Role of Aeromonas hydrophila flagella glycosylation in adhesion to Hep-2 cells, biofilm formation and immune stimulation

Abstract

Polar flagellin proteins from Aeromonas hydrophila strain AH-3 (serotype O34) were found to be O-glycosylated with a heterogeneous heptasaccharide glycan. Two mutants with altered (light and strong) polar flagella glycosylation still able to produce flagella were previously obtained, as well as mutants lacking the O34-antigen lipopolysaccharide (LPS) but with unaltered polar flagella glycosylation. We compared these mutants, altogether with the wild type strain, in different studies to conclude that polar flagella glycosylation is extremely important for A. hydrophila adhesion to Hep-2 cells and biofilm formation. Furthermore, the polar flagella glycosylation is an important factor for the immune stimulation of IL-8 production via toll receptor 5 (TLR5).

Figure 1

(A) Electron microscopy of whole cells from A. hydrophila strains stained according to Experimental Section. Bar represents 1 µm; (B) SDS-PAGE of purified polar flagellins, Coomasie blue stained. St, size standard. 1, A. hydrophila AH-3 (wild type); 2, A. hydrophila AH-3ΔWecP mutant; and 3, A. hydrophila AH-3:gne (AH-2767) mutant.

Figure 2

Schematic representation for the polar flagellin glycosylation of the wild type, and mutants AH-3ΔWecP and AH-2767:gne.

Figure 3

Human embryonic kidney cells (HEK293-null) and cells stably transfected with the human toll receptor 5 (TLR5)-gene (HEK293-hTLR5) were exposed to three different amounts of purified A. hydrophila AH-3 wild type or both mutants (AH-3ΔWecP and AH-2767) polar flagellins and IL-8 release was measured. The data represent the mean +/− SD of three independent experiments carried out in triplicates (n = 9). * p < 0.05, student’s t-test compared to the AH-3.