IsdB antibody–mediated sepsis following S. aureus surgical site infection
Kohei Nishitani, Masahiro Ishikawa, Yugo Morita, Noriaki Yokogawa, Chao Xie, Karen L. de Mesy Bentley, Hiromu Ito, Stephen L. Kates, John L. Daiss, Edward M. Schwarz
Kohei Nishitani, Masahiro Ishikawa, Yugo Morita, Noriaki Yokogawa, Chao Xie, Karen L. de Mesy Bentley, Hiromu Ito, Stephen L. Kates, John L. Daiss, Edward M. Schwarz
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Research Article Bone biology Infectious disease

IsdB antibody–mediated sepsis following S. aureus surgical site infection

Abstract

Staphylococcus aureus is prevalent in surgical site infections (SSI) and leads to death in approximately 1% of patients. Phase IIB/III clinical trial results have demonstrated that vaccination against the iron-regulated surface determinant protein B (IsdB) is associated with an increased mortality rate in patients with SSI. Thus, we hypothesized that S. aureus induces nonneutralizing anti-IsdB antibodies, which facilitate bacterial entry into leukocytes to generate “Trojan horse” leukocytes that disseminate the pathogen. Since hemoglobin (Hb) is the primary target of IsdB, and abundant Hb-haptoglobin (Hb-Hp) complexes in bleeding surgical wounds are normally cleared via CD163-mediated endocytosis by macrophages, we investigated this mechanism in vitro and in vivo. Our results demonstrate that active and passive IsdB immunization of mice renders them susceptible to sepsis following SSI. We also found that a multimolecular complex containing S. aureus protein A–anti-IsdB–IsdB–Hb-Hp mediates CD163-dependent bacterial internalization of macrophages in vitro. Moreover, IsdB-immunized CD163–/– mice are resistant to sepsis following S. aureus SSI, as are normal healthy mice given anti-CD163–neutralizing antibodies. These genetic and biologic CD163 deficiencies did not exacerbate local infection. Thus, anti-IsdB antibodies are a risk factor for S. aureus sepsis following SSI, and disruption of the multimolecular complex and/or CD163 blockade may intervene.

Authors

Kohei Nishitani, Masahiro Ishikawa, Yugo Morita, Noriaki Yokogawa, Chao Xie, Karen L. de Mesy Bentley, Hiromu Ito, Stephen L. Kates, John L. Daiss, Edward M. Schwarz

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Figure 5

All components of the multimolecular complex physically associate and are required for anti-IsdB antibody–mediated S. aureus internalization of macrophages.

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All components of the multimolecular complex physically associate and ar...
(A) A schematic illustration of the 5 proteins in the multimolecular complex with their hypothesized orientation is shown. In this model, anti-IsdB antibody attaches to the bacterial surface via Spa-Fc binding and to soluble IsdB via Fab binding. Antibody-bound IsdB protein also binds to Hb-Hp, such that all 5 proteins are physically associated in the multimolecular complex. (B) Immunoprecipitation-Western blotting was performed to demonstrate specific protein interactions. Coomassie blue–stained SDS-PAGE is shown to illustrate the purity of the input proteins. Lane 1, anti-IsdB mAb; lane 2, recombinant IsdB; lane 3, hemoglobin (Hb); lane 4, haptoglobin (Hp); and lane, 5, Hb-Hp complex. (C) For immunoprecipitation, a combination of anti-IsdB mAb or irrelevant IgG control mAb (50 μg/mL), recombinant IsdB (40 μg/mL), Hb (25 μg/mL), and Hp (35 μg/mL) were incubated with S. aureus protein A–coupled (Spa-coupled) beads. Eluates were assessed for Hp content via Western blot with anti-Hp antibody. Note that Hp detection in this immunoprecipitation-Western assay requires a multimolecular complex that includes Spa, anti-IsdB mAb, IsdB, and the Hb-Hp complex, as omission of any of these proteins results in the loss of detection. In vitro S. aureus internalization assays were performed to quantify GFP+ S. aureus (UAMS-1) in LysoTracker Red–stained primary bone marrow–derived macrophages incubated with the indicated multimolecular complex proteins. Representative fluorescent micrographs were obtained 3 hours later (original magnification, ×10) (D–J) or after 48 hours culture in gentamicin (scale bar: 100 μm) (K and L). (M) Quantification of bacterial internalization was performed via Visiopharm (Supplemental Figure 1), and the data are presented with the mean ± SD. In the protein A–negative group, the UAMS-1ΔSpa strain was used instead of UAMS-1 to show that Spa is required for multimolecular complex–mediated bacterial endocytosis (n = 5, ****P < 0.0001 vs. all other groups via 1-way ANOVA with post hoc Tukey’s test).