Hydrophobin-mediated uptake of posaconazole by Aspergillus fumigatus conidia

Ref ID: 19174

Author:

R. Cerone*, P. Campoli, A.S. Kristof, D.S. Perlin, D.C. Sheppard

Author address:

Montreal, CA; New Jersey, US

Full conference title:

23rd European Congress of Clinical Microbiology and
Infectious Diseases

Date: 27 April 2014

Abstract:

Objectives: The incidence of invasive aspergillosis (IA) has risen significantly in recent years and IA remains a major cause of mortality in high risk hematology patients. Posaconazole (PCZ) prophylaxis has proven to be highly efficacious in preventing IA despite relatively low serum concentrations. The efficacy of this medical triazole has been linked to high tissue concentrations, as lipophilic PCZ concentrates within host cell membranes to levels exceeding those necessary to inhibit fungal growth. However, the mechanism by which host cell-associated PCZ prevents the development of invasive aspergillosis remains largely unknown. Since IA is initiated by the inhalation of hydrophobic conidia, we hypothesized that the PCZ might bind to conidial hydrophobins, and that this binding could contribute to the unique efficacy of this agent in prophylaxis.
Methods: Fluorescent PCZ was synthesized via conjugation with the flurorophore BODIPY (BDP-PCZ). In order to elucidate whether PCZ is transferred from host cells to fungal cells, A549 pulmonary epithelial cells were exposed to BDP-PCZ and co-incubated with Aspergillus fumigatus conidia examined using confocal microscopy. To determine conidial binding of PCZ, conidia were exposed to free BDP-PCZ, washed and analyzed by flow cytometry. To study the effects of conidial hydrophobins, an A. fumigatus mutant deficient in the hydrophobin RodA and which expressed red fluorescent protein was engineered.
Results: Using confocal microscopy, BDP-PCZ was observed to transfer from the cellular membrane of A549 cells to the fungal membrane of wild-type conidia. This transfer was contact dependent, and occurred extremely rapidly (<5 minutes). In contrast, infection of BDP-PCZ cells with a hydrophobin deficient mutant required prolonged incubation (>2 hrs) for transfer of BDP-PCZ to conidia to be observed. This reduced binding of BDP-PCZ was also observed with free drug. Using flow cytometric analysis, conidia of wild-type A. fumigatus bound BDP-PCZ to over 10 fold higher levels than the hydrophobin deficient strain.
Conclusion: These results suggest that conidial cell wall hydrophobins facilitate the fungal uptake of PCZ. Since hydrophobins are largely produced by conidia, these hydrophobic interactions may contribute to the unique efficacy of PCZ in preventing IA.

Abstract Number: P1000

Conference Year: 2013

Link to conference website: http://registration.akm.ch/einsicht.php?XNABSTRACT_ID=166235&XNSPRACHE_ID=2&XNKONGRESS_ID=180&XNMASK

New link: NULL

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