Virulence determinants of the insect pathogen Bacillus thuringiensis; : Molecular characterization of flagellins and the virulence protease immune inhibitor A

Sammanfattning: Bacillus thuringiensis (Bt) is a motile, Gram positive, rod-shaped and spore-forminginsect pathogenic bacterium found on plants and in soil. The main insecticidal activityresides in a proteinacious crystal inclusion body (the S-endotoxin), but also the sporesare important for lethality. Bt is subclassified according to flagellar serotype and the 5-endotoxin they produce. Subspecies that lack 6-endotoxin but still kill insects have beenisolated, proving the importance of other virulence factors. This thesis includes theinvestigation of two such virulence factors: Immune inhibitor A (InA), and flagella andtheir constituent proteins, the flageilins. In addition, an effective method to transformvegetative Bt cells with plasmid DNA is described.InA is a 72 kDa molecular weight, neutral metalloprotease toxic to lepidopteran,coleopteran and dipteran insects. The LD50 dose in Trichoplusia ni is 12 ng/mg oflarval body weight. This thesis presents the cloning and molecular characterization ofInA and the deduced amino acid sequence is compared to that of other neutralmetalloproteases such as elastase, a virulence protease from Pseudomonas aeruginosa.InA was found to be secreted by at least 70% of tested Bt subspecies.Flagella, and motility as such, were found to be important for Bt invasiveness, butthey may also serve additional functions. The results in this thesis suggest that flageilinsare co-expressed with other virulence determinants such as secretion of lecithinase,ability to adhere to and kill Spodoptera frugiperda cells and ability to avoid and survivethe Hyalophora cecropia host defence systems. Flagellin from Bt var. alesti andgelechiae have been purified, and one complete and one partial alesti flagellin genehave been cloned and sequenced. A striking feature of one of these genes is the presence ofa 355 bp direct repeat.