Characterisation of the iron uptake mechanisms of Aeromonas Salmonicida : role in virulence and protective immunity

Abstract

The ability of the bacterial fish pathogen Aeromonas Salmonicida to grow under conditions of iron-restriction In vitro was examined in an attempt to identify and characterise the iron uptake mechanisms of the pathogen. Both typical and atypical strains of A. Salmonicida grew and multiplied in the presence of the synthetic high affinity iron chelators, ethylene diamine di(o-hydroxyphenylacetic acid) (EDDA) and 2,2'dipyridyl (Op) and possessed one or more functional iron uptake mechanisms. The first mechanism involved the inducib1e production of a phenolate siderophore which was detected using chrome azurol S (CAS) agar. This inducible siderophore-mediated mechanism was possessed only by typical strains of A. Salmonicida. A second mechanism was identified which enabled both typical and atypical strains to utilise a number of mammalian sources of transferrins (Tf). Typical strains were able to utilise Tf via a siderophore-mediated mechanism; atypical strains were able to utilise Tf via a mechanism involving the proteolytic degradation of Tf by an extracellular metalloprotease. A third mechanism was identified which enabled virulent strains of A. Salmonicida to utilise haem sources of iron via a constitutive, siderophore-independant mechanism. The mechanism involved a common cell-surface associated haem-binding protein, thought to be the 49-k1lodalton (kOa) A-layer protein capable of binding the sulphonated diazo dye, congo red. Growth under conditions of Iron-restriction resulted In the Increased synthesis of a number of extracellular virulence factors Involved In the pathogenesis of furunculosis, Including haemolysin and protease. Iron-restricted growth also resulted in the expression of four Iron-regulated outer membrane proteins (IROMPs) of molecular weight 82, 77, 72 and 70k0a which were present and immunologically cross-reactive in eighteen strains of A. Salmonicida grown In the presence of an iron chelator. The IROMPs were shown by Immunoblotting techniques to be expressed In vivo during Infection, since A. Salmonicida isolated directly without subculture from the furuncle material of an Infected Atlantic salmon expressed IROMPs. Immunoblotting techniques and an enzyme-linked immunosorbent assay (ELISA) developed to detect the presence of anti-IROMP immunoglobulin (Ig) found the IROMPs to be immunogenic In Atlantic salmon, and in addition, convalescent sera of Atlantic salmon surviving an A. Salmonicida challenge contained antibodies against the IROMPs. Outer membrane proteins ((3MPs) of A. Salmonicida were then evaluated for their ability to Induce protective immunity In Atlantic salmon. Compared with OMP prepared from A. Salmonicida grown under iron-replete conditions, IROMPs conferred protection against both natural and experimental heterologous A. Salmonicida bath challenge. In addition, passive immunisation of Atlantic salmon with an antisera containing antibodies to the IROMPs, and a rabbit anti-IROMP antisera or affinity-purified immunoglobulin 0 (IgO) also conferred protection against heterologous bath challenge. In addition, an Iron-restricted bacterin of A. Salmonicida containing IROMPs was found to be capable of protecting Atlantic salmon against A. Salmonicida bath challenge. Both the Atlantic salmon antisera raised against the IROMPs, and the rabbit anti-IROMP antisera were bactericidal against virulent strains of A. Salmonicida In the presence of complement. Results presented in this thesis Indicate that the IROMPs of A. Salmonicida represent important protective antigens in the vaccination of Atlantic salmon against furunculosis.