Internalization of OspA in rsCD14 complex and aggregated forms

Area of Science:

• Immunology
• Cell Biology
• Microbial Pathogenesis

Background:

• The spirochetal protein OspA activates immune cells via CD14 and Toll-like receptor 2 (TLR2).
• Cellular handling of TLR2 receptor complex ligands like OspA remains poorly understood.
• Understanding OspA internalization is crucial for deciphering its immune-stimulating mechanisms.

Purpose of the Study:

• To investigate the cellular internalization mechanisms of the OspA lipoprotein and its lipohexapeptide.
• To compare OspA uptake with that of acetylated low-density lipoprotein (AcLDL), a known scavenger receptor ligand.
• To determine the effect of serum availability on OspA internalization and localization.

Main Methods:

• Utilized fluorescently derivatized OspA (full-length lipoprotein and lipohexapeptide) and AcLDL.
• Examined internalization in RAW264.7 macrophages and endothelial cells under varying serum conditions.
• Assessed co-localization with cellular organelles (lysosomes, Golgi) and effects of inhibitors (cytochalasin D).

Main Results:

• Both OspA forms were internalized similarly to AcLDL, with competition observed between OspA and AcLDL for uptake.
• OspA co-localized with lysosomes but not the Golgi complex in normal serum conditions.
• Serum starvation altered OspA localization to the Golgi and kept the lipohexapeptide on the cell surface, without affecting TNF induction.

Conclusions:

• OspA internalization pathways resemble those of scavenger receptor ligands like AcLDL.
• Cellular processing of OspA is serum-dependent, influencing its intracellular localization.
• Tumor necrosis factor (TNF) induction by OspA likely occurs via cell surface signaling, independent of internalization.