BhuR, a virulence-associated outer membrane protein of Bordetella avium, is required for the acquisition of iron from heme and hemoproteins

Abstract

Iron (Fe) is an essential element for most organisms which must be obtained from the local environment. In the case of pathogenic bacteria, this fundamental element must be acquired from the fluids and tissues of the infected host. A variety of systems have evolved in bacteria for efficient acquisition of host-bound Fe. The gram-negative bacterium Bordetella avium, upon colonization of the avian upper respiratory tract, produces a disease in birds that has striking similarity to whooping cough, a disease caused by the obligate human pathogen Bordetella pertussis. We describe a B. avium Fe utilization locus comprised of bhuR and six accessory genes (rhuIR and bhuSTUV). Genetic manipulations of B. avium confirmed that bhuR, which encodes a putative outer membrane heme receptor, mediates efficient acquisition of Fe from hemin and hemoproteins (hemoglobin, myoglobin, and catalase). BhuR contains motifs which are common to bacterial heme receptors, including a consensus FRAP domain, an NPNL domain, and two TonB boxes. An N-terminal 32-amino-acid segment, putatively required for rhuIR-dependent regulated expression of bhuR, is present in BhuR but not in other bacterial heme receptors. Two forms of BhuR were observed in the outer membrane of B. avium: a 91-kDa polypeptide consistent in size with the predicted mature protein and a smaller 82-kDa polypeptide which lacks the 104 amino acids found at the N terminus of the 91-kDa form. A mutation in hemA was engineered in B. avium to demonstrate that the bacterium transports heme into the cytoplasm in a BhuR-dependent manner. The role of BhuR in virulence was established in turkey poults by use of a competitive-infection model.

FIG. 1.

Growth curves demonstrating the growth of 4169rif and Pho20 in culture media containing various Fe sources. (A) BHI broth; (B) BHI(EDDHA); (C) BHI(EDDHA) plus 144 μM FeSO₄; (D) BHI(EDDHA) plus 50 μM hemin. While the data in the curves are derived from a single experiment, the growth trends were identical for each strain in two additional replicates. +, 4169rif(pRK415); ▵, Pho20(pRK415).

FIG. 2.

Schematic representation of the entire B. avium heme utilization locus. Each identified promoter is indicated, along with the regulatory proteins known to mediate expression from the given promoter (26).

FIG. 3.

RNase protection assay demonstrating that the insertions into bhuR in Pho20 and 4169rif(bhuR::kan) are nonpolar. Total RNA isolated from each strain was hybridized to probe 1 or probe 2. Probe 1 was homologous to DNA sequences located 5′ of the insertion site of TnphoA in Pho20 and of the insertion of the Kan resistance cassette in 4169(bhuR::kan); probe 2 was homologous to DNA sequences located in the bhuR-bhuS intergenic region. The control lanes contained only the radiolabeled hybridization probes.

FIG. 4.

SDS-PAGE of the OMPs from B. avium. Lanes: 1, 4169rif cultured in BHI broth containing 144 μM FeSO₄; 2, 4169rif cultured in BHI(100 μM EDDHA); 3, Pho20 cultured in BHI(100 μM EDDHA); 4, Pho20(pERM25) cultured in BHI(100 μM EDDHA); 5, 4169rif(bhuR::kan) cultured in BHI(100 μM EDDHA); 6, 4169rif(bhuR::kan)(pERM25) cultured in BHI(100 μM EDDHA). Each lane contained 8 μg of total protein. Molecular masses are indicated in kilodaltons on the left. The positions of the 91-kDa FeRP and the 82-kDa FeRP are indicated by arrows. The gel was stained with colloidal Coomassie brilliant blue.

FIG. 5.

Growth curve analysis to determine the role of bhuR in heme utilization. (A) BHI broth; (B) BHI(EDDHA); (C) BHI(EDDHA) plus 5 μM hemin. The error bars represent the standard deviations obtained during three independent growth experiments. +, 4169rif(pUFR047); ○, 4169rif(bhuR::kan)(pUFR047); ▴, 4169rif(bhuR::kan)(pERM33).

FIG. 6.

End point growth analysis of 4169rif and 4169rif(bhuR::kan) in Fe-depleted media supplemented with various heme-containing Fe sources. Control, BHI (EDDHA); Fe, BHI(EDDHA) plus 144 μM FeSO₄; Hemin, BHI(EDDHA) plus 5 μM heme; tHb, BHI(EDDHA) plus 1.25 μM turkey hemoglobin; hHb, BHI(EDDHA) plus 1.25 μM human hemoglobin; Myo, BHI(EDDHA) plus 5 μM myoglobin; Cat, BHI(EDDHA) plus 5 μM catalase. With the exception of FeSO₄, the concentration of each Fe source was equivalent to 5 μM Fe. The mean (±1 standard deviation from the mean, as indicated by the error bars) is shown for each value. An asterisk indicates that the growth of 4169rif(bhuR::kan) was significantly different (P < 0.05) from the growth of 4169rif cultured under identical conditions, as determined by Student's t test.

FIG. 7.

B. avium transports heme into the cell. (A) BHI(100 μM EDDHA); (B) BHI(100 μM EDDHA) supplemented with 50 μM ALA; (C) BHI(100 μM EDDHA) plus 5 μM hemin; (D) BHI(100 μM EDDHA) plus 10 μM turkey hemoglobin. The error bars represent the standard deviations obtained during three independent growth experiments. •, 4169rif; ▴, 4169rif(bhuR::kan); ○, 4169rifhemA; ▵, 4169rif(bhuR::kan)hemA.