Penicillium phytopathogenic species provoke severe postharvest disease and economic losses. Penicillium expansum is the main pome fruit phytopathogen while Penicillium digitatum and Penicillium italicum cause citrus green and blue mold, respectively. Control strategies rely on the use of synthetic fungicides, but the appearance of resistant strains and safety concerns have led to the search for new antifungals. Here, the potential application of different antifungal proteins (AFPs) including the three Penicillium chrysogenum proteins (PAF, PAFB and PAFC), as well as the Neosartorya fischeri NFAP2 protein to control Penicillium decay, has been evaluated. PAFB was the most potent AFP against P. digitatum, P. italicum and P. expansum, PAFC and NFAP2 showed moderate antifungal activity, whereas PAF was the least active protein. In fruit protection assays, PAFB provoked a reduction of the incidence of infections caused by P. digitatum and P. italicum in oranges and by P. expansum in apples. A combination of AFPs did not result in an increase in the efficacy of disease control. In conclusion, this study expands the antifungal inhibition spectrum of the AFPs evaluated, and demonstrates that AFPs act in a species-specific manner. PAFB is a promising alternative compound to control Penicillium postharvest fruit decay.

Small, cysteine-rich and cationic antimicrobial proteins (AMPs) from filamentous ascomycetes promise treatment alternatives to licensed antifungal drugs. In this study, we characterized the Penicillium chrysogenum Q176 antifungal protein C (PAFC), which is phylogenetically distinct to the other two Penicillium antifungal proteins, PAF and PAFB, that are expressed by this biotechnologically important ascomycete. PAFC is secreted into the culture broth and is co-expressed with PAF and PAFB in the exudates of surface cultures. This observation is in line with the suggested role of AMPs in the adaptive response of the host to endogenous and/or environmental stimuli. The in silico structural model predicted five β-strands stabilized by four intramolecular disulfide bonds in PAFC. The functional characterization of recombinant PAFC provided evidence for a promising new molecule in anti-Candida therapy. The thermotolerant PAFC killed planktonic cells and reduced the metabolic activity of sessile cells in pre-established biofilms of two Candidaalbicans strains, one of which was a fluconazole-resistant clinical isolate showing higher PAFC sensitivity than the fluconazole-sensitive strain. Candidacidal activity was linked to severe cell morphology changes, PAFC internalization, induction of intracellular reactive oxygen species and plasma membrane disintegration. The lack of hemolytic activity further corroborates the potential applicability of PAFC in clinical therapy.

Increasing bacterial resistance to antibiotics is a serious problem worldwide. We sought to record the acquisition of antibiotic-resistant Escherichia coli (E. coli) in healthy infants in Northern Thailand and investigated potential determinants. Stool samples from 142 infants after birth, at ages 2wk, 2mo, 4 to 6mo, and 1y, and parent stool samples were screened for E. coli resistance to tetracycline, ampicillin, co-trimoxazole, and cefazoline by culture, and isolates were further investigated for multiresistance by disc diffusion method. Pulsed-field gel electrophoresis was performed to identify persistent and transmitted strains. Genetic comparison of resistant and transmitted strains was done by multilocus sequence typing (MLST) and strains were further investigated for extra- and intra-intestinal virulence factors by multiplex PCR. Forty-seven (33%) neonatal meconium samples contained resistant E. coli. Prevalence increased continuously: After 1y, resistance proportion (tetracycline 80%, ampicillin 72%, co-trimoxazole 66%, cefazoline 35%) almost matched those in parents. In 8 infants (6%), identical E. coli strains were found in at least 3 sampling time points (suggesting persistence). Transmission of resistant E. coli from parents to child was observed in only 8 families. MLST showed high diversity. We could not identify any virulence genes or factors associated with persistence, or transmission of resistant E. coli. Full-term, vaginal birth and birth in rural hospital were identified as risk factors for early childhood colonization with resistant E. coli. One third of healthy Thai neonates harboured antibiotic-resistant E. coli in meconium. The proportion of resistant E. coli increased during the first year of life almost reaching the value in adults. We hypothesize that enhancement of infection control measures and cautious use of antibiotics may help to control further increase of resistance.

Temporin B (TB) is a short, positively charged peptide secreted by the granular glands of the European frog Rana temporaria. While the antibacterial and antiviral efficacy of TB and some of its improved analogs are well documented, nothing is known about their antifungal potency so far. We dedicated this study to characterize the antifungal potential of the TB analog TB_KKG6K and the newly designed D-Lys_TB_KKG6K, the latter having the L-lysines replaced by the chiral counterpart D-lysines to improve its proteolytic stability. Both peptides inhibited the growth of opportunistic human pathogenic yeasts and killed planktonic and sessile cells of the most prevalent human pathogen, Candida albicans. The anti-yeast efficacy of the peptides coincided with the induction of intracellular reactive oxygen species. Their thermal, cation, pH and serum tolerance were similar, while the proteolytic stability of D-Lys_TB_KKG6K was superior to that of its template peptide. Importantly, both peptides lacked hemolytic activity and showed minimal in vitro cytotoxicity in primary human keratinocytes. The tolerance of both peptides in a reconstructed human epidermis model further supports their potential for topical application. Our results open up an exciting field of research for new anti-Candida therapeutic options based on amphibian TB analogs.

The genome of Penicillium chrysogenum Q176 contains a gene coding for the 88-amino-acid (aa)-long glycine- and cysteine-rich P. chrysogenum antifungal protein C (PAFC). After maturation, the secreted antifungal miniprotein (MP) comprises 64 aa and shares 80% aa identity with the bubble protein (BP) from Penicillium brevicompactum, which has a published X-ray structure. Our team expressed isotope (¹⁵N, ¹³C)-labeled, recombinant PAFC in high yields, which allowed us to determine the solution structure and molecular dynamics by nuclear magnetic resonance (NMR) experiments. The primary structure of PAFC is dominated by 14 glycines, and therefore, whether the four disulfide bonds can stabilize the fold is challenging. Indeed, unlike the few published solution structures of other antifungal MPs from filamentous ascomycetes, the NMR data indicate that PAFC has shorter secondary structure elements and lacks the typical β-barrel structure, though it has a positively charged cavity and a hydrophobic core around the disulfide bonds. Some parts within the two putative γ-core motifs exhibited enhanced dynamics according to a new disorder index presentation of ¹⁵N-NMR relaxation data. Furthermore, we also provided a more detailed insight into the antifungal spectrum of PAFC, with specific emphasis on fungal plant pathogens. Our results suggest that PAFC could be an effective candidate for the development of new antifungal strategies in agriculture.