Early Events in Macrophage Killing of Aspergillus fumigatus Conidia: New Flow Cytometric Viability Assay

AUTOR(ES)

Marr, Kieren A.

FONTE

American Society for Microbiology

RESUMO

Detailed investigations of macrophage phagocytosis and killing of Aspergillus fumigatus conidia have been limited by technical difficulties in quantifying fungal uptake and viability. In order to study early events in cell pathogen ingestion and killing, we developed a new flow cytometry assay that utilizes the fungus-specific viability dye FUN-1. Metabolically active A. fumigatus conidia accumulate orange fluorescence in vacuoles, while dormant or dead conidia stain green. After incubation within THP-1 cells, recovered conidia are costained with propidium iodide (PI) to discriminate between dormant and dead cells. Flow cytometric measurements of FUN-1 metabolism and PI uptake provide indicators of conidial viability, dormancy, and death. Conidial phagocytosis and killing are also assessed by measurement of green and orange FUN-1 fluorescence within the THP-1 cell population. Compared to previously described methods, this assay has less error introduced by membrane permeability changes and serial dilution of filamentous fungal forms. Results suggest that the THP-1 cells kill conidia rapidly (within 6 h) after exposure. Conidia that are preexposed to human serum are ingested and killed more quickly than are nonopsonized conidia.

Documentos Relacionados

• Lack of involvement of nitric oxide in killing of Aspergillus fumigatus conidia by pulmonary alveolar macrophages.
• Accumulation of Amphotericin B in Human Macrophages Enhances Activity against Aspergillus fumigatus Conidia: Quantification of Conidial Kill at the Single-Cell Level
• Binding of human fibronectin to Aspergillus fumigatus conidia.
• Binding of extracellular matrix proteins to Aspergillus fumigatus conidia.
• Killing Activity of Micafungin against Aspergillus fumigatus Hyphae Assessed by Specific Fluorescent Staining for Cell Viability