Specificity for human hemoglobin enhances Staphylococcus aureus infection

Abstract

Iron is required for bacterial proliferation, and Staphylococcus aureus steals this metal from host hemoglobin during invasive infections. This process involves hemoglobin binding to the cell wall of S. aureus, heme extraction, passage through the cell envelope, and degradation to release free iron. Herein, we demonstrate an enhanced ability of S. aureus to bind hemoglobin derived from humans as compared to other mammals. Increased specificity for human hemoglobin (hHb) translates into an improved ability to acquire iron and is entirely dependent on the staphylococcal hemoglobin receptor IsdB. This feature affects host-pathogen interaction as demonstrated by the increased susceptibility of hHb-expressing mice to systemic staphylococcal infection. Interestingly, enhanced utilization of human hemoglobin is not a uniform property of all bacterial pathogens. These results suggest a step in the evolution of S. aureus to better colonize the human host and establish hHb-expressing mice as a model of S. aureus pathogenesis.

Figure 1. S. aureus displays increased binding of hHb as compared to mHb

(A-D) Iron-starved S. aureus strain Newman were incubated with hemoglobin at the indicated concentrations and washed. Captured hemoglobin was eluted, subjected to SDS-PAGE, and silver stained. Representative images are shown in Figure S1B. Bound hemoglobin was quantified based on the relative intensity of Hb bands. Relative quantities of cell-wall bound hemoglobin are expressed as percent of hHb bound by (A) wild type, (B) ΔisdB, and (C) ΔisdB + pisdB. (D) ΔisdB harboring the indicated plasmids were incubated with hemoglobin at 10 μg/ml. Insert below panel is an image of an anti-IsdB immunoblot, demonstrating cell wall IsdB expression. Means and statistical significance were calculated based on logarithmically transformed fractions. Error bars represent confidence intervals (α = 0.05); asterisks denote quantities of bound mHb statistically different from hHb supplemented at the same conditions (Student’s two-tailed t-test, P<0.05). In panel (D) # denotes quantities that are significantly different from hHb and mHb bound by ΔisdB + pisdB (wt). Each graph is a result of three independent experiments.

Figure 2. hHb promotes S. aureus replication in iron-limiting conditions

(A and B) Growth of S. aureus Newman wild type (A) and ΔisdB (B) in liquid medium supplemented with 5 μg/ml hemoglobin as a sole source of iron was measured based on optical density at 600 nm (OD₆₀₀) over 72 hours. The graphs represent a mean of three independent experiments. Error bars represent standard deviation; asterisks denote OD₆₀₀ values upon hHb supplementation significantly different from values upon mHb supplementation at the same time point (Student’s two-tailed t-test, P<0.05). (C) Petri dishes containing iron-restrictive agar were streaked with bacterial cultures. Disks impregnated with 10 μg of hemoglobin were placed on top of the agar and S. aureus growth surrounding the disks was monitored over 72 hours. Opaque gray zones around disks indicate zone of growth. The images are a representative of five independent experiments.

Figure 3. Mice expressing human hemoglobin exhibit increased susceptibility to S. aureus

Number of colony forming units (CFU) of S. aureus Newman isolated from organs of systemically infected C57BL/6J and α^Hβ^A mice 96 hours post-inoculation as determined by serial dilution. Data were logarithmically transformed prior to statistical analyses. Horizontal bars represent the average values of CFU/organ, boxes represent standard deviation. Asterisks denote significantly different values (Student’s two-tailed t-test, P<0.05). The graphs represent combined data acquired from multiple independent experiments.

Figure 4. Bacterial pathogens vary in preference of hHb over mHb

(A and B) Binding of hHb and mHb by S. aureus strains was assessed as in Figure 1. (C and D) Petri dishes containing iron-restrictive agar were streaked with (C) strains of S. aureus, and (D) other bacterial pathogens. Disks impregnated with 10 μg of hHb or mHb were placed on top of the agar and bacterial growth surrounding the disks was measured. The graphs depict growth on mHb as a percentage of growth on hHb in the same conditions (growth on hHb = 100%). The graphs represent a mean of three to four independent experiments. Means and statistical significance were calculated based on logarithmically transformed fractions. Error bars represent confidence intervals (α= 0.05); asterisks denote growth on mHb that is statistically different from growth on hHb supplemented at the same conditions (Student’s two-tailed t-test, P<0.05).