Blad-Containing Oligomer Fungicidal Activity on Human Pathogenic Yeasts. From the Outside to the Inside of the Target Cell.

Ana M Pinheiro,Sara A Monteiro,Filipe Rollo,Rui Fernandes,Alexandra Carreira,Ricardo B Ferreira

doi:10.3389/fmicb.2016.01803

Abstract

Blad polypeptide comprises residues 109–281 of Lupinus albus β-conglutin precursor. It occurs naturally as a major subunit of an edible, 210 kDa oligomer which accumulates to high levels, exclusively in the cotyledons of Lupinus seedlings between the 4th and 14th day after the onset of germination. Blad-containing oligomer (BCO) exhibits a potent and broad spectrum fungicide activity toward plant pathogens and is now on sale in the US under the tradename FractureTM. In this work we demonstrate its antifungal activity toward human pathogens and provide some insights on its mode of action. BCO bioactivity was evaluated in eight yeast species and compared to that of amphotericin B (AMB). BCO behaved similarly to AMB in what concerns both cellular inhibition and cellular death. As a lectin, BCO binds strongly to chitin. In addition, BCO is known to possess ‘exochitinase’ and ‘endochitosanase’ activities. However, no clear disruption was visualized at the cell wall after exposure to a lethal BCO concentration, except in cell buds. Immunofluorescent and immunogold labeling clearly indicate that BCO enters the cell, and membrane destabilization was also demonstrated. The absence of haemolytic activity, its biological origin, and its extraordinary antifungal activity are the major outcomes of this work, and provide a solid background for a future application as a new antifungal therapeutic drug. Furthermore, its predictable multisite mode of action suggests a low risk of inducing resistance mechanisms, which are now a major problem with other currently available antifungal drugs.

Highlights

IntroductionFungal infections have become an important factor of morbidity and mortality and represent an increasing burden on medical systems (Del Poeta, 2010; Huffnagle and Noverr, 2013), being associated with unavoidable high mortality rates, similar to those caused by tuberculosis or malaria (Brown et al, 2012).Candida species are regarded as common components of the body microbiota in healthy humans (Kathiravan et al, 2012) but are responsible for candidaemia, an invasive fungal infection associated with substantial morbidity, mortality and healthcare costs (Zaoutis et al, 2005;A New Natural Fungicidal OligomerBassetti et al, 2015), being among the top ten pathogens causing bloodstream infections
The antifungal activity of three different batches of the Blad-containing oligomer (BCO) was evaluated in six strains belonging to six Candida species and in one strain of C. neoformans, with each strain cultured in two different growth media
C. albicans CBS 562 was tested for resistance to amphotericin B (AMB), one commonly used antifungal drug

Summary

Introduction

Fungal infections have become an important factor of morbidity and mortality and represent an increasing burden on medical systems (Del Poeta, 2010; Huffnagle and Noverr, 2013), being associated with unavoidable high mortality rates, similar to those caused by tuberculosis or malaria (Brown et al, 2012).Candida species are regarded as common components of the body microbiota in healthy humans (Kathiravan et al, 2012) but are responsible for candidaemia, an invasive fungal infection associated with substantial morbidity, mortality and healthcare costs (Zaoutis et al, 2005;A New Natural Fungicidal OligomerBassetti et al, 2015), being among the top ten pathogens causing bloodstream infections. Fungal infections have become an important factor of morbidity and mortality and represent an increasing burden on medical systems (Del Poeta, 2010; Huffnagle and Noverr, 2013), being associated with unavoidable high mortality rates, similar to those caused by tuberculosis or malaria (Brown et al, 2012). C. albicans still remains the most abundant and significant species associated with the disease, other medically important species of Candida are rising (Klepser, 2011; Papon et al, 2013; Sardi et al, 2013; Won et al, 2015) including C. glabrata, C. rugosa, C. parapsilosis, C. tropicalis, C. dubliniensis, C. krusei, and C. lusitaniae ( Kathiravan et al, 2012; Huffnagle and Noverr, 2013; León et al, 2014). Just a few new antifungal agents were unveiled and they were mainly based on the structural modification of already discovered drugs (Rubbiani et al, 2016)

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