Identification of regions of Ail required for the invasion and serum resistance phenotypes

Yersinia enterocolitica is an enteric pathogen that has served as a model s ystem for the study of microbial pathogenesis. Numerous virulence gene have been identified both on the virulence plasmid and on the chromosome. One o f the chromosomal genes that is highly correlated with virulence is all, a gene identified along with inv in a screen for Y. enterocolitica genes that could confer an invasive phenotype to Escherichia coli. Ail also promotes serum resistance in both E. coli and Y. enterocolitica. Several virulence f actors homologous to Ail have been identified in other pathogens, yet very little is known about what constitutes the functional domain(s) of these pr oteins. Proteins in this family are predicted to consist of eight transmemb rane beta -sheets and four cell surface-exposed loops. We constructed and c haracterized a number of insertion, deletion and point mutations in the reg ions of ail predicted to encode the cell surface loops. The results from th e analysis of these mutants indicate that cell surface loops one and four d o not directly promote invasion or serum resistance, whereas mutations in l oop three appear to modulate both phenotypes. Analysis of mutations in loop 2 suggests that this surface-exposed loop contains sequences required for serum resistance and invasion. In addition, a peptide derived from the sequ ence of loop 2 was able specifically to inhibit Ail-mediated invasion in a dose-dependent manner. These results suggest that Ail directly promotes inv asion and that loop 2 contains an active site, perhaps a receptor-binding d omain. Analyses of the mutations also suggest that the serum resistance and invasion phenotypes may be separable, because there are numerous mutations that affect one phenotype but not the other.