Molecular characterization of opportunistic pathogenic zygomycetes

The term zygomycosis refers to a diverse group of mycotic diseases caused by members of the orders Mucorales and Entomophthorales. These infections are frequently associated with diabetic ketoacidosis, deferoxamine treatment, cancer and its therapy, solid organ or bone marrow transplantations, extreme malnutrition and neutropenia. Although these mycoses are relatively rare, their high mortality rate underline the importance of this group of fungal infections. Molecular techniques are widely used to identify the virulence factors of clinically important fungi or to develop useful diagnostic techniques. However, application of these methods to characterize the opportunistic pathogenic nature of zygomycetes started only a few years ago. This review discusses the current state of molecular studies performed on the pathogenicity and diagnosis of zygomycetes causing opportunistic human mycosis

Potential applications of filamentous fungus derived β-defensin-like antifungal proteins in agriculture

Many filamentous fungi are postharvest and destructive plant pathogens and are thus responsible for enormous crop losses worldwide. The antifungal proteins secreted by filamentous fungi are promising agents for prevention of fungal diseases in the agriculture. The extracellular β-defensin-like antifungal proteins derived from ascomycetous filamentous fungal species are especially interesting in this respect because of their chemical and biological properties. The main features of these extracellular proteins are a low molecular mass, a basic character, and the presence of 6-8 cysteine residues and several intramolecular disulfide bonds which provide them with a high stability against protease degradation, high temperature and within a broad pH range. The tertiary structure of these proteins is very similar to the β-defensins, it contains five antiparallel β-sheets connected by three loops. In spite of the fact that they are very different in their amino acid sequences; conserved homologous regions can be identified. Based on it they can be divided into two main groups: peptides which contain the Penicillium chrysogenum antifungal protein (PAF) cluster in their amino acid sequences, and peptides with Penicillium brevicompactum bubble protein (BP) cluster. Both of them have a potent antifungal activity, but the peptides with PAF-cluster are effective against filamentous fungi. These proteins secreted by taxonomical distinct species cause similar symptoms on the susceptible fungus, but they have different mode of action and species specificity, nevertheless, their structure is very similar. They have high stability and efficacy; their limited toxicity and low costs of production could make them suitable for use in practical respects in agricultural fields, especially in plant protection on the field and crop protection after the harvest

Are cysteine and its derivatives applicable for the prevention of fungal diseases?

Many filamentous fungi are destructive pathogens of plants and are thus responsible for enormous crop losses worldwide. Therefore, there is a substantial demand for safely applicable, new compounds with extensive antifungal activity. Cysteine and its derivatives are interesting from this respect, as they have effective inhibitory potential against microorganisms and do not harmful effect on animal and plant cells. Cysteine is a non-essential amino acid, which is an important structural and functional component of many proteins and enzymes. Previous studies demonstrated that cysteine and N-acetyl-cysteine have also been shown to exert antifungal activity. A number of members of the class Zygomycetes are important as postharvest pathogens of agricultural products; Rhizopus, Mucor and Gilbertella species are among the most frequently isolated causative agents of fungal rots. In this study we investigated the in vitro antifungal activity of cysteine (D- and L-cysteine) and its 4 derivatives (L-cysteine-methyl-ester, N-acetyl-cysteine, N-isobutyryl-D-cysteine and N-isobutyryl-L-cysteine) against 20 zygomyceteous fungal isolates representing 16 genera (Actinomucor, Backusella, Gilbertella, Lichtheimia, Micromucor, Mortierella, Mucor, Mycotypha, Phycomyces, Rhizomucor, Rhizopus, Saksenaea, Syncephalastrum, Thamnostylum, Umbellopsis and Zygorynchus). The inhibitory potential of different concentrations (ranging from 0.625 to 10 mM) of the cysteine and its derivatives were investigated on the germination of sporangiospores and on hyphal extension by broth microdilution method and agar plate test. Cysteine and its derivatives showed a strong inhibitory effect against the most studied strains. Treatment with 10 mM of compounds resulted total growth inhibition in case of some isolates. Severe changes in colony morphology and hyphal growth were observed in presence of 10 mM Lcysteine, N-acetyl-cysteine and N-isobutyryl-L-cysteine when a strain was sensitive to them. Ten mM N-acetyl-cysteine induced dramatic modifications in the structural organization of the hyphae in case of Rhizopus stolonifer. The above mentioned features of the investigated compounds could make them favourable antifungal agents against Zygomycetes in agricultural respect in the future, but it is needed further studies to prove their practical efficiency, for example in plant model experiments

A Mortierellales rend (Fungi, Zygomycetes) átfogó filogenetikai és taxonómiai revíziója morfológiai, fiziológiai és molekuláris eljárásokkal = Reconstruction of the phylogeny of the filamentous fungal order Mortierellales (Zygomycetes): a comprehensive taxonomical revision

A projekt célja a Mortierellales fonalas gomba rend átfogó filogenetikai és taxonómiai revíziója volt. E lipid akkumuláló szervezetek nagy jelentőséggel rendelkeznek, mint többszörösen telítetlen zsírsavak termelői és különböző vegyületek biotranszformáló ágensei. A projekt során létrehoztunk egy több, mint 200 törzsből álló gyűjteményt, mely a gombacsoport minden tenyészthető nemzetségét felöleli és mintegy 100 faj típustörzsét is tartalmazza. Az elhelyezett törzsek esetében meghatároztuk a teljes ITS régió, valamint a 18S és a 28S rRNS gén szekvenciáit és ezek alapján filogenetikai elemzést végeztünk. Több mint száz törzs esetében meghatároztuk a tef és RPB1 fehérjekódoló gének egy szakaszát is és az így létrehozott ötgénes adatsor alapján egy teljesebb filogenetikai elemzést végeztünk. A multigén-alapú törzsfák alapján megállapítottuk, hogy a jelenlegi morfológiai alapon definiált Mortierella nemzetség parafiletikus és meghatároztuk a renden belüli monofiletikus fajcsoportokat, egyszersmind egy új, filogenetikai szempontból megfelelőbb rendszerezés alapjait is leraktuk. Az így meghatározott csoportok morfológiai és fiziológiai jellemzését is elvégeztük. A Mortierellales rend részletes elemzése értékes információt szolgáltatott a későbbi bioaktív metabolitok és enzimaktivitások felfedezését célzó alap- és gyakorlati kutatások számára. Ugyanakkor, a nagy szekvencia, morfológiai és fiziológiai adatsorok elemzése alapján tágabb evolúciós következtetéseket is le tudtunk vonni. | The aim of the present project was a comprehensive phylogenetic and taxonomic revision of the fungal order, Mortierellales. These lipid-accumulating organisms have great importance as producers of polyunsaturated fatty acids or biotransforming agents of several chemical compounds. In this project, we established an outstanding strain collection consisting of more than 200 strains. It covers all cultivable genera of the order and includes approximately 100 type strains. Sequences of the complete ITS region and the SSU and LSU RNA genes were determined in case of the deposited strains and were used for phylogenetic analyses. In case of more than 100 strains, the protein coding tef and RPB1 genes were also sequenced and the resulting five-gene dataset was used in a comprehensive phylogenetic analysis. These data showed the genus Mortierella to be paraphyletic. Based on the multigene phylogenies, we proposed new monophyletic species-groups within the order and established the bases of a new phylogenetically correct taxonomy. Besides the molecular analyses, morphological and physiological examination of the newly determined groups was also performed. The detailed characterization of Mortierellales served valuable new information for further studies with the aim of discovering new bioactive metabolites and enzyme activities. At the same time, analysis of the large molecular, morphological and physiological data sets allowed us to examine evolutional problems in a wider context

Antidermatophytic effect of Bacillus mojavensis SZMC 22228 and its secreted chymotrypsin-like protease

The aim of the present study was to investigate the antifungal effect of Bacillus mojavensis SZMC 22228 against different dermatophytes and to isolate the antidermatophytic compound from the bacterial ferment broth. B. mojavensis SZMC 22228 and its cell-free ferment broth effectively inhibited the growth of clinical reference strain of Microsporum canis, Microsporum gypseum, Trichophyton mentagrophytes, Trichophyton rubrum and Trichophyton tonsurans in agar diffusion test in vitro. An antidermatophytic, ~25 kDa protein (B. mojavensis SZMC 22228 antidermatophyitc protein, BMAP) was purified from the antifungally active, cell-free ferment broth using size exclusion and ion-exchange chromatography. BMAP showed antifungal effect against all of the investigated dermatophytes both in agar diffusion and broth microdilution susceptibility tests. M. gypseum proved to be the most susceptible dermatophyte to BMAP (MIC=40 μg/ml), all the other investigated fungi were less susceptible (MIC=80 μg/ml). The enzymatic activity of this protein was investigated in microtiter plate assay using hydrolase specific chromogenic substrates. BMAP showed high proteolytic activity towards N-Succ-Ala-Ala-Pro-Phe-pNA, and proved to be a chymotrypsin-like protease. These results suggest that the antidermatohytic activity of B. mojavensis SZMC 22228 correlates with its chymotrypsin-like protease production. After further investigations, the purified BMAP could be a promising base of a novel antidermatophytic strategy