The extracellular adherence protein (Eap) of Staphylococcus aureus inhibits wound healing by interfering with host defense and repair mechanisms

Abstract

Staphylococcus aureus is a major human pathogen interfering with host-cell functions. Impaired wound healing is often observed in S aureus-infected wounds, yet, the underlying mechanisms are poorly defined. Here, we identify the extracellular adherence protein (Eap) of S aureus to be responsible for impaired wound healing. In a mouse wound-healing model wound closure was inhibited in the presence of wild-type S aureus and this effect was reversible when the wounds were incubated with an isogenic Eap-deficient strain. Isolated Eap also delayed wound closure. In the presence of Eap, recruitment of inflammatory cells to the wound site as well as neovascularization of the wound were prevented. In vitro, Eap significantly reduced intercellular adhesion molecule 1 (ICAM-1)-dependent leukocyte-endothelial interactions and diminished the consequent activation of the proinflammatory transcription factor nuclear factor kappaB (NFkappaB) in leukocytes associated with a decrease in expression of tissue factor. Moreover, Eap blocked alphav-integrin-mediated endothelial-cell migration and capillary tube formation, and neovascularization in matrigels in vivo. Collectively, the potent anti-inflammatory and antiangiogenic properties of Eap provide an underlying mechanism that may explain the impaired wound healing in S aureus-infected wounds. Eap may also serve as a lead compound for new anti-inflammatory and antiangiogenic therapies in several pathologies.

Figure 1.

Detection of Eap in wounds and effect of Eap on wound healing. (A) Eap detection in S aureus–infected wounds. (B) Eap was detected by Western blot analysis in extracts of noninfected (lane 1) or S aureus–infected (lane 2) wounds. Molecular mass markers are indicated along the right margin. (C) The course of wound closure is shown in the absence (buffer; •), or in the presence of wild-type S aureus (▪) or the Eap-deficient strain (□). *P < .05 compared with buffer-treated wounds; #P < .05 compared with wounds that received the Eap-deficient strain; **P < .05 compared with buffer-treated wounds. (D) The course of wound closure is shown in the absence (•) or presence of 20 μg Eap (▪) or protein A (▵). *P < .05 compared with buffer-treated wounds. Wound closure is expressed relative to the wound size of each wound at day 0 that represents 100%. Data are expressed as mean ± SD (n = 8).

Figure 2.

Inhibition of leukocyte infiltration into wounds by Eap. (A-B) Typical photomicrographs of immunohistochemical staining for the detection of neutrophils (anti-MPO) at day 1 following wound generation in the absence (A; buffer) or presence of 20 μg Eap (B) is shown. (C) The expression of MPO at day 1 following wound generation as assessed by Western blot is shown in the absence (□) or in the presence (▪) of Eap. The insert demonstrates a typical blot with staining for MPO. Densitometric data are expressed relative to control (buffer-treated wounds), and are means ± SD of 3 separate experiments. (D-E) Typical photomicrographs of immunohistochemical staining for the detection of macrophages (anti-F4/80) at day 5 following wound generation in the absence (D; buffer) or presence (E) of Eap is shown. (F) The expression of tissue factor at day 5 after wound generation as assessed by Western blot analysis is shown in the absence (□) or presence (▪) of Eap. The insert demonstrates a typical blot. Densitometric data are expressed relative to control (buffer-treated wounds), and are means ± SD of 3 separate experiments.

Figure 3.

Influence of Eap on leukocyte–endothelial-cell interactions and on the activity of NFκB. (A) Adhesion of human neutrophils to endothelial cells is shown in the absence (–) or presence of blocking mAb to CD18, ICAM-1, and CD31 as indicated (each 20 μg/mL), Eap, or protein A (each 20 μg/mL). Cell adhesion is expressed relative to control (in the absence of competitor). Data are means ± SD (n = 3) of a typical experiment; similar results were obtained in 3 separate experiments. (B) The transendothelial migration of human neutrophils in response toward 50 ng/mL MCP-1 is shown in the absence (–) or presence of mAb to CD18, mAb to ICAM-1, mAb to CD29 (each 20 μg/mL), Eap, or protein A (each 20 μg/mL). Neutrophils and inhibitors were coincubated during the whole course of the transmigration experiment (□), or neutrophils were incubated on the endothelial cells for 20 minutes in the absence of competitors in order to facilitate their initial attachment on the endothelial surface, and inhibitors were added thereafter into the wells (▪). Transmigration is presented as percent of control (in the absence of competitor). Data are means ± SD (n = 3) of a typical experiment; similar results were obtained in 3 separate experiments. (C) The DNA-binding activity of NFκB without or with TNFα or TNFα plus Eap in THP-1 cells is shown in the absence (□) or presence () of ICAM-1 (10 μg/mL), as indicated. The insert demonstrates a typical EMSA for NFκB DNA-binding activity (1 indicates control; 2, TNF-α; 3, TNF-α+Eap; 4, ICAM-1; 5, ICAM-1+TNFα; and 6, ICAM-1+TNFα+Eap). (D) The expression of tissue factor without or with TNFα or TNFα plus Eap in THP-1 cells is shown in the absence (□) or presence () of ICAM-1 (10 μg/mL). Densitometric data are expressed relative to control (100% control is represented in the absence of stimuli or competitors), and are means ± SD of 2 separate experiments. *P < .05.

Figure 4.

Eap inhibits neovascularization in wound healing. (A) Typical photomicrographs (× 100 magnification) of immunohistochemical staining for the detection of PECAM-1 (CD31) in association with blood vessels at day 5 following wound generation in the absence (buffer) or presence of 20 μg Eap is shown. Negative control (first antibody omitted) is also shown. (B) Statistical evaluation of the density of CD31-positive blood vessels (expressed as number of vessels/field) in wound sections at day 5 following wound generation in the absence (buffer) or presence of 20 μg Eap is shown. Evaluation of 7 fields per wound from 8 wounds per treatment group is shown. (C) Mice were anesthetized and fluorescent microbeads were injected. The recovered fluorescence per weight of wound tissue is shown, and data expressed relative to control (buffer-treated wounds) are means ± SD of 10 wounds. *P < .01 compared with control.

Figure 5.

Eap interferes with α_vβ₃-integrin–dependent cell function. (A) VEGF (20 ng/mL)–stimulated migration of HUVECs toward VN, FBG, or FN (each 5 μg/mL) is shown in the absence (▪) or presence of the blocking mAb against α_vβ₃-integrin, LM609 (for VN and FBG), the blocking mAb against β₁-integrin, 6S6 (for FN) (20 μg/mL; □), or in the presence of Eap (;20 μg/mL), or protein A (;20 μg/mL). Cell migration is expressed relative to control, which is represented as cell migration in the absence of any stimulus or competitor. Data are means ± SD of 4 experiments performed in triplicate. *P < .05 compared with VEGF-stimulated migration in the absence of competitor. ns indicates nonsignificant. (B-D) Binding of VN to immobilized α_vβ₃-integrin (B), binding of FBG to immobilized α_vβ₃-integrin (C), and binding of FN to immobilized α₅β₁-integrin(D) was performed in the absence (–; ▪) or presence of blocking mAb against α_vβ₃-integrin, LM609 (for VN and FBG), the blocking mAb against β₁-integrin, 6S6 (for FN) (20 μg/mL; □), or increasing concentrations of Eap () as indicated. Specific binding is expressed as absorbance at 405 nm. Data are means ± SD (n = 3) of a typical experiment; similar results were observed in 3 separate experiments.

Figure 6.

Effect of Eap on endothelial-cell proliferation and capillary sprout formation. (A) HUVEC proliferation. HUVECs were incubated without (□) or with (▪) VEGF (10 ng/mL) in the absence (–) or presence of increasing concentrations of Eap as indicated. Proliferation of HUVECs is expressed as a percentage of control, defined as cell proliferation in the absence of any stimulus or competitor. Data are means ± SD (n = 3) of 1 experiment typical of 3 separate experiments performed. (B-C) Capillary sprout formation. (B) HUVECs were incubated for 24 hours in the absence (–; □) or presence (▪) of VEGF (25 ng/mL) with or without increasing concentrations of Eap, as indicated. Capillary-like tube formation is expressed as capillary sprout length in micrometers. (C) HUVECs were incubated for 24 hours in the presence of VEGF (25 ng/mL) with or without (–) Eap, protein A (each 20 μg/mL), cyclic RGD peptide (cRGD; 20 μg/mL), or the mAb against α_vβ₃-integrin, LM609 (20 μg/mL), as indicated. Capillary-like tube formation is expressed relative to control, which is represented as sprout formation in the presence of VEGF and in the absence of any competitor. Data are means ± SD (n = 10) of a representative experiment. Similar results were obtained in 3 independent experiments.

Figure 7.

Inhibition of angiogenesis by Eap in the in vivo matrigel plug assay. (A) Neovascularization in the matrigel plug assay was performed without (buffer) or with VEGF (100 ng/mL) in the absence or presence of 10 μg or 30 μg Eap as indicated. Photographs of a typical experiment performed in triplicates are shown; similar results were obtained in 3 separate experiments. (B) The quantitation of neovascularization in the matrigels was performed by measuring the hemoglobin concentration. Hemoglobin concentration was expressed as milligrams of hemoglobin per grams of wet tissue. Data are expressed as a percentage of the maximum (VEGF treatment in the absence of competitors). Data are means ± SD (n = 3) of a typical experiment; similar results were observed in 3 separate experiments.