Staphylococcus aureus activates type I IFN signaling in mice and humans through the Xr repeated sequences of protein A

Abstract

The activation of type I IFN signaling is a major component of host defense against viral infection, but it is not typically associated with immune responses to extracellular bacterial pathogens. Using mouse and human airway epithelial cells, we have demonstrated that Staphylococcus aureus activates type I IFN signaling, which contributes to its virulence as a respiratory pathogen. This response was dependent on the expression of protein A and, more specifically, the Xr domain, a short sequence-repeat region encoded by DNA that consists of repeated 24-bp sequences that are the basis of an internationally used epidemiological typing scheme. Protein A was endocytosed by airway epithelial cells and subsequently induced IFN-beta expression, JAK-STAT signaling, and IL-6 production. Mice lacking IFN-alpha/beta receptor 1 (IFNAR-deficient mice), which are incapable of responding to type I IFNs, were substantially protected against lethal S. aureus pneumonia compared with wild-type control mice. The profound immunological consequences of IFN-beta signaling, particularly in the lung, may help to explain the conservation of multiple copies of the Xr domain of protein A in S. aureus strains and the importance of protein A as a virulence factor in the pathogenesis of staphylococcal pneumonia.

Figure 1. S. aureus strain USA300 induces type I IFN signaling in airway epithelial cells that is dependent on the expression of protein A.

(A) Primary mouse airway epithelial cells were incubated with S. aureus USA300 or a SpA deletion mutant, and induction of IFN-β, Mx-1, IL-6, LIF, and PKR was detected by real-time PCR. Data are represented as fold increases over unstimulated controls and are representative of 3 independent experiments. *P < 0.05 as compared with the spa-null strain, Student’s t test. Error bars represent standard deviations. (B) p-STAT1, p-STAT2, and p-STAT3 were detected by immunoblot in primary airway epithelial cell cultures treated with USA300 (+) or spa-null (–). Corresponding total STATs are shown for loading controls.

Figure 2. Type I IFN signaling increases susceptibility to staphylococcal respiratory infection.

Adult WT or Ifnar^–/– mice were intranasally infected with (A) 5 × 10⁸ CFUs of S. aureus USA300 and mortality determined at 24 hours. *P = 0.0055, Fisher’s exact test; n = 10 mice per group. Mice were infected with 5 × 10⁷ CFUs of S. aureus USA300 and at 4 and 24 hours after infection (B), bacterial loads were determined in lungs and spleens and lung suspensions and cells obtained by BAL fluid stained and analyzed by flow cytometry to enumerate (C) CD45⁺Gr1⁺ PMNs and (D) CD11c⁺CD11b⁺CD45⁺ DCs. Data are represented as percentage of total CD45 population. (E) TNF-α and IL-6 were detected in BAL fluid and blood by ELISA. Each point represents 1 animal; horizontal lines represent median values.

Figure 3. SpA is internalized and induces type 1 IFN signaling in airway epithelial cells.

(A–E) Airway cells were incubated with SpA or SpA Xr and (A) internalization monitored by flow cytometry (ΔMFI, change in mean fluorescence intensity) or (B) by confocal microscopy for SpA (green) and transferrin (red) at 1 hour (original magnification, ×80). (C) SpA-induced IFN-β production was measured at 2 hours by real-time PCR in the presence of Dynasore (Dyn), (D) in response to transfection of an SpA Xr plasmid (Xr) or empty vector (CV), and (E) in the presence of 50 μg/ml polymixin B. (F) STAT1 and STAT3 phosphorylation in response to SpA (S) or LPS (L) after 1 and 2 hours was monitored in murine nasal epithelial cells from WT or trif^–/– mice. (G) STAT1 and STAT3 phosphorylation was monitored in airway cells in response to SpA Xr (30 min., n = 2; 60 and 120 min., n = 3), in the presence of Dyn (n = 3), pan-JAK inhibitor (Pan, n = 3), JAK2 inhibitor (JAK2, n = 3), or neutralizing antibody to IFN-β (n = 2). Thin vertical lines between bands indicate data spliced together from original blot. Red arrows indicate that the same bands from the 120 min. Xr treatment were used as the control for pan-JAK and JAK2 inhibitor treatments. U, M, or Unstim. indicates unstimulated control. Unless otherwise indicated, data shows mean values of triplicate samples from 1 representative experiment out of 3, and error bars indicate standard deviations. *P < 0.001; **P < 0.05.

Figure 4. SpA Xr induces IFN-β– and STAT3-dependent IL-6 production.

(A) Airway epithelial cells in primary culture were stimulated with SpA Xr for the times indicated or for 2 hours in the absence or presence of dynasore, and IL-6 induction was detected by real-time PCR (black bars, left y axis) or ELISA (white bars, right y axis). Untreated cells or cells stimulated with SpA Xr were incubated with IFN-β–neutralizing antibody, and IL-6 induction was measured by real-time PCR. IL-6 mRNA production is expressed as the fold induction compared with that of unstimulated cells. (B) Airway epithelial cells from WT or Stat1^–/– mice were stimulated with SpA Xr in the absence (control) or presence of the inhibitors indicated, and IL-6 production was measured by real-time PCR. IL-6 induction is expressed as a percentage of that induced by SpA Xr in cells from WT mice. (A and B) Data represent the mean and SD of 3 wells from 1 representative experiment out of 3. *P < 0.05; **P < 0.001, Student’s t test performed on the raw data. Immunoblots demonstrating phosphorylation of STAT3 in airway cells from Stat1^–/– mice and inhibition in the presence of STAT3 inhibitor are shown in B.

Figure 5. SpA Xr activates STAT1/3 in vivo.

(A) C57BL/6 mice were intranasally inoculated with SpA Xr, S. aureus, or PBS (control). 4 hours later, p-STAT1 (Tyr701), p-STAT3 (Tyr705), and β-actin were detected in lung lysates by immunoblot. Data from 2 representative mice out of 6 are shown for both left quadrants and bottom right quadrant. Data from 2 representative mice out of 3 are shown for top right quadrant. The thin vertical line between bands within a group indicate data spliced from the original blot. (B) IL-6 mRNA in the lung of SpA Xr–treated mice was detected by real-time PCR, and the percentage of PMNs among total leukocytes in the lung was determined by flow cytometry following intranasal inoculation of SpA Xr in untreated or JAK inhibitor–treated mice (Inh). Each dot represents an individual mouse, and horizontal lines show the median value in each group. (C) IL-6 mRNA in the lung of S. aureus–infected mice was detected by real-time PCR. The median values of the percentage of PMNs corresponding to those mice are shown.