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 (InlC) 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‐ inl FE. The two proteins encoded by this locus, i‐InlE and i‐InlF, belong to the group of small, secreted internalins. Southern blot analyses show that the i‐ inl 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 inl C encodes the only small internalin. The mature i‐InlE 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‐ inl E mRNA and i‐InlE protein are expressed at high levels. As shown by Northern blot analysis and primer extension, transcription of the tandemly arranged i‐ inl F and i‐ inl E genes is dependent on the virulence regulator PrfA, and characteristic palindromic sequences (‘PrfA‐boxes’) were identified in the promoter regions of i‐ inl F and i‐ inl E. Non‐polar i‐ inl E and i‐ inl F deletion mutants and an i‐ inl 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 50 for the parental strain was determined as 4 × 10 7 bacteria per mouse. These data suggest an important role for i‐InlE and i‐InlF in L. ivanovii virulence.