TnphoA Salmonella abortusovis Mutants Unable To Adhere to Epithelial Cells and with Reduced Virulence in Mice

Abstract

SalmoneUla abortusovis is a pathogenic bacterium highly specific to sheep, causing spontaneous abortion. In order to understand the role of genes involved in pathogenicity, we investigated S. abortusovis with the random mutagenic TnphoA transposon. A total of 95 S. abortusovis TnphoA mutants yielding alkaline phosphatase active fusion protein were obtained. In this way we created a bank of strains in order to identify any phenotypic modification which could affect the periplasmic and/or exported proteins involved in virulence. The TnphoA mutants were screened for the ability to adhere to epithelial cells: a total of 23 mutant strains lost this phenotypic feature. To detect the chromosomal TnphoA insertions, DNA was restricted by the enzyme EcoRV, which does not cleave the TnphoA sequence. Southern blotting analysis revealed the existence of four classes of integration. Colonies of adhesiveless mutants appear to be as smooth as the S. abortusovis wild type, and electrophoretic analysis indicates a normal lipopolysaccharide profile. To identify mutations affecting genes encoding for outer membrane proteins (OMPs), the alkaline phosphatase portion of the fusion proteins was revealed 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 involved in pathogenicity in mice, since a group of mutant strains has shown reduced virulence in mice and one mutant is completely avirulent. Furthermore, after mice were exposed orally to these mutants, significant protection against oral challenge with the parental virulent strain resulted.