The gamma-core motif determines the antifungal mode of action of small,
cysteine-rich, cationic proteins from Ascomycetes
(OTKA ANN 134141)
Principal Investigator: László Galgóczy (in bilateral cooperation with
Florentine Marx from the Medical University of Innsbruck)
Duration: 40 moths
The increasing incidence of fungal infections and contaminations
due to drug-resistant filamentous fungi in medicine, agriculture and food
industry urges the development of new antifungal strategies.
The highly stable, extracellular, cysteine-rich antifungal proteins from filamentous
Ascomycetes (AFPs) offer an alternative, safely applicable solution. Our in
silico investigations revealed that all isolated AFPs contain an
evolutionary conserved [GXC]-[X3-9]-[C] consensus γ-core motif. Our
preliminary results demonstrated that the antifungal efficacy of AFPs
possibly depends on the physical and chemical properties of the γ-core
constituting amino acids.
Based on this we hypothesize that the antifungal efficacy of AFPs is improvable with rational design of the γ-core motif, and
the improved AFPs and γ-core peptides are applicable as antifungal drugs,
biopesticides and crop preservatives. We address our hypotheses on the basis
of the intensively studied AFPs, the PAF from Penicillium chrysogenum and
the NFAP from Neosartorya fischeri. We plan to change the amino acid
sequence of the γ -core motif of these two proteins to create new AFP
variants with improved antifungal efficiency. We further investigate the
structure and function of designed synthetic γ -core peptides that show
enhanced activity. Finally, we provide a proof-of-principle for the
applicability of the best candidates of engineered AFPs and rationally
designed γ -core peptides to inhibit plant-pathogen infection and mycotoxin
production in crops and dermatophytosis.
The achievements in this project
allow further steps towards the biotechnological application of AFPs.
Supported by Hungarian National Research, Development and Innovation Office;
and FWF Austrian Science Fund.
Application of cysteine-rich antifungal
proteins secreted by Neosartorya fischeri NRRL 181 as biopesticides and crop
(OTKA PD 135248, connected to OTKA ANN 134141)
Principal Investigator: László Galgóczy
Duration: 36 moths (2016-2019)
Presumably as a consequence of climate changes the occurrence of resistant phyto-, pre- and
postharvest pathogenic fungi and the number of mycotoxin contaminated feeds
and foods are continuously increasing in Europe in the last years causing
loss of billions of Euro and posing severe risks to human and animal health.
These facts urge the development of new, and more effective antifungal
The highly stable, extracellular, cysteine-rich antifungal
proteins from filamentous Ascomycetes (crAFPs) could offer an alternative,
safely applicable solution. Our in silico investigations revealed that all
isolated crAFPs contain an evolutionary conserved [GXC]-[X3-9]-[C] consensus
γ-core motif. Our preliminary results demonstrated that the antifungal
efficacy of crAFPs depends on the physical and chemical properties of the
γ-core constituting amino acids.
Based on this we hypothesize that the
antifungal efficacy of crAFPs is improvable with rational design of the
γ-core motif, and the improved crAFPs are applicable as biopesticides and
crop preservatives in the agriculture and food industry. We address this
question by investigating different crAFPs produced by Neosartorya fischeri,
their γ-core improved variants, and their γ-core peptides by antifungal
susceptibility, haemolytic activity, cytotoxicity tests, plant and crop
The achievements in this project allow further steps
towards the biotechnological application of crAFPs, and open up a completely
new avenue for the development of bioactive proteins and peptides with
worldwide economic and societal impact on pest control and crop
Supported by Hungarian National Research, Development and
New antifungal strategies: structure and function of NFAP
(FWF M 1776-B20)
Principal Investigator: László Galgóczy (Co-applicant: Florentine Marx)
Duration: 24 moths (2014-2016)
The increased incidence of severe fungal infections and the fast
development of drug resistant filamentous fungi causing mycoses,
plant infections or damage of cultural heritages
strongly demand for the development of new antifungal strategies.
Small, cysteine-rich, highly stable antifungal proteins secreted by filamentous
Ascomycetes have great potential for application in these fields. The
antifungal protein NFAP from the Neosartorya fischeri NRRL 181 isolate is a
novel representative of this protein group.
In our previous work we
demonstrated that NFAP effectively inhibits the growth of numerous
filamentous Ascomycetes including potential human and plant pathogens, and
its antifungal effect is dose-dependent and strongly influenced by the
extracellular mono- and divalent cation concentration. In susceptible fungi,
NFAP causes damage to the cell wall by destructing chitin filaments and
triggers apoptotic-necrotic pathways by intracellular accumulation of
reactive oxygen species. However, we could also show that NFAP differs in
its antifungal spectrum, its antifungal mode of action and its tertiary
structure from the two most investigated NFAP-related proteins, the
Aspergillus giganteus antifungal protein AFP and the Penicillium chrysogenum
antifungal protein PAF. Further efforts are needed to characterize in detail
the solution structure of NFAP, its structure-function relation, the primary
targets and the antifungal mode of action, which have not been investigated
in detail so far.
The present project aims to clarify two aspects: (1) the
connection between the protein structure and the antifungal properties; and
(2) the identification of target molecules of NFAP. We address the first
question by investigating the role of structural features of NFAP by nuclear
magnetic resonance, thermal unfolding experiments and antimicrobial
susceptibility tests. To this end distinct recombinant NFAP protein mutants
are generated for structure-function investigations. The second question we
address by molecular screening of (i) potential lipid targets of NFAP via in
vitro protein-lipid overlay assays and (ii) protein receptors by chemical
cross-linking, affinity purification, and differential mass spectrometry.
The results significantly contribute to the understanding of the mode of
action and structure-function relation not only of NFAP but also of other
cysteine-rich antifungal proteins from Ascomycetes in general. Taking into
account the wide distribution of antimicrobial proteins in nature, our
project helps to solve problems that are also relevant for related
A detailed insight into the structure, function,
interaction with target molecules and antifungal mechanisms of more members
of this new protein group is an essential prerequisite for the
identification of protein motifs with specific functions. This ultimately
enables the construction of synthetic or chimeric proteins with improved and
specific antimicrobial potential for medical therapy, pest control, food
preservation and conservation of cultural heritages in the near future and
the outcome of our project promises patent registration.
Supported by FWF Austrian Science Fund.
Identification and characterization of defensin-like
antimicrobial proteins secreted by Neosartorya fischeri and related species
(OTKA PD 83355)
Principal Investigator: László Galgóczy
Duration: 36 moths (2010-2013)
Defensins and similar antimicrobial proteins are widely
distributed in the nature. Five similar proteins have been identified among
filamentous fungi. These proteins secreted by taxonomical distinct species
have different mode of action and species specificity, nevertheless their
structure is very similar.
Neosartorya fischeri antimicrobial protein (NFAP) from N. fischeri (anamorph:
Aspergillus fischerianus) is a hypothetical antifungal protein derived from
genomic database, its presence is confirmed by in silico investigation only.
Its cloning, isolating, antifungal spectrum, mode of action and biological role
has not been described yet.
The goal of the proposed project is to increase the knowledge of defensin-like
antimicrobial proteins derived from filamentous fungi by investigation of
NFAP. The main aims of the research project are the followings: (1)
determination of the presence of NFAP and its orthologues from Neosartorya
fischeri (NRRL 181) and related species; (2) investigation of the
antimicrobial properties and mode of the action in case of NFAP; (3)
creation a heterologous expression system for production of NFAP; (4)
examination of the connection between the structure of NAFP and its
efficacy; (5) observation of biological role of NFAP.
The final results provide information about the distribution of defensin-like
antimicrobial proteins among filamentous fungi. The results about the structure-efficacy
connection will be the bases of further theoretical and practical studies to
develop effective antimicrobial drugs. Achievements of this project provide
important information in addition to microbiology.
Supported by the Hungarian Scientific Research Fund.