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Isolation of Pneumocystis carinii gp120 by fibronectin affinity: evidence for manganese dependence
P Wisniowski1, R Pasula, W J Martin
1Indiana University School of Medicine, Department of Internal Medicine, Indianapolis.
Abstract:
Pneumocystis carinii is a major opportunistic lung pathogen and a leading cause of death among patients with the human immunodeficiency virus. Adherence of P. carinii to type I alveolar epithelial cells is essential for growth and replication and has been shown to be mediated in part by fibronectin (Fn). To better understand the mechanisms underlying this attachment, P. carinii-Fn interaction was characterized with respect to divalent and monovalent ion concentration and pH using an 125I-Fn binding assay to P. carinii. The results suggest that P. carinii has a receptor for Fn that was partially dependent on Ca2+, enhanced by Mn2+, and diminished somewhat by Mg2+. Additional data demonstrated that P. carinii-Fn interaction was sensitive to ionic strength. The pH profile revealed that P. carinii-Fn interaction increased with decreasing pH. The results from the binding assay provided the rationale for a simple isolation of the Fn receptor from P. carinii using a Fn-affinity column involving nondenaturing conditions. The isolated receptor appeared highly purified by SDS-PAGE analysis, with apparent molecular weights of 114 to 118 kD and 66 kD. Western blot analysis indicated that this receptor was gp120, a major surface glycoprotein of P. carinii. Furthermore, the isolated receptor inhibited Fn binding to P. carinii. Finally, a monoclonal antibody raised against the affinity-purified gp120 blocked Fn binding to P. carinii.
Insights
Pneumocystis carinii attaches to lung cells via fibronectin (Fn). Researchers identified gp120 as the specific receptor responsible for this interaction, crucial for understanding infections in immunocompromised patients.
Area of Science:
- Medical Mycology
- Immunology
- Cell Biology
Background:
- Pneumocystis carinii is an opportunistic pathogen causing fatal pneumonia in HIV/AIDS patients.
- P. carinii adherence to alveolar cells is vital for its proliferation and is partly mediated by fibronectin (Fn).
Purpose of the Study:
- To investigate the molecular mechanisms of P. carinii-fibronectin interaction.
- To identify the specific receptor on P. carinii responsible for fibronectin binding.
Main Methods:
- Utilized an 125I-Fn binding assay to analyze P. carinii-Fn interactions under varying ionic conditions and pH.
- Employed Fn-affinity chromatography for receptor isolation.
- Characterized the isolated receptor using SDS-PAGE and Western blot analysis.
- Tested the inhibitory effect of the isolated receptor and a monoclonal antibody on Fn binding.
Main Results:
- P. carinii-Fn binding is influenced by divalent cations (Ca2+, Mn2+, Mg2+), ionic strength, and pH.
- A P. carinii surface glycoprotein, gp120, was identified as the fibronectin receptor.
- The isolated gp120 and an anti-gp120 monoclonal antibody effectively inhibited P. carinii-Fn binding.
Conclusions:
- P. carinii utilizes its surface glycoprotein gp120 to bind fibronectin.
- Understanding this interaction is critical for developing therapeutic strategies against P. carinii infections, particularly in HIV/AIDS patients.