Figure 7. Minihepcidin PR73 acts in serum by slowing bacterial growth.

Serum was collected from iron-depleted or iron-loaded Hamp1^−/− mice that were injected with PR73 or solvent, and from iron-depleted or iron-loaded WT mice (not treated with PR73). Serum iron concentrations are shown in Table S2. V. vulnificus carrying the non-replicating marker plasmid pGTR905 was incubated in these sera in vitro for 2 h, and CFU were determined either on plates without chloramphenicol (total bacteria) or plates with chloramphenicol and arabinose (allows growth of only plasmid-containing bacteria). (A) PR73 greatly reduced total bacterial yield, which reflects the sum of bacteriostatic and bactericidal effects. (B) PR73 only slightly reduced the yield of plasmid-containing bacteria indicating only a small bactericidal effect. (C) The number of total bacteria was much higher in serum from iron-loaded WT mice than in serum from iron-depleted mice, as expected. (D) Different serum iron concentrations did not affect the yield of plasmid-containing bacteria indicating that hypoferremia by itself does not have a bactericidal effect. Each vertical bar represents the mean, and error bars represent standard deviation for 3 independent experiments (with 3 replicates in each experiment). The black dotted line represents the number of plasmid-containing bacteria after growth that diluted plasmid copy number to 1 plasmid per bacterium (thus bacteria in the original inoculum carried 5 plasmids per bacterium). Statistical significance was assessed using student’s t test (*p<0.05; **p<0.01; ***p<0.001). See also Figure S7.