Candida albicans is the most common fungal pathogen of humans, forming both commensal and opportunistic pathogenic interactions, causing a variety of skin and soft tissue infections in healthy people. In immunocompromised patients C. albicans can cause invasive, systemic infections that are associated with a high incidence of mortality.

 

Propolis, a natural product of plant resins, is used by the bees to seal holes in their honeycombs and protect the hive entrance. However, propolis has also been used in the folk medicine for centuries in order to treat several diseases mainly because of its antimicrobial, anti-inflammatory, antiviral and others important properties.

 

Our research group has been developing  some therapeutics aiming to treat inflammations, healings and Candidiasis. Previous results of our research group demonstrated the efficacy of a propolis mucoadhesive gel to treat vulvovaginal candidiasis (Berretta et al. 2013).

 

Subsequently, we applied the power of Saccharomyces cerevisiae as a model organism for studies of genetics, cell biology, and genomics aiming to understand how propolis affects fungi at cellular level. Propolis is able to induce an apoptosis cell death response. However, increased exposure to propolis provides a corresponding increase in the necrosis response. We showed that cytochrome c but not endonuclease G Nuc1p, is involved in the propolis-mediated cell death in S. cerevisiae. We also observed that the metacaspase YCA1 gene is important for propolis-mediated cell death.

 

  To elucidate the gene functions that may be required for propolis-sensitivity in eukaryotes, the full collection of about 4,800 haploid S. cerevisiae deletion strains was screened for propolis-sensitivity. We were able to identify 138 deletion strains that have different degrees of propolis-sensitivity when compared to the corresponding wild-type strain. Systems biology revealed enrichment for genes involved in mitochondrial electron transport chain, vacuolar acidification, negative regulation of transcription from RNA polymerase II promoter, regulation of macroautophagy associated with protein target to vacuole and cellular response to starvation. Validation studies indicated that propolis sensitivity is dependent on the mitochondrial function, and vacuolar acidification and autophagy are important for yeast cell death caused by propolis. 

 

The information obtained in this step was published in the journal Eukaryotic Cell (from the American Society of Microbiology) by our team composed by Patrícia Alves de Castro, Marcela Savoldi, Diego Bonatto, Mário Henrique Barros, Maria Helena S. Goldman, Andresa A. Berretta, and Gustavo Henrique Goldman, members of the University of São Paulo, Federal University of Rio Grande do Sul, and  Apis Flora Co.

 

After obtaining the results showing that propolis sensitivity is dependent on the mitochondrial function and that vacuolar acidification and autophagy are important for yeast cell death caused by propolis, we extended our understanding of propolis mediated cell death in the yeast Saccharomyces cerevisiae by applying microarray hybridization analysis and systems biology tools to analyze the transcriptional profiling of cells exposed to propolis. In summary, our data indicated that propolis is largely affecting several pathways in the eukaryotic cell. However, the most prominent pathways are related to oxidative stress, mitochondrial electron transport chain, vacuolar acidification, regulation of macroautophagyassociated with protein target to vacuole, cellular response to starvation, and negative regulation of transcription from RNA polymerase II promoter. Our work emphasizes again the importance of S. cerevisiae as a model system to understand at molecular level the mechanism by which propolis causes cell death in this organism. Our study is the first one that investigates systematically by using functional genomics how propolis influences and modulates the mRNA abundance of an organism and may stimulate further work on the propolis-mediated cell death mechanisms in fungi. These data was previously published in BMC Complementary and Alternative Medicine by basically the same group.

 

Finally, after using S. cerevisiae like a model to understand important points that were involved in cell death mediated by propolis, we demonstrated the fungicidal activity of propolis against all three morphogenetic types of C. albicans and that propolis-induced cell death was mediated via metacaspase

and Ras signaling in this microrganism. To identify genes that were involved in propolis tolerance, we screened 800 C. albicans homozygous deletion mutants for decreased tolerance to propolis. Fifty-one mutant strains were identified as being hypersensitive to propolis including seventeen genes involved in cell adhesion, biofilm formation, filamentous growth, phenotypic switching and pathogenesis (HST7, GIN4, VPS34, HOG1, ISW2, SUV3, MDS3, HDA2, KAR3, YHB1, NUP85, CDC10, MNN9, ACE2, FKH2, and SNF5). We validated these results by showing that propolis inhibited the transition from yeast-like to hyphal growth. Propolis was shown to contain compounds that conferred fluorescent properties to C. albicans cells. Moreover, we have shown that a topical pharmaceutical preparation, based upon propolis, was able to control C. albicans infections in a mouse model for vulvovaginal candidiasis. Our results strongly indicate that propolis could be used as a strategy for controlling candidiasis.

 

The research group involved in vulvovaginal Candidiasis therapeutics using propolis like a medicinal strategy mainly led by Andresa A. Berretta and Gustavo Henrique Goldman, is grateful to Apis Flora Co., Fapesp, Finep and  University of São Paulo for financial support and laboratorial structure.

 

*Andresa A. Berretta é  PhD e R&D Manager Apis Flora Co.