Numerous reports indicate that the activity of antifungal proteins can be antagonized by the external addition of Ca2+ ions to the test medium [[15, selleck inhibitor 17–21]] pointing towards the induction of adaptive responses which may be triggered by Ca2+ signalling [15, 17]. The aim of this study was to characterize
in more detail the mode of action of the A. giganteus AFP variant protein CHIR98014 nmr AFPNN5353 and to investigate the pathways that might be affected/modulated by this antifungal protein. Therefore, we focussed our interest on the involvement of the CWIP and the Ca2+ signalling in the toxicity of AFPNN5353. To address these questions, we used the highly AFPNN5353 sensitive model organisms A. nidulans and A. niger for which appropriate mutant strains were available. Results In silico analysis of AFPNN5353 CLUSTALW amino acid (aa) sequence analysis of AFPNN5353 with other known antifungal proteins revealed that AFPNN5353 from A. giganteus strain A3274 is Adriamycin mouse a protein homologous to AFP from A. giganteus strain MDH 18894 [8, 22]. AFPNN5353 exhibits > 90% identity with AFP, but only 42% identity with the P. chrysogenum PAF and 27% identity with the A. niger ANAFP. In fact, the secreted mature form of AFPNN5353 consists of 51 aa and differs only in 5 aa from AFP (Figure 1). Three aa exchanges belong to structurally related aa,
one aa exhibits weak similarity and one aa is different (position 4). These aa exchanges do not influence the theoretical isoelectric point (pI) of AFPNN5353, which is the same as for AFP (pI 9.3, http://expasy.org/tools/protparam.html). Most importantly, AFPNN5353 still contains the putative chitin-binding domain CKYKAQ present in AFP but not in PAF or ANAFP and also harbors all conserved cysteine residues important for protein stabilization why [10, 23]. Figure 1 Clustalw sequence alignment http://www.ebi.ac.uk/Tools/msa/clustalw2/ of the antifungal
proteins AFP NN5353 and AFP from A. giganteus , ANAFP from A. niger and PAF from P. chrysogenum. Identical amino acids (aa) are marked with (*), aa with strong similarity are indicated with (:) and aa with weak similarity are marked with (.). Antifungal activity of the protein AFPNN5353 To investigate the antifungal specificity of AFPNN5353, fifteen filamentous fungi were tested for their susceptibility to the protein. Since antifungal proteins might be useful for biotechnological applications, filamentous human and plant pathogenic fungi were selected as test organisms (e.g. Fusarium oxysporum, Botrytis cinerea, Mucor sp. and A. fumigatus) in addition to the model organisms A. nidulans and A. niger. As shown in Table 1, thirteen out of fifteen tested moulds were found to be sensitive against AFPNN5353. A. nidulans wild type, N. crassa wild type and A. niger wild type were the most sensitive strains to AFPNN5353.