V antigen-polyhistidine fusion peptide: Binding to LcrH and active immunity against plague

The structural gene for V antigen (lcrV) is known to be encoded within the lcrGVH-yopBD operon of the -70-kb low-calcium-response or Lcr plasmid of Yersinia pestis. This 37-kDa monomeric peptide was reported to provide active immunity in mice, suppress inflammatory cytokines, and regulate expression of the low calcium response (Lcr⁺). Here we describe pVHB62, encoding a polyhistidine-V antigen fusion peptide (V(h)) and linked LcrH. V(h) underwent degradation from both the C terminus and N terminus during classical chromatographic fractionation but remained intact within two compartments during Ni²⁺ affinity chromatography. The first was homogeneous, capable of active immunization (mouse intravenous 50% lethal dose, > 10⁷ bacteria), and stable at 4°C. The second remained bound to the affinity column but could be eluted as a mixture of V(h), LcrH, and low-molecular-weight material by application of 6 M guanidine · HCl. This mixture was dialyzed, denatured in 8 M urea, and again applied to the affinity column, which then hound V(h) but not LcrH. The latter was recovered and renatured, and low-molecular-weight material was removed by biochemical fractionation. The resulting homogeneous LcrH bound protein A-V antigen fusion peptide but not protein A in a sandwich enzyme-linked immunosorbent assay, and this reaction was inhibited by V(h). These observations indicate that LcrH normally binds V antigen in bacterial cytoplasm and suggest that only free LcrH down-regulates expression of the low calcium response.