD 21201, USA

In this study, we delineated the role of N-acylhomoserine lactone(s) (AHLs)-mediated quorum sensing (QS) in the virulence of diarrhoeal isolate SSU of Aeromonas hydrophila by generating a double knockout ahyRI mutant. Protease production was substantially reduced in the ahyRI mutant when compared with that in the wild-type (WT) strain. Importantly, based on Western blot analysis, the ahyRI mutant was unable to secrete type VI secretion system (T6SS)-associated effectors, namely haemolysin coregulated protein and the valine-glycine repeat family of proteins, while significant levels of these effectors were detected in the culture supernatant of the WT A. hydrophila. In contrast, the production and translocation of the type III secretion system (T3SS) effector AexU in human colonic epithelial cells were not affected when the ahyRI genes were deleted. Solid surface-associated biofilm formation was significantly reduced in the ahyRI mutant when compared with that in the WT strain, as determined by a crystal violet staining assay. Scanning electron microscopic observations revealed that the ahyRI mutant was also defective in the formation of structured biofilm, as it was less filamentous and produced a distinct exopolysaccharide on its surface when compared with the structured biofilm produced by the WT strain. These effects of AhyRI could be complemented either by expressing the ahyRI genes in trans or by the exogeneous addition of AHLs to the ahyRI/ahyR⁺ complemented strain. In a mouse lethality experiment, 50 % attenuation was observed when we deleted the ahyRI genes from the parental strain of A. hydrophila. Together, our data suggest that AHL-mediated QS modulates the virulence of A. hydrophila SSU by regulating the T6SS, metalloprotease production and biofilm formation.

Correspondence
Ashok K. Chopra
achopra{at}utmb.edu

The GenBank/EMBL/DDBJ accession number for the ahyRI sequence of A. hydrophila SSU is DQ398101.

Four supplementary figures, showing cross streaks of A. hydrophila strains against a Chromobacterium violaceum CV026 reporter strain, Western blot analysis showing production of Act in the supernatant from A. hydrophila strains, measurement of biofilm mass by crystal violet staining formed on polystyrene by strains of A. hydrophila, and Western blot analysis showing production of Hcp2, are available with the online version of this paper.