Figure 3. Multiple Ail substitutions required to reveal a serum sensitivity phenotype comparable to Δail deletion.

A) Amino acid substitutions of Y. pestis Ail residues corresponding to homologous residues that no longer confer full serum resistance in Yersinia enterocolitica Ail (Miller et al., 2001) and Salmonella enterica Rck (Cirillo et al., 1996). B) Resistance of Y. pestis KIM5 Δail expressing plasmid-borne Ail or Ail derivatives, to killing by normal human serum (NHS). ~7.5 × 10⁵ CFU of mid-log culture grown with 500μM IPTG (to induce Ail expression) was added to 80% heat-inactivated serum (HIS) or 80% NHS for one hour at 37^oC. Surviving bacteria were plated and enumerated by colony counting. Percent serum resistance was calculated by (number of surviving colonies in NHS or NHS-AP/HIS) x 100 and is displayed on a logarithmic scale. Strains were tested a minimum of 3 times in separate experiments. Ail expression and stability was determined by Coomassie staining shown beneath the graph. Significance was assessed using the one-way ANOVA with Tukey’s post hoc test. *, p-value < 0.05 when compared to serum resistance of a strain expressing wild-type Ail.