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Induction of macrophage-mediated production of tumor necrosis factor alpha by an L-form derived from Staphylococcus
K Kuwano1, A Akashi, I Matsu-ura
1Department of Microbiology, Kurume University School of Medicine, Japan.
Abstract:
We investigated the capability of an L-form derived from Staphylococcus aureus to induce tumor necrosis factor alpha (TNF-alpha) production in murine peritoneal macrophages. The activity for TNF-alpha induction was found in the membrane fraction of the L-form but not in the cytoplasmal fraction purified by the sucrose step gradient centrifugation. TNF-alpha mRNA was also detected in macrophages stimulated with L-form membranes. L-form induced TNF-alpha production in macrophages from both lipopolysaccharide-responsive and -unresponsive mouse strains. Regardless of the presence of polymyxin B, the activity of TNF-alpha induction of L-form was mostly found in the phenol layer, but not in the aqueous layer, both of which were prepared by phenol extraction method. Fractions of L-form membranes representing molecular masses of approximately between 29 and 36 kDa were primarily responsible for inducing the production of TNF-alpha consistently. Moreover, this stimulatory effect was abolished by digestion with Streptomyces griseus protease. In Western blot (immunoblot) analysis with anti-lipoteichoic acid antibody, two bands (65 and 45 kDa) were observed in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phenol layer, whereas one band (14 kDa) was observed in either the aqueous layer or lipoteichoic acid of S. aureus. These results suggest that the component in the membrane of the L-form, distinct from cell wall components such as teichoic acid or lipopolysaccharide, possesses the capability to stimulate TNF-alpha production by macrophages.
Insights
Staphylococcus aureus L-forms can trigger tumor necrosis factor alpha (TNF-alpha) production in macrophages. This activity originates from specific membrane components, distinct from typical bacterial cell wall molecules.
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- Staphylococcus aureus L-forms are cell wall-deficient bacteria with altered pathogenic potential.
- Tumor necrosis factor alpha (TNF-alpha) is a key cytokine in inflammatory responses.
- Understanding microbial triggers of TNF-alpha is crucial for immune modulation.
Purpose of the Study:
- To identify the bacterial components responsible for TNF-alpha induction by Staphylococcus aureus L-forms.
- To investigate the cellular location and molecular characteristics of the TNF-alpha inducing factor.
Main Methods:
- Isolation and fractionation of Staphylococcus aureus L-forms.
- Stimulation of murine peritoneal macrophages with L-form fractions.
- Detection of TNF-alpha mRNA and protein.
- Biochemical characterization including phenol extraction and protease digestion.
- Western blot analysis using anti-lipoteichoic acid antibodies.
Main Results:
- TNF-alpha induction activity was localized to the L-form membrane fraction.
- Stimulation occurred in macrophages from both LPS-responsive and -unresponsive mice.
- The active component was primarily in the phenol layer after extraction and had a molecular mass of 29-36 kDa.
- Protease digestion abolished the stimulatory effect.
- Western blot analysis indicated the active component is distinct from lipoteichoic acid.
Conclusions:
- A specific component within the Staphylococcus aureus L-form membrane, not typical cell wall components, induces TNF-alpha production.
- This membrane-associated factor plays a role in macrophage activation by L-forms.
- Further characterization of this novel immune-stimulatory molecule is warranted.