Resolving the Mechanism of Anti-Fungal Therapeutics

ABOVE: Neutron reflectivity profile illustrating the effect of AmB on supported membranes formed from P. pastoris lipid extracts. P. pastoris h-lipids before (black symbols) and after 1 mM AmB injection in D2O (red squares), 66% D2O (green circles) and H2O (blue diamonds). IMAGE: A. de Ghellinck et al.

LUND—Treating patients with serious fungal and parasitic infections remains an uphill battle for medical professionals due to the limited number of effective drug treatments available today. Part of the issue lies in the poorly understood mechanism of the action existing drugs have on fungus.

 

A recent study of the potent, broad-spectrum drug Amphotericin B (AmB), however, shows promise to more accurately explain how the drug alters and disrupts fungal membrane function with sometimes toxic side effects in humans. The investigation was conducted by scientists from the Institut Laue Langevin (ILL) in France, the University of Copenhagen, the French national research agencies CNRS and CEA, the Université Libre de Bruxelles, and the European Spallation Source (ESS).

 

ESS instrument scientist Hanna Wacklin. PHOTO courtesy of Hanna Wacklin

In-situ measurements of the yeast Pichia pastoris were made using the instrument FIGARO, a neutron reflectometer at ILL. It was found that a higher presence of polyunsaturated lipids in the fungal membrane significantly increases the efficacy of the AmB drug. Moreover, this phospholipid composition—and not only the drug interaction with ergosterol—may be the conditional factor that allows the drug to structurally modify the membrane. A major component of fungal membranes, the plant sterol ergosterol, has been widely regarded as the key target for anti-fungal drug development for decades. The study results also showed changes to ergosterol-free membranes, suggesting that the AmB-ergosterol interaction is not completely specific.

 

“Amphotericin B represents a class of pharmaceuticals whose mechanism is difficult to investigate due to the complex nature of the cell membrane environment,” says ESS Instrument Scientist for Neutron Reflectometry Hanna Wacklin, who was involved in the study. “By using neutron reflectometry we could probe the location of the drug in the presence of a realistic membrane target extracted from fungal cells at the same time as observing the structural changes induced in the lipid matrix.”

 

An animation showing the orientation of AmB corresponding to the layer thickness in an ergosterol-containing h-lipid membrane. IMAGE: A. de Ghellinck et al.

 

The neutron scattering experiment on FIGARO revealed the density profile of the fungal membrane from hydrogenated and deuterated samples containing varying amounts of ergosterol. This was the first time a measurement of the structure of a native-like lipid membrane under physiologically relevant conditions has been made, and one of the first studies utilising per-deuterated lipids extracted at their natural occurrence from yeast cells.

 

The research suggests that anti-fungal drug AmB may be improved with the addition of polyunsaturated lipids to the prescribed formula. “Our current investigations are now taking the next step by using selective deuteration of the lipid components and ergosterol to understand their individual roles in antibiotic susceptibility and resistance in fungi, as well as the toxic side effects of AmB,” says Wacklin.