IsdB antibody–mediated sepsis following S. aureus surgical site infection
Kohei Nishitani, … , John L. Daiss, Edward M. Schwarz
Kohei Nishitani, … , John L. Daiss, Edward M. Schwarz
Published October 2, 2020
Citation Information: JCI Insight. 2020;5(19):e141164. https://doi.org/10.1172/jci.insight.141164.
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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 2

Development of a nonneutralizing anti-IsdB mAb.

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Development of a nonneutralizing anti-IsdB mAb. Mice (n = 5) were immuni...
Mice (n = 5) were immunized with recombinant IsdB protein (rIsdB), and their spleen cells were used to make hybridomas, which were screened for anti-IsdB antibodies via ELISA, as described in Methods. Sixteen anti-rIsdB antibody–producing hybridoma pools were obtained for single-cell cloning. (A) Twelve anti-IsdB hybridoma cell lines were successfully established and further screened to assess their cross-reactivity with IsdA and IsdH via ELISA. (B and C) To identify a lead “nonneutralizing” anti-IsdB mAb, which has high avidity to rIsdB without disrupting rIsdB binding to hemoglobin (Hb), we performed sandwich ELISA studies that assessed mAb inhibition of rIsdB binding to Hb and hemin versus an irrelevant anti–S. aureus amidase (Amd) 1.11 isotype mAb control. Based on these results, anti-IsdB 1.5 was selected as the promising non-neutralizing mAb, whereas anti-IsdB is a 1.12 neutralizing mAb. (D) To assess mAb-binding capacity to native IsdB, ELISA was performed with bacterial extract from lysostaphin and lysozyme-digested S. aureus, which demonstrated dose-dependent binding of all 3 mAbs versus mouse IgG1 negative control. (E) Western blot analysis also confirmed specific anti-IsdB 1.5 mAb binding to 83 kDa rIsdB (lane 1) and 80 kDa endogenous IsdB (arrowhead) in S. aureus lysostaphin and lysozyme extract (lane 2, USA300ΔSpa surface protein extract; lane 3, UAMS-1ΔSpa surface protein extract). (F) To further confirm IsdB specificity, lysostaphin and lysozyme protein extract from USA300 ΔSpa was immunoprecipitated with anti-IsdB 1.5 mAb–protein G beads, separated via reducing SDS-PAGE, and the 80 kDa reverse stain band was excised, digested with trypsin, and analyzed by mass spectrometry. The results identified 70 unique peptide sequences (Supplemental Table 1), which cover 70.7% of IsdB (green-labeled sequence; O = oxidized amino acid).