Abstract

Small, cysteine-rich and cationic proteins with antimicrobial activity are produced by diverse organisms of all kingdoms and represent promising molecules for drug development. The ancestor of all industrial penicillin producing strains, the ascomycete Penicillium chryosgenum Q176, secretes the extensively studied antifungal protein PAF. However, the genome of this strain harbours at least two more genes that code for other small, cysteine-rich and cationic proteins with potential antifungal activity. In this study, we characterized the pafB gene product that shows high similarity to PgAFP from P. chrysogenum R42C. Although abundant and timely regulated pafB gene transcripts were detected, we could not identify PAFB in the culture broth of P. chrysogenum Q176. Therefore, we applied a P. chrysogenum-based expression system to produce sufficient amounts of recombinant PAFB to address unanswered questions concerning the structure and antimicrobial function. Nuclear magnetic resonance (NMR)-based analyses revealed a compact β-folded structure, comprising five β-strands connected by four solvent exposed and flexible loops and an “abcabc” disulphide bond pattern. We identified PAFB as an inhibitor of growth of human pathogenic moulds and yeasts. Furthermore, we document for the first time an anti-viral activity for two members of the small, cysteine-rich and cationic protein group from ascomycetes.

Highlights

  • The increasing incidence of fatal microbial infections due to the development of resistance against licensed antimicrobial drugs raises a strong demand for new antimicrobial treatment strategies

  • P. chrysogenum Q176 grown under standard conditions (Fig. 1)

  • We found that pafB mRNA was detectable in P. chrysogenum Q176 shaking cultures with an expression maximum at 48 h after inoculation and a subsequent decrease in transcript amount upon further incubation time (Fig. 1a)

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Summary

Introduction

The increasing incidence of fatal microbial infections due to the development of resistance against licensed antimicrobial drugs raises a strong demand for new antimicrobial treatment strategies. PAF represents a promising bio-molecule for novel antifungal drug development as www.nature.com/scientificreports/ It is stable against proteolytic degradation, thermo-resistant and active within a broad pH range[9], and shows no cytotoxic effects in vitro[10] or in vivo[11,12]. Genome mining in the strain Wisconsin 54-1255 for nucleotide (nt) sequences coding for other small, cysteine-rich and cationic proteins revealed that this P. chrysogenum strain harbours - apart from the paf gene The genome of P. chrysogenum Wisconsin 54-1255 and its progenitor strain Q176 contain at least three genes that code for proteins with reported antifungal activity. This represents a unique chance to investigate their function within one organism. Phylogenetic analyses suggested a classification of the three proteins into different groups, whereby Pc-Arctin is grouped more distantly from PAF and PgAFP1,18

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