Immunostimulatory activity of inactivated environmental Bacillus isolates and their endospores

Abstract The spore-forming capacity of Bacillus spp. enables environmental persistence and stable product formulations, yet the interactions of environmental Bacillus spores and vegetative cells with the human immune system are not fully understood. We investigated the immunostimulatory potential of...

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Main Authors: Max Dekeukeleire, Dieter Vandenheuvel, Tippapron Khondee, Lize Delanghe, Tim Van Rillaer, Sofie Thys, Jean-Pierre Timmermans, Sarah Lebeer, Irina Spacova
Format: Article
Language:English
Published: Nature Portfolio 2025-08-01
Series:Scientific Reports
Online Access:https://doi.org/10.1038/s41598-025-12833-7
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author Max Dekeukeleire
Dieter Vandenheuvel
Tippapron Khondee
Lize Delanghe
Tim Van Rillaer
Sofie Thys
Jean-Pierre Timmermans
Sarah Lebeer
Irina Spacova
author_facet Max Dekeukeleire
Dieter Vandenheuvel
Tippapron Khondee
Lize Delanghe
Tim Van Rillaer
Sofie Thys
Jean-Pierre Timmermans
Sarah Lebeer
Irina Spacova
author_sort Max Dekeukeleire
collection DOAJ
description Abstract The spore-forming capacity of Bacillus spp. enables environmental persistence and stable product formulations, yet the interactions of environmental Bacillus spores and vegetative cells with the human immune system are not fully understood. We investigated the immunostimulatory potential of seven environmental Bacillus isolates (B. subtilis, B. velezensis, B. licheniformis, B. pumilus) and optimized their inactivation methods to preserve surface integrity and immunostimulatory activity. Inactivation was evaluated using heat/UV-C and 10–100% formalin, followed by scanning electron microscopy and human reporter cell assays (THP1-Dual monocytes, HEK-Blue hTLR2/6 and hTLR4). Heat/UV-C treatment retained Bacillus morphology and enhanced immune activation compared to 10–100% formalin treatment. Spores and vegetative cells activated key immune transcription factors (nuclear factor kappa B and interferon regulatory factors) in human monocytes, with spores inducing 1.5- to 8-fold lower responses compared to vegetative cells, and strain- and species-dependent effects observed. All tested Bacillus isolates significantly activated Toll-like receptor TLR2/6, but not TLR4. Genome analysis identified Bacillus spore envelope components (SpsAEIK, CotBCGQ) that may influence differences in immune responses. Our findings improve understanding of Bacillus-human immune interactions, revealing strain- and species-specific immunostimulatory activity. These results support further exploration of Bacillus isolates for immune-related and environmental applications.
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spelling doaj-art-7b4368a1a42e453397a7ee6237bb66082025-08-24T11:28:33ZengNature PortfolioScientific Reports2045-23222025-08-0115111610.1038/s41598-025-12833-7Immunostimulatory activity of inactivated environmental Bacillus isolates and their endosporesMax Dekeukeleire0Dieter Vandenheuvel1Tippapron Khondee2Lize Delanghe3Tim Van Rillaer4Sofie Thys5Jean-Pierre Timmermans6Sarah Lebeer7Irina Spacova8Laboratory of Applied Microbiology and Biotechnology (LAMB), Department of Bioscience Engineering, University of AntwerpLaboratory of Applied Microbiology and Biotechnology (LAMB), Department of Bioscience Engineering, University of AntwerpLaboratory of Applied Microbiology and Biotechnology (LAMB), Department of Bioscience Engineering, University of AntwerpLaboratory of Applied Microbiology and Biotechnology (LAMB), Department of Bioscience Engineering, University of AntwerpLaboratory of Applied Microbiology and Biotechnology (LAMB), Department of Bioscience Engineering, University of AntwerpLaboratory of Cell Biology and Histology (CHB), Department of Pharmaceutical, Biomedical and Veterinary Sciences, University of AntwerpLaboratory of Cell Biology and Histology (CHB), Department of Pharmaceutical, Biomedical and Veterinary Sciences, University of AntwerpLaboratory of Applied Microbiology and Biotechnology (LAMB), Department of Bioscience Engineering, University of AntwerpLaboratory of Applied Microbiology and Biotechnology (LAMB), Department of Bioscience Engineering, University of AntwerpAbstract The spore-forming capacity of Bacillus spp. enables environmental persistence and stable product formulations, yet the interactions of environmental Bacillus spores and vegetative cells with the human immune system are not fully understood. We investigated the immunostimulatory potential of seven environmental Bacillus isolates (B. subtilis, B. velezensis, B. licheniformis, B. pumilus) and optimized their inactivation methods to preserve surface integrity and immunostimulatory activity. Inactivation was evaluated using heat/UV-C and 10–100% formalin, followed by scanning electron microscopy and human reporter cell assays (THP1-Dual monocytes, HEK-Blue hTLR2/6 and hTLR4). Heat/UV-C treatment retained Bacillus morphology and enhanced immune activation compared to 10–100% formalin treatment. Spores and vegetative cells activated key immune transcription factors (nuclear factor kappa B and interferon regulatory factors) in human monocytes, with spores inducing 1.5- to 8-fold lower responses compared to vegetative cells, and strain- and species-dependent effects observed. All tested Bacillus isolates significantly activated Toll-like receptor TLR2/6, but not TLR4. Genome analysis identified Bacillus spore envelope components (SpsAEIK, CotBCGQ) that may influence differences in immune responses. Our findings improve understanding of Bacillus-human immune interactions, revealing strain- and species-specific immunostimulatory activity. These results support further exploration of Bacillus isolates for immune-related and environmental applications.https://doi.org/10.1038/s41598-025-12833-7
spellingShingle Max Dekeukeleire
Dieter Vandenheuvel
Tippapron Khondee
Lize Delanghe
Tim Van Rillaer
Sofie Thys
Jean-Pierre Timmermans
Sarah Lebeer
Irina Spacova
Immunostimulatory activity of inactivated environmental Bacillus isolates and their endospores
Scientific Reports
title Immunostimulatory activity of inactivated environmental Bacillus isolates and their endospores
title_full Immunostimulatory activity of inactivated environmental Bacillus isolates and their endospores
title_fullStr Immunostimulatory activity of inactivated environmental Bacillus isolates and their endospores
title_full_unstemmed Immunostimulatory activity of inactivated environmental Bacillus isolates and their endospores
title_short Immunostimulatory activity of inactivated environmental Bacillus isolates and their endospores
title_sort immunostimulatory activity of inactivated environmental bacillus isolates and their endospores
url https://doi.org/10.1038/s41598-025-12833-7
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