Pneumonia infection in mice reveals the involvement of the feoA gene in the pathogenesis of Acinetobacter baumannii

Abstract

Acinetobacter baumannii has emerged in the last decade as an important nosocomial pathogen. To identify genes involved in the course of a pneumonia infection, gene expression profiles were obtained from A. baumannii ATCC 17978 grown in mouse infected lungs and in culture medium. Gene expression analysis allowed us to determine a gene, the A1S_0242 gene (feoA), over-expressed during the pneumonia infection. In the present work, we evaluate the role of this gene, involved in iron uptake. The inactivation of the A1S_0242 gene resulted in an increase susceptibility to oxidative stress and a decrease in biofilm formation, in adherence to A549 cells and in fitness. In addition, infection of G. mellonella and pneumonia in mice showed that the virulence of the Δ0242 mutant was significantly attenuated. Data presented in this work indicated that the A1S_0242 gene from A. baumannii ATCC 17978 strain plays a role in fitness, adhesion, biofilm formation, growth, and, definitively, in virulence. Taken together, these observations show the implication of the feoA gene plays in the pathogenesis of A. baumannii and highlight its value as a potential therapeutic target.

Keywords: Acinetobacter baumannii; animal infection models; iron uptake; virulence.

Figure 1.

cDNA amplification of genes from the A1S_0242–0244 operon of A. baumannii ATCC 17978 strain. The intergenic regions from genes A1S_0242–0243 and A1S_0243–0244 are shown in lanes 8 and 9, respectively. The intergenic regions from genes A1S_0241–0242 and A1S_0244–0245 are shown in lanes 7 and 10, respectively (negative controls). Genomic DNA was used as template for positive control (lanes 1 to 5, respectively). Lanes 5 and 11 show the gyrB amplification from DNA and cDNA, respectively (positive controls). Lane 6 shows GeneRuler 1 Kb Plus DNA Ladder (Thermo Fisher Scientific).

Figure 2.

Growth curves of the ATCC 17978 strain and the isogenic mutant derivative strains Δ0242, Δ3850 and Δ0242/Δ3850 in presence and absence of the iron chelator 2,2′-bipyridyl (BIP). Data correspond to the mean of three replicates and bars represent the standard deviations.

Figure 3.

Quantification of biofilm formation by the A. baumannii ATCC 17978 strain, the mutant derivative strain Δ0242, the mutant derivative strain Δ3850, the double mutant derivative strain Δ0242/Δ3850, the ATCC 17978 harboring the empty vector pWH1266-Km (ATCC 17978 + pWH1266-Km), the mutant derivative strain harboring the empty vector pWH1266-Km (Δ0242 + pWH1266-Km) and the mutant derivative Δ0242 over-expressing the A1S_0242 gene from the pWH1266-Km plasmid (Δ0242 complemented).

Figure 4.

Quantification of bacterial adhesion to A549 cells by the A. baumannii ATCC 17978 strain, the mutant derivative strain Δ0242, the mutant derivative strain Δ3850 and the double mutant strain Δ0242/Δ3850.

Figure 5.

Survival of Galleria mellonella larvae (n = 10 per group) after infection with A. baumannii ATCC 17978, Δ0242, Δ3850 and Δ0242/Δ3850 strains. Survival was significantly higher in caterpillars infected with the Δ0242 mutant than those infected with the wild type strain (p < 0.05). No deaths were observed in any of the two control groups (not injected and injected with sterile PBS).

Figure 6.

Pneumonia infection in mice. A) Survival of BALB/c (n  = 10 per group) mice after pneumonia infection with A. baumannii ATCC 17978 and Δ0242 strains. Survival was significantly higher in mice infected with the Δ0242 mutant (p < 0.01). B) Bacterial load determination in lungs of mice infected with the ATCC 17978 strain and the Δ0242 mutant. Bacterial load (p < 0.01) was significantly lower in mice infected with the Δ0242 mutant.