Brucella abortus requires the heme transporter BhuA for maintenance of chronic infection in BALB/c mice

Abstract

The gene annotated BAB2_1150 in the Brucella abortus 2308 genome sequence is predicted to encode a homolog of the well-characterized heme transporter ShuA of Shigella dysenteriae and accordingly has been given the designation bhuA (Brucella heme utilization). Phenotypic analysis of an isogenic bhuA mutant derived from B. abortus 2308 verified that there is a link between BhuA and the ability of the parent strain to use heme as an iron source in in vitro assays. Maximum expression of bhuA in B. abortus 2308 is observed during stationary phase when this strain in cultivated in low-iron minimal medium, and a comparison of the growth characteristics of the B. abortus bhuA mutant and 2308 in this medium suggested that heme serves as an important iron source for the parent strain during stationary phase. The B. abortus bhuA mutant HR1703 exhibits significant attenuation in cultured murine macrophages compared to strain 2308, and unlike its parent strain, the B. abortus bhuA mutant is unable to maintain a chronic spleen infection in experimentally infected BALB/c mice. These experimental findings suggest that heme and/or heme-containing proteins represent important iron sources for B. abortus 2308 during its residence in the mammalian host and that BhuA is required for efficient utilization of these iron sources.

FIG. 1.

Growth and viability of B. abortus 2308 (diamonds), HR1703 (2308 bhuA) (squares), and HR1703RC (HR1703 bhuA⁺) (triangles) in (A) low-iron minimal medium and (B) low-iron minimal medium containing 50 μM FeCl₃. The data are means and standard deviations for triplicate determinations from a single flask for each strain at each experimental time point in a single experiment. An asterisk indicates that the P value is <0.05 for a comparison of the data obtained for HR1703 and the data obtained for 2308 and HR1703RC. The data are representative of multiple (≥3) experiments from which equivalent results and statistical trends were obtained.

FIG. 2.

Growth and viability of the B. abortus bhuA mutant HR1703 in low-iron minimal medium (squares) or in this medium supplemented with 50 μM FeCl₃ (diamonds) or 50 μM Fe(NH₄)₂(SO₄)₂ (triangles) at 96 h postinoculation. The arrow indicates the time of addition of FeCl₃ and Fe(NH₄)₂(SO₄)₂ to the bacterial cultures. The data are means and standard deviations for triplicate determinations from a single flask for each strain at each experimental time point in a single experiment. An asterisk indicates that the P value is <0.05 for a comparison of the unsupplemented HR1703 culture and a culture of this strain supplemented with FeCl₃ or Fe(NH₄)₂(SO₄)₂. The data are representative of multiple (≥3) experiments from which equivalent results and statistical trends were obtained.

FIG. 3.

Expression of a bhuA-lacZ fusion in B. abortus 2308 during growth in low-iron minimal medium (filled bars) and low-iron minimal medium supplemented with 50 μM FeCl₃ (open bars). β-Galactosidase activity is expressed on the y axis in Miller units (27). The data are means and standard deviations for triplicate determinations from a single culture for each strain at each experimental time point in a single experiment. The data are representative of multiple (≥3) experiments from which equivalent results and statistical trends were obtained. The β-galactosidase levels produced by the base pMR15 plasmid in B. abortus 2308 during growth in low-iron minimal medium or in this medium supplemented with 50 μM FeCl₃ never exceeded 200 Miller units in the assays (data not shown).

FIG. 4.

Elevated levels of bhuA transcripts were present in B. abortus 2308 in response to (A) iron deprivation and (B) the transition into stationary phase during growth under iron-limiting conditions. The fold induction in response to iron deprivation represents the difference between the levels of bhuA, dhbC, and BAB1_0370 transcripts detected by real-time RT-PCR in RNA preparations from B. abortus 2308 cultures after 96 h of growth in low-iron minimal medium or low-iron minimal medium supplemented with 50 μM FeCl₃. The open bars in panel B indicate the levels of BAB1_0370 transcripts, and the cross-hatched bars indicate the levels of bhuA transcripts detected by real-time RT-PCR in RNA preparations from B. abortus 2308 after 72, 96, and 120 h of growth in low-iron minimal medium compared to the levels of these transcripts detected in RNA preparations obtained from this strain after 48 h of cultivation in low-iron minimal medium. The data are means and standard deviations for triplicate determinations for each gene at each experimental time point in a single experiment. The data are representative of three separate experiments from which equivalent results were obtained.

FIG. 5.

Survival and replication of B. abortus 2308 (triangles), HR1703 (2308 bhuA) (squares), and HR1703RC (HR1703 bhuA⁺) (diamonds) in cultured resident peritoneal macrophages from BALB/c mice. The data are means and standard deviations for the number of intracellular brucellae recovered for each strain from three separate wells of cultured macrophages at each experimental time point in a single experiment. One asterisk indicates that the P value is <0.05 and two asterisks indicate that the P value is <0.01 for comparisons of the data obtained for HR1703 with the data obtained for 2308 and HR1703RC. The data are representative of multiple (≥3) experiments from which equivalent results and statistical trends were obtained.

FIG. 6.

Spleen colonization profiles for B. abortus 2308 (diamonds), HR1703 (2308 bhuA) (squares), and HR1703RC (HR1703 bhuA⁺) (triangles) in experimentally infected BALB/c mice. The data are means and standard deviations for the number of brucellae detected in the spleens of five mice infected with each strain at each experimental time point in a single experiment. Two asterisks indicate that the P value is <0.01 for comparisons of the data obtained for HR1703 with the data obtained for 2308 and HR1703RC.