Objectives: Infections due to Aspergillus fumigatus remain a critical concern in high risk hematology patients. Posaconazole (PCZ) prophylaxis has proven highly effective in preventing these infections, despite relatively low serum concentrations. We have previously shown that PCZ concentrates within host cell membranes to a high enough concentration to both inhibit growth and preventfungal damage. However, the specific subcellular location of membrane associated PCZ within host and fungal cells is unknown. The objectives of this study are to determine where PCZ localizes in both epithelial cells and fungi in order to better understand its mechanism of action. Methods: Fluorescent PCZ was synthesized by conjugation with the fluorophore BOPIDY (BOPIDY-PCZ). A549 epithelial cells were exposed to varying concentrations of BOPIDY-PCZ and examined using confocal microscopy. Cells were costained with DAPI and an endoplasmic reticulum (ER) specific anti-ERP57 antibody to facilitate visualization of cell microstructures. In parallel, these experiments were also conducted with A. fumigatus hyphae. Finally, to confirm the specificity of these findings, competitive inhibition assays using unlabelled PCZ and voriconazole (VCZ) were performed. Results: A549 epithelial cells exposed to BOPIDY-PCZ exhibited increased total cell fluorescence, centred around the peri-nuclear area. Similar findings were observed with hyphae of A. fumigatus, with increased fluorescence localizing to the perinuclear area, corresponding to the location of the ER. To confirm this finding, A549 cells were immunostained for ER protein 57 (ERP57). Staining for this protein demonstrated co-localization with BOPIDY-PCZ in the perinuclear region, suggesting that posaconazole concentrates within the membrane rich endoplasmic reticulum. Co-culture of host and fungal cells with BOPIDY-PCZ and unlabelled PCZ resulted in a decreased in fluorescence of the cells, while the addition of VCZ had little effect on ER concentration of PCZ, suggesting that the concentration of PCZ within the ER membranes is specific to this azole. Conclusion: These results suggest that PCZ concentrates specifically to the endoplasmic reticulum within host and fungal cells. Since the target of PCZ, CYP51A, is found within the ER, this finding suggests that the ability of PCZ to concentrate to high concentrations in the endoplasmic reticulum may contribute to its antifungal activity.
Full conference title:
22nd European Congress of Clinical Microbiology and Infectious Diseases
- ECCMID 22nd (2012)