The role of A-layer in polyunsaturated fatty acid (PUFA)-mediated effects on Aeromonas salmonicida subsp. salmonicida

Aeromonas salmonicida subsp. salmonicida is a Gram-negative bacterium that infects salmonids and non-salmonids worldwide leading to an infection known as furunculosis, which is characterized by skin lesions and hemorrhages of the fish epidermis. This infection is carried out by a A+ (virulent strain) of A. salmonicida containing an important virulent factor known as the A-layer, which is a 2D paracrystalline structure that binds to the basement membrane and functions to promote adherence to host membranes and resistance to host defense. The purpose of this study was to examine the ability of the A+ A. salmonicida to incorporate exogenous fatty acids into its lipid membrane and explore the phenotypic outcomes. A. salmonicida A+ and A- strains were differentiated using the Congo Red plating method. The A+ A. salmonicida cultures were grown in CM9 supplemented with the presence or absence of 300µM exogenous polyunsaturated fatty acids (PUFAs). Lipids were extracted and analyzed for membrane assimilation by thin-layer chromatography and ultra performance liquid chromatography mass spectrometry and showed the ability of A. salmonicida to incorporate exogenous fatty acids into its lipid profile. The phenotypic outcomes were examined using a series of assays for membrane permeability, antimicrobial peptide susceptibility, and biofilm formation. The fatty acid 20:5 significantly (p \u3c 0.001) decreased biofilm formation consistently across the temperatures tested. A general decrease in biofilm formation was seen at a higher temperature of 28ºC. The PUFAs (18:3γ 20:3, 20:4, 20:5, 22:6) significantly (p \u3c 0.001) permeabilized the membrane by 40% when compared to the control tested in the crystal violet uptake. In addition, the PUFAs (18:3γ, 20:3) provided significant (p \u3c 0.001) protection against the antimicrobial peptide polymyxin B (PMB). The A+ strain when compared to the A- displayed similar phenotypic effects as a fish pathogen. In fact, both A+ and A- strains preferred the fatty acid 22:6 which may indicate a role omega-3 fatty acids play in A. salmonicida being a fish pathogen