Structure-based minimization of a high-mobility group protein B1 (HMGB1) B box peptide uncouples anti-NETotic activity and anti-biofilm function
- 1Center for Microbe and Immunity Research, Abigail Wexner Research Institute at Nationwide Children’s Hospital
- 2Department of Pediatrics, College of Medicine, Ohio State University
Abstract
Introduction: We previously showed that human HMGB1 has anti-biofilm activity localized to a 97-amino-acid B-Box-derived peptide (mB Box-97), engineered with a C106S mutation to reduce HMGB1’s proinflammatory properties while retaining anti-biofilm function. This peptide also suppresses NETosis by inhibiting neutrophil protein kinase C (PKC) activity. Here, we tested whether the anti-biofilm and anti-NETotic activities of mB Box-97 could be separated into distinct shorter peptides by constructing truncated variants containing Helix 1 of the B-Box (with the C106S mutation), with or without the A Box–B Box linker region, and by introducing mutations at key DNA-binding residues.
Methods: Anti-NETotic activity was assessed in human neutrophils, including peptide binding to PKC, effects on PKC activity, and downstream NADPH oxidase (NOX) activation, reactive oxygen species (ROS) generation, and NETosis. Biofilm disruption and bacterial growth effects were tested against pathogenic bacteria.
Results: Peptides combining the linker with Helix 1 and C106S significantly inhibited NETosis while preserving anti-biofilm activity, via PKC binding/inhibition, reduced NOX activation, and lower ROS. Removing the linker preserved anti-biofilm activity but abolished anti-NETotic activity, yielding a 29-amino-acid anti-biofilm-only peptide. Conversely, adding DNA-binding mutations to the linker–Helix 1 peptide abolished anti-biofilm activity while retaining anti-NETotic function.
Conclusions: Anti-NETotic and anti-biofilm activities of HMGB1-derived peptides can be structurally separated: the linker plus Helix 1 are required for anti-NETotic activity, while Helix 1 alone suffices for anti-biofilm activity. These compact 42- and 29-amino-acid peptides are readily synthesizable, promising candidates for biofilm- and NET-mediated inflammatory diseases, and further support PKC as a target for NETosis inhibition.
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BibTeX
@article{more2026structurebased,
title = {{Structure-based minimization of a high-mobility group protein B1 (HMGB1) B box peptide uncouples anti-NETotic activity and anti-biofilm function}},
author = {Kunal R. More and Jaime D. Rhodes and Lauren O. Bakaletz and Steven D. Goodman},
journal = {Frontiers in Immunology},
year = {2026},
month = oct,
doi = {10.3389/fimmu.2026.1970789},
url = {https://www.frontiersin.org/articles/10.3389/fimmu.2026.1970789/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Structure-based minimization of a high-mobility group protein B1 (HMGB1) B box peptide uncouples anti-NETotic activity and anti-biofilm function
AU - Kunal R. More
AU - Jaime D. Rhodes
AU - Lauren O. Bakaletz
AU - Steven D. Goodman
PY - 2026
DA - 2026/10/16
JO - Frontiers in Immunology
DO - 10.3389/fimmu.2026.1970789
UR - https://www.frontiersin.org/articles/10.3389/fimmu.2026.1970789/full
AB - Introduction: We previously showed that human HMGB1 has anti-biofilm activity localized to a 97-amino-acid B-Box-derived peptide (mB Box-97), engineered with a C106S mutation to reduce HMGB1’s proinflammatory properties while retaining anti-biofilm function. This peptide also suppresses NETosis by inhibiting neutrophil protein kinase C (PKC) activity. Here, we tested whether the anti-biofilm and anti-NETotic activities of mB Box-97 could be separated into distinct shorter peptides by constructing truncated variants containing Helix 1 of the B-Box (with the C106S mutation), with or without the A Box–B Box linker region, and by introducing mutations at key DNA-binding residues. Methods: Anti-NETotic activity was assessed in human neutrophils, including peptide binding to PKC, effects on PKC activity, and downstream NADPH oxidase (NOX) activation, reactive oxygen species (ROS) generation, and NETosis. Biofilm disruption and bacterial growth effects were tested against pathogenic bacteria. Results: Peptides combining the linker with Helix 1 and C106S significantly inhibited NETosis while preserving anti-biofilm activity, via PKC binding/inhibition, reduced NOX activation, and lower ROS. Removing the linker preserved anti-biofilm activity but abolished anti-NETotic activity, yielding a 29-amino-acid anti-biofilm-only peptide. Conversely, adding DNA-binding mutations to the linker–Helix 1 peptide abolished anti-biofilm activity while retaining anti-NETotic function. Conclusions: Anti-NETotic and anti-biofilm activities of HMGB1-derived peptides can be structurally separated: the linker plus Helix 1 are required for anti-NETotic activity, while Helix 1 alone suffices for anti-biofilm activity. These compact 42- and 29-amino-acid peptides are readily synthesizable, promising candidates for biofilm- and NET-mediated inflammatory diseases, and further support PKC as a target for NETosis inhibition.
SN - 1664-3224
ER -