Staphylococcus aureus fur regulates the expression of virulence factors that contribute to the pathogenesis of pneumonia

Abstract

The tremendous success of Staphylococcus aureus as a pathogen is due to the controlled expression of a diverse array of virulence factors. The effects of host environments on the expression of virulence factors and the mechanisms by which S. aureus adapts to colonize distinct host tissues are largely unknown. Vertebrates have evolved to sequester nutrient iron from invading bacteria, and iron availability is a signal that alerts pathogenic microorganisms when they enter the hostile host environment. Consistent with this, we report here that S. aureus senses alterations in the iron status via the ferric uptake regulator (Fur) and alters the abundance of a large number of virulence factors. These Fur-mediated changes protect S. aureus against killing by neutrophils, and Fur is required for full staphylococcal virulence in a murine model of infection. A potential mechanistic explanation for the impact of Fur on virulence is provided by the observation that Fur coordinates the reciprocal expression of cytolysins and a subset of immunomodulatory proteins. More specifically, S. aureus lacking fur exhibits decreased expression of immunomodulatory proteins and increased expression of cytolysins. These findings reveal that Fur is involved in initiating a regulatory program that organizes the expression of virulence factors during the pathogenesis of S. aureus pneumonia.

FIG. 1.

S. aureus senses iron availability via Fur to modulate the production of exoproteins. (A) Representative growth curves for the S. aureus wild-type (WT) and Δfur (Δfur) strains grown in iron-sufficient medium and for the S. aureus wild-type strain grown in iron-depleted medium (medium containing 400 μM 2,2′ dipyridyl) (WT+DIP). (B) Exoprotein profiles of the S. aureus wild-type (WT) and Δfur (Δfur) strains grown to early exponential phase (OD₅₉₀, ∼0.3), mid-exponential phase (OD₅₉₀, ∼1.0), early stationary phase (OD₅₉₀, ∼1.6), and mid-stationary phase (OD₅₉₀, ∼1.7) in iron-sufficient medium and exoprotein profiles of the S. aureus wild-type strain in iron-depleted medium (medium containing 400 μM 2,2′-dipyridyl) (WT+DIP). The results for culture supernatants were normalized based on optical density and CFU data, and exoproteins were precipitated with TCA, separated using SDS-PAGE, and stained with Coomassie blue. (C) Exoprotein profiles of the S. aureus wild-type strain (WT), the Δfur strain (Δfur), and the Δfur strain transformed with a fur complementation plasmid (Δfur/pfur) grown to mid-exponential phase (OD₅₉₀, ∼1.0) and stationary phase (OD₅₉₀, ∼1.7) in iron-sufficient medium, exoprotein profiles of the S. aureus wild-type strain grown in iron-depleted medium (medium containing 400 μM DIP) (WT+DIP), and exoprotein profiles of the S. aureus wild-type strain grown in medium supplemented with DIP and excess iron chloride (medium containing 400 μM DIP and 45 μM FeCl₃) (WT+DIP+Fe). The results for supernatants were normalized and analyzed as described above for panel B. (D) Exoprotein profiles for different S. aureus strains grown to stationary phase in iron-sufficient medium and iron-limited medium (medium containing 300 μM 2,2′-dipyridyl) analyzed as described above for panel B.

FIG. 2.

S. aureus senses iron availability via Fur to modulate hemolytic and cytotoxic activities. (A) Hemolytic activity exhibited by the S. aureus wild-type strain (WT), the S. aureus Δfur strain (Δfur), and the S. aureus Δfur strain harboring a fur complementation plasmid (Δfur/pfur) after 24 h of growth on sheep blood-TSA plates. (B) Hemolysis of erythrocytes intoxicated with exoproteins harvested from the wild-type and S. aureus Δfur strains grown in iron-sufficient medium, from the wild-type strain grown in iron-limited medium (medium containing 300 μM 2,2′-dipyridyl) (DIP), and from the wild-type strain grown in medium containing DIP and 10 μM iron chloride (DIP+Fe). The data are the means and standard deviations for triplicate determinations. Asterisks indicate statistically significant differences between the samples indicated, as determined by Student's t test (P < 0.05). (C) Hemolytic activity exhibited by the S. aureus wild-type strain (WT), an isogenic strain lacking fur (Δfur), a double-mutant isogenic strain lacking fur and hla (Δfur hla), and the fur hla double-mutant strain harboring an hla complementation plasmid (Δfur hla/phla) after 24 h of growth on sheep blood-TSA plates. (D) Viability of HL-60 mammalian cells intoxicated with staphylococcal exoproteins harvested from the wild-type strain (WT) grown in iron-sufficient and iron-limited medium (DIP) or with exoproteins harvested from S. aureus lacking fur (Δfur). Cell viability was measured using CellTiter (Promega). The data are expressed as percentages determined by comparing the number of viable cells with the number of cells grown with medium alone. The data are the means and standard deviations for triplicate determinations. Asterisks indicate statistically significant differences between the samples indicated as determined by Student's t test (P < 0.05). (E) Exoproteins secreted by the S. aureus wild-type strain (WT) and an isogenic strain lacking fur (Δfur) were collected at exponential phase and late exponential phase, and the data were normalized based on optical density. Exoproteins were precipitated with TCA, separated using SDS-PAGE, and transferred onto nitrocellulose membranes. The membranes were immunoblotted with anti-Hla antibodies (44). A nonspecific band that cross-reacted with the anti-Hla antisera was used as a loading control.

FIG. 3.

2D-DIGE analysis of exoproteins secreted by the S. aureus wild-type strain and an isogenic strain lacking fur: false-color representative gel from a three-gel set containing differentially labeled samples. Secreted proteins from three independent cultures of the S. aureus wild-type strain (WT) and an isogenic fur mutant (Δfur strain) were collected and concentrated, and proteins were labeled with fluorescent dye as described in Materials and Methods. Samples were separated using a pI 4 to 7 gel. For the proteins indicated there were statistically significant differences in expression between the wild-type and Δfur strains, and the proteins were identified by a mass spectrometry-based protein identification method.

FIG. 4.

Global analysis of exoproteins secreted by the S. aureus wild-type strain (WT) and an isogenic strain lacking fur (Δfur). The differences in abundance of exoproteins produced by the S. aureus wild-type and isogenic strains lacking fur were determined by liquid chromatography-MS/MS and spectral counting. Exoprotein profiles were subdivided into profiles for iron acquisition (A), cytotoxins and hemolysins (B), and immunomodulatory proteins (C). The data are the means and standard errors of the means for three independent samples. The statistical significance of differences between the samples was analyzed by Student's t test (*, P < 0.05).

FIG. 5.

Inactivation of fur alters S. aureus virulence. (A) Peritoneal neutrophils were infected with the wild-type (WT) and Δfur strains, and the survival of S. aureus, expressed as a percentage, was determined by plating. The value for the wild-type strain was defined as 100%. The data are the means and standard errors of the means for at least three independent experiments. An asterisk indicates that the value is statistically significantly different from the value for the wild-type strain as determined by Student's t test (P < 0.05). (B) Primary murine peritoneal neutrophils were infected with washed wild-type S. aureus supplemented with medium (−) or with staphylococcal exoproteins from stationary cultures of the wild-type strain (WT) or the isogenic fur mutant strain (Δfur). The S. aureus burden was determined by plating, and the value for the sample supplemented with exoproteins produced by the wild-type strain was defined as 100%. The data are the means and standard errors of the means for at least three independent experiments. An asterisk indicates that the value is statistically significantly different from the value for the sample supplemented with exoproteins produced by the wild-type strain as determined by Student's t test (P < 0.05). (C to F) C57BL/6J mice were infected for 6 and 18 h via the intranasal (i.n.) route with the S. aureus wild-type strain (WT) and an isogenic strain lacking fur (Δfur). (C) Photographs of lungs dissected from uninfected animals (−) and from animals infected with the wild-type strain and the fur mutant for 18 h. (D) C57BL/6J mice were infected with the wild-type strain (3.78 × 10⁸ CFU) or the isogenic strain lacking fur (3.51 × 10⁸ CFU) as described above for panel C. Eighteen hours postinfection lungs were harvested, and the S. aureus burden was measured. Each circle represents one infected animal, and each horizontal line indicates the mean of the log values. The asterisk indicates that the data for the two strains are statistically significantly different as determined by Student's t test (P ≤ 0.05). (E) C57BL/6J animals were not infected (−) or were infected as described above, the lungs were dissected and homogenized, and the infiltration of neutrophils (B220⁻ CD11b⁺ Ly6G⁺) was determined by multiparametric FACS analysis. The data are the means and standard errors of the means for three independent experiments in which at least two animals were used for each experiment. The asterisk indicates that the data for the two strains are statistically significantly different as determined by Student's t test (P < 0.05). (F) C57BL/6J mice were made neutropenic by treatment with IgG2b anti-Gr-1 MAb RB6-8C5 and infected with the wild-type strain (2.9 × 10⁷ CFU) or the isogenic strain lacking fur (1.5 × 10⁷ CFU) as described above for panel C. Eighteen hours postinfection lungs were harvested, and the S. aureus burden was measured. Each circle represents one infected animal, and each horizontal line indicates the mean of the log values. The observed differences were not statistically significant in this analysis (P ≥ 0.05).