Detection and analysis of iron uptake components expressed by Acinetobacter baumannii clinical isolates

Abstract

The Acinetobacter baumannii 19606 prototype strain produces a 78-kDa iron-regulated outer membrane protein immunologically related to FatA, which is required for iron acquisition by the fish pathogen Vibrio anguillarum via the anguibactin-mediated system. This A. baumannii strain also secretes histamine, a biosynthetic precursor of the siderophore anguibactin. In contrast, the A. baumannii 8399 clinical strain isolated in Oregon produces a siderophore and a putative 73-kDa iron-regulated outer membrane (OM73) receptor that are different from those produced by V. anguillarum and A. baumannii 19606. These observations suggest that different A. baumannii clinical isolates express unrelated iron uptake systems. This hypothesis is supported by differences in outer membrane protein profiles among A. baumannii isolates obtained from Oregon and Europe. The 19606 isolate and some European isolates expressed a FatA-like protein, while neither 19606 nor any of the European isolates expressed proteins related to OM73. Some European isolates failed to express FatA- and OM73-like proteins. All but one of the Oregon isolates expressed OM73-like proteins, while none of them contained a FatA-like protein. The presence of these proteins always correlated with the presence of the om73- and fatA-like genes in the cognate strains. While 19606 and a few European isolates produced histamine, none of the Oregon isolates had this capability. Interestingly, one strain each from the Oregon and European isolates did not express any of these products involved in iron acquisition, indicating that they could acquire iron through siderophore-mediated transport systems different from those expressed by the 19606 and 8399 clinical isolates.

FIG. 1.

SDS-PAGE analysis of outer membrane proteins produced by A. baumannii clinical isolates. (A) Oregon strains. Lanes: 2, 9235; 3, 8971; 4, 8143; 5, 8114; 6, 8637; 7, 7133; 8, 7138; 9, 8399; 10, 9606; 11, 7931; 12, 9124; and 13, 9397. (B) European strains. Lanes: 2, BM4420; 3, BM4421; 4, BM4422; 5, BM4424; 6, BM4427; 7, BM4430; 8, BM4432; 9, BM4436; 10, BM4439; and 11, 19606. Lanes 1 in both panels, molecular weight markers. Membrane fractions used for this analysis were isolated from cells cultured under iron-deficient conditions.

FIG. 2.

Detection of protein expression by Western blot analysis. (A) Detection of OM73 in the Oregon strains. Lanes: 1, 19606; 2, 9235; 3, 8971; 4, 8143; 5, 8114; 6; 8637; 7, 7133; 8, 7138; 9, 9606; 10, 7931; 11, 9124; 12, 9397; and 13, 8399. (B) Detection of FatA-like protein in the European isolates. Lanes: 1, 19606; 2, BM4439; 3, BM4436; 4, BM4432; 5, BM4430; 6, BM4427; 7, BM4424; 8, BM4422; 9, BM4421; and 10, BM4420.

FIG. 3.

Southern blot analysis of total DNA obtained from the A. baumannii clinical strains. (A) Detection of om73 in HindIII-digested DNA isolated from the Oregon (lanes 3 and 5 to 15) and European (lanes 16 to 24) isolates. Lanes: 3, 8399; 4, 19606; 5, 9397; 6, 9124; 7, 7931; 8, 9606; 9, 7138; 10, 7133; 11, 8637; 12, 8114; 13, 8143; 14, 8971; 15, 9235; 16, BM4439; 17, BM4436; 18, BM4432; 19, BM4430; 20, BM4427; 21, BM4424; 22, BM4422; 23, BM4421; and 24, BM4420. (B) Detection of fatA-like gene in EcoRV-digested DNA isolated from the Oregon (lanes 4 to 15) and European (lanes 16 to 24) isolates. Lanes: 3, 19606; 4, 9397; 5, 9124; 6, 7931; 7, 9606; 8, 8399; 9, 7138; 10, 7133; 11, 8637; 12, 8114; 13, 8143; 14, 8971; 15, 9235; 16, BM4439; 17, BM4436; 18, BM4432; 19, BM4430; 20, BM4427; 21, BM4424; 22, BM4422; 23, BM4421; and 24, BM4420. Lanes 1, HindIII-digested λ DNA; lanes 2, empty.