A Chimeric Peptide Derived from a Bacterial Effector Protein Attenuates TLR-2/4-Mediated Production of Pro-Inflammatory Cytokines and Enhances the Cellular Availability of Gentamicin
Binita Roy Nandi,1,2 Biswaranjan Patra,1,2 Girish K Radhakrishnan1 1Laboratory of Bacterial Pathogenesis, National Institute of Animal Biotechnology, Hyderabad, Telangana, 500032, India; 2Regional Centre for Biotechnology (RCB), Faridabad, 121001, IndiaCorrespondence: Girish K Radhakrishnan, Email g...
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2025-08-01
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| author | Nandi BR Patra B Radhakrishnan GK |
| author_facet | Nandi BR Patra B Radhakrishnan GK |
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| description | Binita Roy Nandi,1,2 Biswaranjan Patra,1,2 Girish K Radhakrishnan1 1Laboratory of Bacterial Pathogenesis, National Institute of Animal Biotechnology, Hyderabad, Telangana, 500032, India; 2Regional Centre for Biotechnology (RCB), Faridabad, 121001, IndiaCorrespondence: Girish K Radhakrishnan, Email girish@niab.org.inIntroduction: Toll-like receptors (TLRs) are critical components of innate immunity, recognizing microbe-derived molecules and triggering pro-inflammatory cytokine production for pathogen clearance. However, TLR hyperactivation can cause excessive inflammation, contributing to disorders such as sepsis. Thus, modulating TLR signalling is a promising therapeutic strategy. The intracellular bacterial pathogen, Brucella, encodes TcpB, a cell-permeable anti-inflammatory protein that inhibits TLR2/4 signalling by targeting adaptor proteins TIRAP and MyD88. TcpB has an N-terminal phospholipid-binding motif conferring cell permeability and a C-terminal TIR domain for TLR interference. This study explores TcpB-derived peptides and evaluates their potential for therapeutic and research applications.Methods: We synthesized TcpB-derived peptides and evaluated their cytotoxicity, cell permeability, and anti-inflammatory effects using LDH assay, fluorescence microscopy, ELISA, and qRT-PCR. A chimeric peptide, TB4-BBL2, was then generated and evaluated for cellular uptake, interaction with TLR adaptors, and functional activities. Binding of FAM-labelled TB4-BBL2 to FLAG-tagged TIRAP/MYD88 was confirmed via co-immunoprecipitation and fluorescence analysis, while degradation of adaptors was assessed by Western blotting. The anti-inflammatory efficacy of TB4-BBL2 was evaluated in a murine endotoxemia model by measuring serum TNF-α and IL-6 levels using ELISA, and cytokine transcript levels in major organs using qRT-PCR. Additionally, TcpB-derived peptides conjugated to gentamicin were analyzed for enhanced intracellular delivery, bacterial clearance, and anti-inflammatory effects using CFU enumeration, ELISA, and qRT-PCR in both in vitro and in vivo.Results: TB4-BBL2 peptide was efficiently internalized by macrophages via endocytosis and promoted the degradation of TIRAP and MYD88, thereby attenuating TLR2/4-mediated pro-inflammatory cytokine production and oxidative stress. In vivo, TB4-BBL2 treatment resulted in over a 70% reduction in serum TNF-α levels, which was also reflected by decreased transcript levels in spleen tissue. Conjugation of TcpB-derived peptides to gentamicin enhanced its cell permeability and facilitated efficient elimination of intracellular bacteria. This was corroborated by enhanced bacterial clearance in CFU assays, showing over a 2-Log reduction in vivo along with more than an 80% decrease in splenic TNF-α transcripts in the murine model of brucellosis.Discussion: Our findings highlight the therapeutic potential of TcpB-derived peptides in modulating TLR-mediated inflammation and enhancing intracellular antibiotic delivery. This dual-functional strategy offers a promising novel approach for treating inflammatory diseases and persistent intracellular infections, warranting further investigation toward clinical applications. Keywords: inflammation, TLR2/4, pro-inflammatory cytokines, inflammatory disorders, peptide-based drugs, Brucella, endotoxemia, cell-permeable antibiotics |
| format | Article |
| id | doaj-art-8ff8c810a7c640c794fb37a7b8c87279 |
| institution | Kabale University |
| issn | 1178-7031 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Dove Medical Press |
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| series | Journal of Inflammation Research |
| spelling | doaj-art-8ff8c810a7c640c794fb37a7b8c872792025-08-20T03:40:25ZengDove Medical PressJournal of Inflammation Research1178-70312025-08-01Volume 18Issue 11075110775105626A Chimeric Peptide Derived from a Bacterial Effector Protein Attenuates TLR-2/4-Mediated Production of Pro-Inflammatory Cytokines and Enhances the Cellular Availability of GentamicinNandi BR0Patra B1Radhakrishnan GK2Laboratory of Bacterial Pathogenesis, Department of Infectious DiseasesLaboratory of Bacterial Pathogenesis, Department of Infectious DiseasesLaboratory of Bacterial Pathogenesis, Department of Infectious DiseasesBinita Roy Nandi,1,2 Biswaranjan Patra,1,2 Girish K Radhakrishnan1 1Laboratory of Bacterial Pathogenesis, National Institute of Animal Biotechnology, Hyderabad, Telangana, 500032, India; 2Regional Centre for Biotechnology (RCB), Faridabad, 121001, IndiaCorrespondence: Girish K Radhakrishnan, Email girish@niab.org.inIntroduction: Toll-like receptors (TLRs) are critical components of innate immunity, recognizing microbe-derived molecules and triggering pro-inflammatory cytokine production for pathogen clearance. However, TLR hyperactivation can cause excessive inflammation, contributing to disorders such as sepsis. Thus, modulating TLR signalling is a promising therapeutic strategy. The intracellular bacterial pathogen, Brucella, encodes TcpB, a cell-permeable anti-inflammatory protein that inhibits TLR2/4 signalling by targeting adaptor proteins TIRAP and MyD88. TcpB has an N-terminal phospholipid-binding motif conferring cell permeability and a C-terminal TIR domain for TLR interference. This study explores TcpB-derived peptides and evaluates their potential for therapeutic and research applications.Methods: We synthesized TcpB-derived peptides and evaluated their cytotoxicity, cell permeability, and anti-inflammatory effects using LDH assay, fluorescence microscopy, ELISA, and qRT-PCR. A chimeric peptide, TB4-BBL2, was then generated and evaluated for cellular uptake, interaction with TLR adaptors, and functional activities. Binding of FAM-labelled TB4-BBL2 to FLAG-tagged TIRAP/MYD88 was confirmed via co-immunoprecipitation and fluorescence analysis, while degradation of adaptors was assessed by Western blotting. The anti-inflammatory efficacy of TB4-BBL2 was evaluated in a murine endotoxemia model by measuring serum TNF-α and IL-6 levels using ELISA, and cytokine transcript levels in major organs using qRT-PCR. Additionally, TcpB-derived peptides conjugated to gentamicin were analyzed for enhanced intracellular delivery, bacterial clearance, and anti-inflammatory effects using CFU enumeration, ELISA, and qRT-PCR in both in vitro and in vivo.Results: TB4-BBL2 peptide was efficiently internalized by macrophages via endocytosis and promoted the degradation of TIRAP and MYD88, thereby attenuating TLR2/4-mediated pro-inflammatory cytokine production and oxidative stress. In vivo, TB4-BBL2 treatment resulted in over a 70% reduction in serum TNF-α levels, which was also reflected by decreased transcript levels in spleen tissue. Conjugation of TcpB-derived peptides to gentamicin enhanced its cell permeability and facilitated efficient elimination of intracellular bacteria. This was corroborated by enhanced bacterial clearance in CFU assays, showing over a 2-Log reduction in vivo along with more than an 80% decrease in splenic TNF-α transcripts in the murine model of brucellosis.Discussion: Our findings highlight the therapeutic potential of TcpB-derived peptides in modulating TLR-mediated inflammation and enhancing intracellular antibiotic delivery. This dual-functional strategy offers a promising novel approach for treating inflammatory diseases and persistent intracellular infections, warranting further investigation toward clinical applications. Keywords: inflammation, TLR2/4, pro-inflammatory cytokines, inflammatory disorders, peptide-based drugs, Brucella, endotoxemia, cell-permeable antibioticshttps://www.dovepress.com/a-chimeric-peptide-derived-from-a-bacterial-effector-protein-attenuate-peer-reviewed-fulltext-article-JIRInflammationTLR2/4pro-inflammatory cytokinesinflammatory disorderspeptide-based drugsBrucella |
| spellingShingle | Nandi BR Patra B Radhakrishnan GK A Chimeric Peptide Derived from a Bacterial Effector Protein Attenuates TLR-2/4-Mediated Production of Pro-Inflammatory Cytokines and Enhances the Cellular Availability of Gentamicin Journal of Inflammation Research Inflammation TLR2/4 pro-inflammatory cytokines inflammatory disorders peptide-based drugs Brucella |
| title | A Chimeric Peptide Derived from a Bacterial Effector Protein Attenuates TLR-2/4-Mediated Production of Pro-Inflammatory Cytokines and Enhances the Cellular Availability of Gentamicin |
| title_full | A Chimeric Peptide Derived from a Bacterial Effector Protein Attenuates TLR-2/4-Mediated Production of Pro-Inflammatory Cytokines and Enhances the Cellular Availability of Gentamicin |
| title_fullStr | A Chimeric Peptide Derived from a Bacterial Effector Protein Attenuates TLR-2/4-Mediated Production of Pro-Inflammatory Cytokines and Enhances the Cellular Availability of Gentamicin |
| title_full_unstemmed | A Chimeric Peptide Derived from a Bacterial Effector Protein Attenuates TLR-2/4-Mediated Production of Pro-Inflammatory Cytokines and Enhances the Cellular Availability of Gentamicin |
| title_short | A Chimeric Peptide Derived from a Bacterial Effector Protein Attenuates TLR-2/4-Mediated Production of Pro-Inflammatory Cytokines and Enhances the Cellular Availability of Gentamicin |
| title_sort | chimeric peptide derived from a bacterial effector protein attenuates tlr 2 4 mediated production of pro inflammatory cytokines and enhances the cellular availability of gentamicin |
| topic | Inflammation TLR2/4 pro-inflammatory cytokines inflammatory disorders peptide-based drugs Brucella |
| url | https://www.dovepress.com/a-chimeric-peptide-derived-from-a-bacterial-effector-protein-attenuate-peer-reviewed-fulltext-article-JIR |
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