A novel PrfA-regulated chromosomal locus, which is specific for Listeria ivanovii, encodes two small, secreted internalins and contributes to virulence in mice

Several large, cell wall-associated internalins and one small, secreted internalin (InIC) have been described previously in Listeria monocytogenes, Using degenerate primers derived from sequenced peptides of an L. ivanovii major secreted protein, we identified a new 4.25 kb internalin locus of L, ivanovii, termed i-in/FE, The two proteins encoded by this locus, i-InIE and i-InIF, belong to the group of small, secreted internalins, Southern blot analyses show that the i-in/FE locus does not occur in L. monocytogenes. These data also indicate that six genes encoding small, secreted internalins are present in L, ivanovii, in contrast to L. monocytogenes, in which in/C encodes the only small internalin. The mature i-InIE protein (198 amino acids) is secreted in large amounts into the brain-heart infusion (BHI) culture medium in the stationary growth phase, In minimum essential medium (MEM), which has been used previously to induce PrfA-dependent gene transcription, i-in/E mRNA and i-InIE protein are expressed at high levers, As shown by Northern blot analysis and primer extension, transcription of the tandemly arranged i-in/F and i-in/E genes is dependent on the virulence regulator PrfA, and characteristic palindromic sequences ('PrfA-boxes') were identified in the promoter regions of i-in/F and i-in/E. Non-polar i-in/E and i-in/F deletion mutants and an i-in/FE double deletion mutant were constructed and tested in the mouse infection model, After intravenous infection, all three mutants entirely failed to kill C57BL/6 mice even at high infectious doses of 10(9) bacteria per. mouse, whereas the LD,, for the parental strain was determined as 4 x 10(7) bacteria per mouse. These data suggest an important role for i-InIE and i-InIF in L. ivanovii virulence.