Characterization of Aeromonas salmonicida variants with altered cell surfaces and their use in studying surface protein assembly

Aeromonas salmonicida variants were characterized for alterations in their cell surface structure and used to examine reconstitution of the surface protein layer (A-layer). Variants lacking outer membrane O-polysaccharide were devoid of A-layer and excreted stainable floret-like material of the surface protein (A-protein). One variant, showing partial loss of O-polysaccharide, was associated with a disrupted A-layer and excretion of some A-protein. Variants lacking A-protein but possessing O-polysaccharide rapidly absorbed and concentrated sufficient excreted A-protein at the cell surface to coat the cells with a single confluent layer. Although differences in electrophoretic mobilities of A-proteins and O-polysaccharides from typical and atypical strains were evident, the different A-proteins and A-protein-deficient variants were interchangeable for reconstitution of a surface protein layer. No association of A-protein with cell surfaces of unrelated gram-negative bacteria was observed.Abbreviations A-layer additional surface protein layer - A-protein surface protein - Ast Aeromonas salmonicida typical - Asa Aeromonas salmonicida atypical - A^- phenotypically A-protein-negative variant - O^- phenotypically O-polysaccharide-negative variant - O^wk phenotypically O-polysaccharide weak variant - BHI brain heart infusion - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - TEM transmission electron microscopy     

Distribution of intravenously injected A-layer protein and lipopolysaccharide (LPS) from Aeromonas salmonicida in Atlantic salmon, Salmo salar L.

The distribution of intravenously injected A-layer protein and lipopolysaccharide (LPS) purified from the outer surface of the fish pathogen Aeromonas salmonicida, was studied in Atlantic salmon. Radiolabelling was achieved by conjugating the antigens to tyramine cellobiose (TC) or fluorescein isothiocyanate (FITC) which were radioiodinated either before or after conjugation. Since both TC and FITC are trapped intralysosomally at the cellular site of uptake, the ligands are advantageous in studies on tissue distribution of antigens. Injection of TC-A-layer protein and TC-LPS resulted in high specific radioactivity (cpm g⁻¹tissue) in both head kidney and trunk kidney. In contrast, only low specific radioactivity was recovered in spleen, heart and liver. Surprisingly, use of FITC-LPS as the antigen changed the uptake to be high in both spleen and head kidney. Radiolabelled (¹²⁵I-TC-) LPS and A-protein, administered by a dorsal aorta catheterisation technique, were cleared from the blood within 24 h. In immunised fish, the antibody activity against the A-layer protein was diminished even within 10 min after administration, in contrast to the level of anti-LPS antibodies which remained high. These results suggest that immune-complex formation took place at least with the A-layer protein, but the uptake of A-layer protein in the various tissues did not differ significantly in vaccinated (A. salmonicida bacterin) and non-vaccinated fish.