Identification and analysis of the virulence factors in Serratia entomophila causing amber disease to the grass grub Costelytra zealandica : A molecular genetics approach
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Amber disease caused by Serratia entomophila to larvae of Costelytra zealandica (Coleoptera: Scarabaeidae), is characterized by the production of two symptoms: anti-feeding effect (AFE) and amber coloration (AC). This study was aimed to identify and characterize the virulence factors involved in the disease. Three factors were identified: i) MRE-HA fimbriae; ii) an extracellular protease and, iii) an anti-feeding toxin. i) Fimbriae type 1, 3 and MRE-HA were identified and characterized in S. entomophila by haemagglutination tests and electron microscopy. Analysis of nonpathogenic mutants suggested that the MRE-HA fimbriae were associated with pathogenicity. ii) The locus coding for the extracellular protease of S. entomophila was identified and cloned. Examination and complementation assays of pathogenic and nonpathogenic strains showed that the protease is not directly involved, but it might potentiate the disease. It was suggested that the protease might be linked with pathogenicity by a common regulator factor. iii) A locus named amb2 was identified, isolated and cloned. Genetic evidence and complementation assays with nonpathogenic mutants demonstrated that amb2 is responsible for the AFE. SDS-PAGE analysis of the amb2 gene products expressed in minicells showed the synthesis of two proteins of 21 and 25 kDa, named AnfA and AnfB. The genes encoding these proteins were mapped by deletion analysis and lacZ-gene fusions. DNA sequencing of the anfA gene revealed that another protein of ~12 kDa (AnfA2) was also encoded by amb2. Consensus sequences with homology to the binding sites of the bacterial regulators CAP, Fur and ToxR were identified in the promoter regions. Homology of 50% was found between a hydrophobic motif of the δ-endotoxin of Bacillus thuringiensis and AnfA1, The results suggest that AnfA1 , AnfA2 and AnfB might be subunits of a toxin causing the AFE. It was concluded that virulence determinants in S. entomophila including the MRE-HA fimbriae, the extracellular protease and the anti-feeding toxin act in collaboration to produce amber disease.