Passively released heme from hemoglobin and myoglobin is a potential source of nutrient iron for Bordetella bronchiseptica

Abstract

Colonization by Bordetella bronchiseptica results in a variety of inflammatory respiratory infections, including canine kennel cough, porcine atrophic rhinitis, and a whooping cough-like disease in humans. For successful colonization, B. bronchiseptica must acquire iron (Fe) from the infected host. A vast amount of Fe within the host is sequestered within heme, a metalloporphyrin which is coordinately bound in hemoglobin and myoglobin. Utilization of hemoglobin and myoglobin as sources of nutrient Fe by B. bronchiseptica requires expression of BhuR, an outer membrane protein. We hypothesize that hemin is acquired by B. bronchiseptica in a BhuR-dependent manner after spontaneous loss of the metalloporphyrin from hemoglobin and/or myoglobin. Sequestration experiments demonstrated that direct contact with hemoglobin or myoglobin was not required to support growth of B. bronchiseptica in an Fe-limiting environment. Mutant myoglobins, each exhibiting a different affinity for heme, were employed to demonstrate that the rate of growth of B. bronchiseptica was directly correlated with the rate at which heme was lost from the hemoprotein. Finally, Escherichia coli cells expressing recombinant BhuR had the capacity to remove hemin from solution. Collectively, these experiments provided strong experimental support for the model that BhuR is a hemin receptor and B. bronchiseptica likely acquires heme during infection after passive loss of the metalloporphyrin from hemoglobin and/or myoglobin. These results also suggest that spontaneous hemin loss by hemoglobin and myoglobin may be a common mechanism by which many pathogenic bacteria acquire heme and heme-bound Fe.

FIG. 1.

B. bronchiseptica RB50 utilizes a variety of hemoproteins as sources of nutrient Fe. Fe-stressed strain RB50 and the isogenic bhuR mutant RBB4 were inoculated into BHI broth supplemented with 500 μM EDDHA containing 5 μM hemin or an equimolar concentration (with respect to hemin) of hemoproteins. Relative cell densities of the cultures grown to stationary phase were determined spectrophotometrically by measuring the absorbance at 600 nm. All conditions were replicated in triplicate. The error bars indicate one standard deviation from the mean. Fe+, untreated BHI broth; Fe−, BHI broth supplemented with 500 μM EDDHA; pHb, porcine Hb; rHb, rabbit Hb; hHb, human Hb; swMb, sperm whale Mb; cCyto c, canine cytochrome c; bCat, bovine catalase.

FIG. 2.

Direct cellular contact with Hb or Mb is not required to support growth of RB50. Flasks containing 20 ml of Fe-depleted BHI broth supplemented with 500 μM EDDHA were inoculated with a 1:100 dilution of stationary-phase, Fe-stressed RB50. A 1-ml aliquot of a solution containing an equimolar amount (5 μM with respect to Fe) of hemin, human Hb (hHb), or sperm whale Mb (Mb) or a control solution without Fe supplementation (500 mM EDDHA) (−Fe) was added to a 3,000-molecular-weight cutoff dialysis bag which was suspended in the culture. After overnight incubation at 37°C, the cell density of each culture was determined spectrophotometrically by measuring the absorbance at 600 nm. All conditions were replicated in triplicate. The error bars indicate one standard deviation from the mean.

FIG. 3.

Hemin-dependent growth of RB50 is dependent upon spontaneous loss of hemin from Mb. Ten milliliters of Fe-depleted BHI broth supplemented with 500 μM EDDHA containing either no supplement (−Fe), hemin, wild-type Mb (Mb), Mb^H64L, or Mb^V68T was inoculated with a 1:100 dilution of stationary-phase Fe-stressed RB50. The relative affinities of the Mb for heme were Mb^V68T > Mb > Mb^H64L (24, 25, 27). The cell densities of the stationary-phase cultures were determined spectrophotometrically at a wavelength of 600 nm at various time points. All conditions were replicated in triplicate. The error bars indicate one standard deviation from the mean.

FIG. 4.

Recombinant BhuR is surface exposed in BL21(DE3)(pMOX14.1) and adsorbs hemin from solution. (A) Absorption of hemin from solution by recombinant BhuR. Stationary-phase BL21(DE3)(pET21a) and BL21(DE3)(pMOX14.1) were added to a solution of 20 μM hemin dissolved in 10% DMSO and PBS (pH 7.4) (final DMSO concentration, 2%). After 1 h of incubation, cells were removed from the suspension, and the amount of hemin remaining in solution was determined spectrophotometrically at a wavelength of 400 nm. The data are the averages of three independent experiments with three replicates per experiment (n = 9). The differences in the absorbance values of BL21(DE3)(pET21a) and BL21(DE3)(pMOX14.1) are highly significant (three asterisks, P < 0.001). (B) Recombinant BhuR is expressed in the outer membrane of BL21(DE3)(pMOX14.1). Ten micrograms of membranes isolated from BL21(DE3)(pET21a) and BL21(DE3)(pMOX14.1) was resolved in a 12.5% SDS-PAGE gel. An immunoblot of the gel was probed with a 1:500 dilution of anti-BhuR antiserum. Lane 1, total membranes of BL21(DE3)(pET21a); lane 2, total membranes of BL21(DE3)(pMOX14.1); lane 3, enriched outer membranes of BL21(DE3)(pET21a); lane 4, enriched outer membranes of BL21(DE3)(pMOX14.1). The arrow indicates the position of BhuR. Molecular masses (in kilodaltons) are indicated on the left. (C) BhuR expressed by BL21(DE3)(pMOX14.1) is susceptible to digestion by extracellular trypsin. BL21(DE3)(pMOX14.1) cells were digested with increasing amounts of trypsin (1, 10, 100, and 1,000 U). Recombinant cells adjusted to an OD₆₀₀ of 0.8 were pelleted and resuspended in 1-ml portions of the trypsin solutions. Cells were digested at 37°C for 15 min. The trypsin in the cell suspensions was neutralized with a protease inhibitor cocktail. After the reaction mixtures were diluted with electrophoresis sample buffer, equivalent amounts of solubilized cells were resolved on a 12.5% SDS-PAGE gel, and the proteins were immunoblotted with a 1:500 dilution of anti-BhuR antiserum. (D) Treatment of BL21(DE3)(pMOX14.1) with trypsin did not degrade periplasmic MBP. BL21(DE3)(pET21a) and BL21(DE3)(pMOX14.1) cells were digested with increasing amounts of trypsin (1, 10, 100, and 1,000 U) for 15 min at 37°C. Proteins in the cells were resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted with a 1:2,000 dilution of anti-MBP antiserum (New England Biolabs).