TNPHOA SALMONELLA-ABORTUSOVIS MUTANTS UNABLE TO ADHERE TO EPITHELIAL-CELLS AND WITH REDUCED VIRULENCE IN MICE

Salmonella abortusovis is a pathogenic bacterium highly specific to sh eep, causing spontaneous abortion. In order to understand the role of genes involved in pathogenicity, we investigated S. abortusovis with t he random mutagenic TnphoA transposon. A total of 95 S. abortusovis Tn phoA mutants yielding alkaline phosphatase active fusion protein were obtained. In this way we created a bank of strains in order to identif y any phenotypic modification which could affect the periplasmic and/o r exported proteins involved in virulence. The TnphoA mutants were scr eened for the ability to adhere to epithelial cells: a total of 23 mut ant strains lost this phenotypic feature. To detect the chromosomal Tn phoA insertions, DNA was restricted by the enzyme EcoRV, which does no t cleave the TnphoA sequence. Southern blotting analysis revealed the existence of four classes of integration. Colonies of adhesiveless mut ants appear to be as smooth as the S. abonusovis wild type, and electr ophoretic analysis indicates a normal lipopolysaccharide profile. To i dentify mutations affecting genes encoding for outer membrane proteins (OMPs), the alkaline phosphatase portion of the fusion proteins was r evealed in TnphoA mutants by immunoblotting with specific antibodies. A mutation in OMPs was detected in seven mutants. Restriction analysis identified in four mutants a common region of 2 kb where alterations in genes coding for OMPs occur. We suggested that this region is invol ved in pathogenicity in mice, since a group of mutant strains has show n reduced virulence in mice and one mutant is completely avirulent. Fu rthermore, after mice were exposed orally to these mutants, significan t protection against oral challenge with the parental virulent strain resulted.