Fig 1.
Genetic loci associated with siderophore biosynthesis and transport in A. baumannii ATCC 17978.
Gene clusters for acinetobactin (A), baumannoferrin (B), and fimsbactins (C) are shown. Genes for the reductive release of iron from the siderophore, biosynthesis, import, export, and regulation are shown in the colors indicated in the corresponding legend. Putative Fur boxes are represented by black arrows, and the scale bar for each panel represents 1 kb. Key biosynthetic genes that were disrupted to elucidate siderophore function in this study are highlighted with thick black borders. A schematic highlighting the known and predicted siderophore biosynthetic and transport proteins in A. baumannii ATCC 17978, using the same color scheme as in A-C (D).
Fig 2.
Siderophore biosynthetic and transport genes are upregulated in metal deplete conditions.
WT A. baumannii was grown in metal-restricted media with or without the addition of exogenous iron or zinc. RNA was extracted and transcriptional changes in the expression of siderophore-associated genes were assessed by qRT-PCR in iron-deplete vs. replete conditions at 4 (A) and 12 h (B), and zinc-deplete vs. replete at 4 (C) and 12 (D) conditions, where expression was normalized to the expression of rpoB. * p < 0.05 and ** p < 0.01, as determined by Student’s t test relative to a hypothetical value of 1. Data are the means combined from two independent experiments, each with three biological replicates.
Fig 3.
Iron, but not zinc, represses the overall siderophore activity of A. baumannii.
Chrome Azurol S (CAS) assays to assess for total siderophore activity were performed on the spent culture supernatants of WT A. baumannii grown in cTMS media for 12 h with or without the addition of exogenous metals, as indicated. The colorimetric changes observed in the CAS assay are shown, where blue indicates an absence of siderophore activity and orange indicates that an iron chelator capable of mobilizing iron from CAS is present. Total siderophore activity is expressed as the percent activity of WT A. baumannii grown in the absence of exogenously added metals. **** p < 0.0001, as determined by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test. Data are the means of three biological replicates and are representative of three independent assays.
Fig 4.
Disrupting a single siderophore biosynthetic pathway does not drastically impact overall siderophore activity in A. baumannii.
Wild-type (WT) A. baumannii and its isogenic acinetobactin (ΔbasG), baumannoferrin (ΔbfnL) and fimsbactins (ΔfbsE) biosynthetic mutants were grown for 12 h in cTMS, and CAS assays to assess overall siderophore activity were performed on the spent culture supernatants. Total siderophore activity is expressed as the percent activity of WT A. baumannii, where data are the means of three biological replicates and are representative of three independent assays. ** p < 0.01, as determined by one-way analysis ANOVA with Dunnett’s multiple comparisons test.
Fig 5.
Acinetobactin biosynthetic mutants are impaired for growth under iron restriction.
Wild-type (WT) A. baumannii and its isogenic acinetobactin (ΔbasG), baumannoferrin (ΔbfnL) and fimsbactins (ΔfbsE) biosynthetic mutants were grown in cTMS media with 20% human serum (A), 180 mg/dL human transferrin (B), no added iron source (C), or 30 μM FeCl3 (D). Bacterial growth was assessed by determining the optical density at 600 nm (OD600), at the time points indicated. Data are representative of three independent experiments, and error bars represent the standard error of the mean. Where error bars are not visible, they are shorter than the height of the symbol. Statistical analysis is given for the endpoint growth, as performed by repeated measures two-way ANOVA, where *p < 0.05 and *** p < 0.001. Additional analyses of growth kinetics for these data can be found in S2 Fig.
Fig 6.
Disruption to all three siderophore biosynthetic pathways in A. baumannii is required to severely attenuate overall siderophore activity.
Wild-type (WT) A. baumannii and its isogenic combinatorial mutants were grown for 12 h in cTMS, and CAS assays to assess overall siderophore production were performed on the spent culture supernatants. Total siderophore activity is expressed as the percent activity of WT A. baumannii, where data are the means of three biological replicates and are representative of three independent assays. **** p < 0.0001, as determined by one-way ANOVA with Dunnett’s multiple comparisons test.
Fig 7.
Siderophores are required for A. baumannii to utilize human serum and transferrin as iron sources to support growth.
Wild-type (WT) A. baumannii and its isogenic combinatorial mutants were grown in cTMS media with 20% human serum (A), 180 mg/dL human transferrin (B), no added iron source (C), or 30 μM FeCl3 (D). Bacterial growth was assessed by determining OD600nm, at the time points indicated. Data are representative of three independent experiments, and error bars represent the standard error of the mean. Statistical analysis is given for the endpoint growth, as performed by repeated measures two-way ANOVA, where *p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Additional analyses of growth kinetics for these data can be found in S7 Fig.
Fig 8.
Siderophore biosynthetic and transport genes are upregulated in vivo.
Mice were systemically infected with WT A. baumannii and sacrificed at 24 h post-infection. Organs were harvested, RNA extracted, and gene expression changes relative to growth in vitro were determined in the heart (A) and lungs (B) using NanoString technology. Genes are clustered by known or predicted function, as indicated.
Fig 9.
Acinetobactin biosynthetic mutant is severely attenuated for survival and proliferation within the host.
Mice were systemically infected with WT A. baumannii or its isogenic siderophore biosynthetic mutants, as indicated. After 24 h, mice were sacrificed and the bacterial burdens of the kidneys, heart, liver, spleen, lungs, and blood were determined by plating for viable cell counts on lysogeny agar. Each symbol represents the A. baumannii count in the corresponding organ of one animal. Data are compiled from three independent experiments. Statistical significance was determined by Kruskal-Wallis with Dunn’s multiple comparisons test, where *p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.
Table 1.
Strains and plasmids employed in this study.
Table 2.
Primers employed in this study.