Blad-containing oligomer: a novel fungicide used in crop protection as an alternative treatment for tinea pedis and tinea versicolor.

PURPOSE: The lack of novel antifungal drugs and the increasing incidence and severity of fungal infections are major concerns worldwide. Herein, we tested the activity of the Blad-containing oligomer (BCO), a new antifungal molecule already in use for agriculture, on Malassezia spp. and dermatophytes, the causal agents of human tinea versicolor and tinea pedis. Given the lack of a standard method for Malassezia susceptibility testing and the plethora of published methods, we also developed an improved method for this genus. METHODOLOGY: The efficacy of BCO was assessed in vitro and compared to that of the drugs currently utilized in the treatment of tinea versicolor (fluconazole and itraconazole) and tinea pedis (itraconazole and terbinafine). For dermatophytes, the standard microdilution broth-based method was used, with small adjustments, and several broth formulations and inocula sizes were tested to develop an improved susceptibility method for Malassezia spp. RESULTS: We successfully developed a microdilution broth-based method with considerable advantages over other available methods, and used it for all in vitro susceptibility tests of Malassezia spp. isolates. We report that, on a molar basis, BCO was more effective than fluconazole or itraconazole on most strains of Malassezia spp. isolated from clinical samples (n=29). By contrast, BCO was less effective than itraconazole or terbinafine on the common dermatophytes Trichophyton rubrum and Trichophyton interdigitale. CONCLUSION: These data place BCO as a promising drug for the treatment of Malassezia-associated skin diseases. Further in vivo studies are now required to ascertain its applicability in the clinical setting.

Fusion proteins towards fungi and bacteria in plant protection.

In agriculture, although fungi are considered the foremost problem, infections by bacteria also cause significant economical losses. The presence of different diseases in crops often leads to a misuse of the proper therapeutic, or the combination of different diseases forces the use of more than one pesticide. This work concerns the development of a 'super-Blad': a chimeric protein consisting of Blad polypeptide, the active ingredient of a biological fungicide already on the market, and two selected peptides, SP10-5 and Sub5, proven to possess biological potential as antibacterial agents. The resulting chimeric protein obtained from the fusion of Blad with SP10-5 not only maintained strong antibacterial activity, especially against Xanthomonas spp. and Pseudomonas syringae, but was also able to retain the ability to inhibit the growth of both yeast and filamentous fungi. However, the antibacterial activity of Sub5 was considerably diminished when fused with Blad, which seems to indicate that not all fusion proteins behave equally. These newly designed drugs can be considered promising compounds for use in plant protection. A deeper and focused development of an appropriate formulation may result in a potent biopesticide that can replace, per se, two conventional chemistries with less impact on the environment.

Bridging the Gap to Non-toxic Fungal Control: Lupinus-Derived Blad-Containing Oligomer as a Novel Candidate to Combat Human Pathogenic Fungi.

The lack of antifungal drugs with novel modes of action reaching the clinic is a serious concern. Recently a novel antifungal protein referred to as Blad-containing oligomer (BCO) has received regulatory approval as an agricultural antifungal agent. Interestingly its spectrum of antifungal activity includes human pathogens such as Candida albicans, however, its mode of action has yet to be elucidated. Here we demonstrate that BCO exerts its antifungal activity through inhibition of metal ion homeostasis which results in apoptotic cell death in C. albicans. HIP HOP profiling in Saccharomyces cerevisiae using a panel of signature strains that are characteristic for common modes of action identified hypersensitivity in yeast lacking the iron-dependent transcription factor Aft1 suggesting restricted iron uptake as a mode of action. Furthermore, global transcriptome profiling in C. albicans also identified disruption of metal ion homeostasis as a potential mode of action. Experiments were carried out to assess the effect of divalent metal ions on the antifungal activity of BCO revealing that BCO activity is antagonized by metal ions such as Mn2+, Zn2+, and Fe2+. The transcriptome profile also implicated sterol synthesis as a possible secondary mode of action which was subsequently confirmed in sterol synthesis assays in C. albicans. Animal models for toxicity showed that BCO is generally well tolerated and presents a promising safety profile as a topical applied agent. Given its potent broad spectrum antifungal activity and novel multitarget mode of action, we propose BCO as a promising new antifungal agent for the topical treatment of fungal infections.

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

Blad polypeptide comprises residues 109-281 of Lupinus albus ß-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.

A nontoxic polypeptide oligomer with a fungicide potency under agricultural conditions which is equal or greater than that of their chemical counterparts.

There are literally hundreds of polypeptides described in the literature which exhibit fungicide activity. Tens of them have had attempted protection by patent applications but none, as far as we are aware, have found application under real agricultural conditions. The reasons behind may be multiple where the sensitivity to the Sun UV radiation can come in first place. Here we describe a multifunctional glyco-oligomer with 210 kDa which is mainly composed by a 20 kDa polypeptide termed Blad that has been previously shown to be a stable intermediary product of ß-conglutin catabolism. This oligomer accumulates exclusively in the cotyledons of Lupinus species, between days 4 and 12 after the onset of germination. Blad-oligomer reveals a plethora of biochemical properties, like lectin and catalytic activities, which are not unusual per si, but are remarkable when found to coexist in the same protein molecule. With this vast range of chemical characteristics, antifungal activity arises almost as a natural consequence. The biological significance and potential technological applications of Blad-oligomer as a plant fungicide to agriculture, its uniqueness stems from being of polypeptidic in nature, and with efficacies which are either equal or greater than the top fungicides currently in the market are addressed.

Influence of selected factors on browning of Camembert cheese.

Experimental Camembert cheeses were made to investigate the effects on browning of the following factors: inoculation with Yarrowia lipolytica, the use of Penicillium candidum strains with different proteolytic activity, the addition of tyrosine, and the addition of Mn2+ thus leading to 16 different variants of cheese. Two physical colour parameters were used to describe browning, depending on the location in the cheeses: a whiteness index for the outside browning (mould mycelium), and a brownness index for the inside browning (surface of the cheese body). Mn2+ promoted a significant increase of browning at both locations, whereas Yar. lipolytica had the opposite effect. Outside browning was significantly more intense when using the Pen. candidum strain with higher proteolytic activity. A significant interaction was found between Yar. lipolytica and Pen. candidum. The yeast had no effect in combination with a low proteolytic strain of Pen. candidum, but significantly reduced proteolysis and browning in combination with a high proteolytic strain of Pen. candidum. We further confirmed that both strains of Pen. candidum were able to produce brown pigments from tyrosine and thus both are presumably responsible for the browning activity in this type of cheese.