title: Collagenases in an ether extract of bacterial metabolites used as an immunostimulator induces TNF-α and IFN-γ.
abstract: Non-specific immunostimulation by bacterial extracts and their components are widely accepted for the prevention and treatment of several infectious diseases. An ether extract of the metabolites of ß-streptococcus, Staphylcoccus albus, Staphylcoccus aureus, Escherichia coli, Haemophilus influenza, Moraxella caterhalis, Salmonella typhi (standard O & H), Salmonella paratyphi (A & B) and Diptheroid bacilli along with bile lipids is used as a licensed drug for immunostimulation. While characterizing the drug, we observed gelatinolytic/collagenolytic activity in the ether extract by zymography. This activity was contributed by each bacterial species as observed by collagen zymography of individual extract. Immuno-blot also confirmed the presence of collagenases in the pooled extract whose activity was estimated to be 0.081 U/ml ± 0.005 by DQ-gelatin assay. The enzyme was purified by immuno-affinity chromatography. Homogeneity of the preparation was demonstrated by SDS-PAGE and SE-HPLC. Degradation of collagen by purified collagenases was visualized by atomic force microscopy and transmission electron microscopy wherein, fragmentation of collagen leading to loss of network structure occurred under physiological conditions. Results indicated that purified collagenases can trigger the release of pro-inflammatory cytokines TNF-α and IFN-γ in-vitro and in-vivo without inducing detectable stress and toxicity on both models. The findings suggest that bacterial collagenases remain stable and biological functional in an organic solvent validating its potential for industrial and medical applications as the enzymes are key regulators of inflammatory and immune responses.
authors.affiliation: Division of Structural Biology and Bioinformatics, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata 700032, India. Electronic address: namratasingh819@gmail.com.
authors.forename: Namrata
authors.initials: N
authors.lastname: Singh
authors.affiliation: Division of Structural Biology and Bioinformatics, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata 700032, India. Electronic address: debasish@iicb.res.in.
authors.forename: Debasish
authors.initials: D
authors.lastname: Bhattacharyya
mesh_terms: D000276:Adjuvants, Immunologic / Q000008:administration & dosage* / Q000009:adverse effects / Q000302:isolation & purification; D000818:Animals; D001419:Bacteria / Q000201:enzymology / Q000276:immunology*; D001426:Bacterial Proteins / Q000276:immunology* / Q000302:isolation & purification; D002460:Cell Line; D003094:Collagen / Q000378:metabolism* / Q000648:ultrastructure; D017364:Collagenases / Q000008:administration & dosage* / Q000009:adverse effects / Q000302:isolation & purification; D005260:Female; D006801:Humans; D007114:Immunization; D007163:Immunosorbent Techniques; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism; D017737:Macrophages, Peritoneal / Q000276:immunology* / Q000382:microbiology; D051379:Mice; D008807:Mice, Inbred BALB C; D018625:Microscopy, Atomic Force; D046529:Microscopy, Electron, Transmission; D051381:Rats; D017207:Rats, Sprague-Dawley; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
chemical_list: D000276:Adjuvants, Immunologic; D001426:Bacterial Proteins; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D003094:Collagen; D017364:Collagenases
keywords: Bacterial metabolites; Collagenases; Ether extraction; Immunostimulation; TNF-α and IFN-γ