Regulation of gliotoxin biosynthesis and protection in Aspergillus species.

Aspergillus fumigatus causes a range of human and animal diseases collectively known as aspergillosis. A. fumigatus possesses and expresses a range of genetic determinants of virulence, which facilitate colonisation and disease progression, including the secretion of mycotoxins. Gliotoxin (GT) is the best studied A. fumigatus mycotoxin with a wide range of known toxic effects that impair human immune cell function. GT is also highly toxic to A. fumigatus and this fungus has evolved self-protection mechanisms that include (i) the GT efflux pump GliA, (ii) the GT neutralising enzyme GliT, and (iii) the negative regulation of GT biosynthesis by the bis-thiomethyltransferase GtmA. The transcription factor (TF) RglT is the main regulator of GliT and this GT protection mechanism also occurs in the non-GT producing fungus A. nidulans. However, the A. nidulans genome does not encode GtmA and GliA. This work aimed at analysing the transcriptional response to exogenous GT in A. fumigatus and A. nidulans, two distantly related Aspergillus species, and to identify additional components required for GT protection. RNA-sequencing shows a highly different transcriptional response to exogenous GT with the RglT-dependent regulon also significantly differing between A. fumigatus and A. nidulans. However, we were able to observe homologs whose expression pattern was similar in both species (43 RglT-independent and 11 RglT-dependent). Based on this approach, we identified a novel RglT-dependent methyltranferase, MtrA, involved in GT protection. Taking into consideration the occurrence of RglT-independent modulated genes, we screened an A. fumigatus deletion library of 484 transcription factors (TFs) for sensitivity to GT and identified 15 TFs important for GT self-protection. Of these, the TF KojR, which is essential for kojic acid biosynthesis in Aspergillus oryzae, was also essential for virulence and GT biosynthesis in A. fumigatus, and for GT protection in A. fumigatus, A. nidulans, and A. oryzae. KojR regulates rglT, gliT, gliJ expression and sulfur metabolism in Aspergillus species. Together, this study identified conserved components required for GT protection in Aspergillus species.

Chromatin profiling reveals heterogeneity in clinical isolates of the human pathogen Aspergillus fumigatus.

Invasive Pulmonary Aspergillosis, which is caused by the filamentous fungus Aspergillus fumigatus, is a life-threatening infection for immunosuppressed patients. Chromatin structure regulation is important for genome stability maintenance and has the potential to drive genome rearrangements and affect virulence and pathogenesis of pathogens. Here, we performed the first A. fumigatus global chromatin profiling of two histone modifications, H3K4me3 and H3K9me3, focusing on the two most investigated A. fumigatus clinical isolates, Af293 and CEA17. In eukaryotes, H3K4me3 is associated with active transcription, while H3K9me3 often marks silent genes, DNA repeats, and transposons. We found that H3K4me3 deposition is similar between the two isolates, while H3K9me3 is more variable and does not always represent transcriptional silencing. Our work uncovered striking differences in the number, locations, and expression of transposable elements between Af293 and CEA17, and the differences are correlated with H3K9me3 modifications and higher genomic variations among strains of Af293 background. Moreover, we further showed that the Af293 strains from different laboratories actually differ in their genome contents and found a frequently lost region in chromosome VIII. For one such Af293 variant, we identified the chromosomal changes and demonstrated their impacts on its secondary metabolites production, growth and virulence. Overall, our findings not only emphasize the influence of genome heterogeneity on A. fumigatus fitness, but also caution about unnoticed chromosomal variations among common laboratory strains.

The Aspergillus fumigatus transcription factor RglT is important for gliotoxin biosynthesis and self-protection, and virulence.

Aspergillus fumigatus is an opportunistic fungal pathogen that secretes an array of immune-modulatory molecules, including secondary metabolites (SMs), which contribute to enhancing fungal fitness and growth within the mammalian host. Gliotoxin (GT) is a SM that interferes with the function and recruitment of innate immune cells, which are essential for eliminating A. fumigatus during invasive infections. We identified a C6 Zn cluster-type transcription factor (TF), subsequently named RglT, important for A. fumigatus oxidative stress resistance, GT biosynthesis and self-protection. RglT regulates the expression of several gli genes of the GT biosynthetic gene cluster, including the oxidoreductase-encoding gene gliT, by directly binding to their respective promoter regions. Subsequently, RglT was shown to be important for virulence in a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA). Homologues of RglT and GliT are present in eurotiomycete and sordariomycete fungi, including the non-GT-producing fungus A. nidulans, where a conservation of function was described. Phylogenetically informed model testing led to an evolutionary scenario in which the GliT-based resistance mechanism is ancestral and RglT-mediated regulation of GliT occurred subsequently. In conclusion, this work describes the function of a previously uncharacterised TF in oxidative stress resistance, GT biosynthesis and self-protection in both GT-producing and non-producing Aspergillus species.

Aspergillus fumigatus calcium-responsive transcription factors regulate cell wall architecture promoting stress tolerance, virulence and caspofungin resistance.

Aspergillus fumigatus causes invasive aspergillosis, the most common life-threatening fungal disease of immuno-compromised humans. The treatment of disseminated infections with antifungal drugs, including echinocandin cell wall biosynthesis inhibitors, is increasingly challenging due to the rise of drug-resistant pathogens. The fungal calcium responsive calcineurin-CrzA pathway influences cell morphology, cell wall composition, virulence, and echinocandin resistance. A screen of 395 A. fumigatus transcription factor mutants identified nine transcription factors important to calcium stress tolerance, including CrzA and ZipD. Here, comparative transcriptomics revealed CrzA and ZipD regulated the expression of shared and unique gene networks, suggesting they participate in both converged and distinct stress response mechanisms. CrzA and ZipD additively promoted calcium stress tolerance. However, ZipD also regulated cell wall organization, osmotic stress tolerance and echinocandin resistance. The absence of ZipD in A. fumigatus caused a significant virulence reduction in immunodeficient and immunocompetent mice. The ΔzipD mutant displayed altered cell wall organization and composition, while being more susceptible to macrophage killing and eliciting an increased pro-inflammatory cytokine response. A higher number of neutrophils, macrophages and activated macrophages were found in ΔzipD infected mice lungs. Collectively, this shows that ZipD-mediated regulation of the fungal cell wall contributes to the evasion of pro-inflammatory responses and tolerance of echinocandin antifungals, and in turn promoting virulence and complicating treatment options.

Aspergillus fumigatus protein phosphatase PpzA is involved in iron assimilation, secondary metabolite production, and virulence.

Metal restriction imposed by mammalian hosts during an infection is a common mechanism of defence to reduce or avoid the pathogen infection. Metals are essential for organism survival due to its involvement in several biological processes. Aspergillus fumigatus causes invasive aspergillosis, a disease that typically manifests in immunocompromised patients. A. fumigatus PpzA, the catalytic subunit of protein phosphatase Z (PPZ), has been recently identified as associated with iron assimilation. A. fumigatus has 2 high-affinity mechanisms of iron acquisition during infection: reductive iron assimilation and siderophore-mediated iron uptake. It has been shown that siderophore production is important for A. fumigatus virulence, differently to the reductive iron uptake system. Transcriptomic and proteomic comparisons between ∆ppzA and wild-type strains under iron starvation showed that PpzA has a broad influence on genes involved in secondary metabolism. Liquid chromatography-mass spectrometry under standard and iron starvation conditions confirmed that the ΔppzA mutant had reduced production of pyripyropene A, fumagillin, fumiquinazoline A, triacetyl-fusarinine C, and helvolic acid. The ΔppzA was shown to be avirulent in a neutropenic murine model of invasive pulmonary aspergillosis. PpzA plays an important role at the interface between iron starvation, regulation of SM production, and pathogenicity in A. fumigatus.

Genome-wide transcriptome analysis of Aspergillus fumigatus exposed to osmotic stress reveals regulators of osmotic and cell wall stresses that are SakAHOG1 and MpkC dependent.

Invasive aspergillosis is predominantly caused by Aspergillus fumigatus, and adaptations to stresses experienced within the human host are a prerequisite for the survival and virulence strategies of the pathogen. The central signal transduction pathway operating during hyperosmotic stress is the high osmolarity glycerol mitogen-activated protein kinase cascade. A. fumigatus MpkC and SakA, orthologues of the Saccharomyces cerevisiae Hog1p, constitute the primary regulator of the hyperosmotic stress response. We compared A. fumigatus wild-type transcriptional response to osmotic stress with the ΔmpkC, ΔsakA, and ΔmpkC ΔsakA strains. Our results strongly indicate that MpkC and SakA have independent and collaborative functions during the transcriptional response to transient osmotic stress. We have identified and characterized null mutants for four A. fumigatus basic leucine zipper proteins transcription factors. The atfA and atfB have comparable expression levels with the wild-type in ΔmpkC but are repressed in ΔsakA and ΔmpkC ΔsakA post-osmotic stress. The atfC and atfD have reduced expression levels in all mutants post-osmotic stress. The atfA-D null mutants displayed several phenotypes related to osmotic, oxidative, and cell wall stresses. The ΔatfA and ΔatfB were shown to be avirulent and to have attenuated virulence, respectively, in both Galleria mellonella and a neutropenic murine model of invasive pulmonary aspergillosis.

The Aspergillus fumigatus SchASCH9 kinase modulates SakAHOG1 MAP kinase activity and it is essential for virulence.

The serine-threonine kinase TOR, the Target of Rapamycin, is an important regulator of nutrient, energy and stress signaling in eukaryotes. Sch9, a Ser/Thr kinase of AGC family (the cAMP-dependent PKA, cGMP- dependent protein kinase G and phospholipid-dependent protein kinase C family), is a substrate of TOR. Here, we characterized the fungal opportunistic pathogen Aspergillus fumigatus Sch9 homologue (SchA). The schA null mutant was sensitive to rapamycin, high concentrations of calcium, hyperosmotic stress and SchA was involved in iron metabolism. The ΔschA null mutant showed increased phosphorylation of SakA, the A. fumigatus Hog1 homologue. The schA null mutant has increased and decreased trehalose and glycerol accumulation, respectively, suggesting SchA performs different roles for glycerol and trehalose accumulation during osmotic stress. The schA was transcriptionally regulated by osmotic stress and this response was dependent on SakA and MpkC. The double ΔschA ΔsakA and ΔschA ΔmpkC mutants were more sensitive to osmotic stress than the corresponding parental strains. Transcriptomics and proteomics identified direct and indirect targets of SchA post-exposure to hyperosmotic stress. Finally, ΔschA was avirulent in a low dose murine infection model. Our results suggest there is a complex network of interactions amongst the A. fumigatus TOR, SakA and SchA pathways.

Mitogen activated protein kinases SakA(HOG1) and MpkC collaborate for Aspergillus fumigatus virulence.

Here, we investigated which stress responses were influenced by the MpkC and SakA mitogen-activated protein kinases of the high-osmolarity glycerol (HOG) pathway in the fungal pathogen Aspergillus fumigatus. The ΔsakA and the double ΔmpkC ΔsakA mutants were more sensitive to osmotic and oxidative stresses, and to cell wall damaging agents. Both MpkC::GFP and SakA::GFP translocated to the nucleus upon osmotic stress and cell wall damage, with SakA::GFP showing a quicker response. The phosphorylation state of MpkA was determined post exposure to high concentrations of congo red and Sorbitol. In the wild-type strain, MpkA phosphorylation levels progressively increased in both treatments. In contrast, the ΔsakA mutant had reduced MpkA phosphorylation, and surprisingly, the double ΔmpkC ΔsakA had no detectable MpkA phosphorylation. A. fumigatus ΔsakA and ΔmpkC were virulent in mouse survival experiments, but they had a 40% reduction in fungal burden. In contrast, the ΔmpkC ΔsakA double mutant showed highly attenuated virulence, with approximately 50% mice surviving and a 75% reduction in fungal burden. We propose that both cell wall integrity (CWI) and HOG pathways collaborate, and that MpkC could act by modulating SakA activity upon exposure to several types of stresses and during CW biosynthesis.

High osmolarity glycerol response PtcB phosphatase is important for Aspergillus fumigatus virulence.

Aspergillus fumigatus is a fungal pathogen that is capable of adapting to different host niches and to avoid host defenses. An enhanced understanding of how, and which, A. fumigatus signal transduction pathways are engaged in the regulation of these processes is essential for the development of improved disease control strategies. Protein phosphatases are central to numerous signal transduction pathways. To comprehend the functions of protein phosphatases in A. fumigatus, 32 phosphatase catalytic subunit encoding genes were identified. We have recognized PtcB as one of the phosphatases involved in the high osmolarity glycerol response (HOG) pathway. The ΔptcB mutant has both increased phosphorylation of the p38 MAPK (SakA) and expression of osmo-dependent genes. The ΔptcB strain was more sensitive to cell wall damaging agents, had increased chitin and ß-1,3-glucan, and impaired biofilm formation. The ΔptcB strain was avirulent in a murine model of invasive pulmonary aspergillosis. These results stress the importance of the HOG pathway in the regulation of pathogenicity determinants and virulence in A. fumigatus.

The Aspergillus fumigatus sitA Phosphatase Homologue Is Important for Adhesion, Cell Wall Integrity, Biofilm Formation, and Virulence.

Aspergillus fumigatus is an opportunistic pathogenic fungus able to infect immunocompromised patients, eventually causing disseminated infections that are difficult to control and lead to high mortality rates. It is important to understand how the signaling pathways that regulate these factors involved in virulence are orchestrated. Protein phosphatases are central to numerous signal transduction pathways. Here, we characterize the A. fumigatus protein phosphatase 2A SitA, the Saccharomyces cerevisiae Sit4p homologue. The sitA gene is not an essential gene, and we were able to construct an A. fumigatus null mutant. The ΔsitA strain had decreased MpkA phosphorylation levels, was more sensitive to cell wall-damaging agents, had increased ß-(1,3)-glucan and chitin, was impaired in biofilm formation, and had decreased protein kinase C activity. The ΔsitA strain is more sensitive to several metals and ions, such as MnCl2, CaCl2, and LiCl, but it is more resistant to ZnSO4. The ΔsitA strain was avirulent in a murine model of invasive pulmonary aspergillosis and induces an augmented tumor necrosis factor alpha (TNF-α) response in mouse macrophages. These results stress the importance of A. fumigatus SitA as a possible modulator of PkcA/MpkA activity and its involvement in the cell wall integrity pathway.

ChIP-seq reveals a role for CrzA in the Aspergillus fumigatus high-osmolarity glycerol response (HOG) signalling pathway.

Aspergillus fumigatus is an opportunistic pathogen and allergen of mammals. Calcium signalling is essential for A. fumigatus pathogenicity and is regulated by the CrzA transcription factor. We used ChIP-seq (Chromatin Immunoprecipitation DNA sequencing) to explore CrzA gene targets in A. fumigatus. In total, 165 potential binding peaks including 102 directly regulated genes were identified, resulting in the prediction of the A[GT][CG]CA[AC][AG] CrzA-binding motif. The 102 CrzA putatively regulated genes exhibited a diverse array of functions. The phkB (Afu3g12530) histidine kinase and the sskB (Afu1g10940) MAP kinase kinase kinase of the HOG (high-osmolarity glycerol response) pathway were regulated by CrzA. Several members of the two-component system (TCS) and the HOG pathway were more sensitive to calcium. CrzA::GFP was translocated to the nucleus upon osmotic stress. CrzA is important for the phosphorylation of the SakA MAPK in response to osmotic shock. The ΔsskB was more sensitive to CaCl2 , NaCl, and paraquat stress, while being avirulent in a murine model of invasive pulmonary aspergillosis. The presence of CaCl2 and osmotic stresses resulted in synergistic inhibition of ΔcrzA and ΔsskB growth. These results suggest there is a genetic interaction between the A. fumigatus calcineurin-CrzA and HOG pathway that is essential for full virulence.

Brilacidin, a host defense peptide mimetic, potentiates ibrexafungerp antifungal activity against the human pathogenic fungus Aspergillus fumigatus.

Aspergillus fumigatus is the primary etiological agent of aspergillosis. Here, we show that the host defense peptide mimetic, brilacidin (BRI) can potentiate ibrexafungerp (IBX) against clinical isolates of A. fumigatus. CAS-resistant strains with mutations in fks1 that encodes the 1,3-ß-D-glucan synthase are not IBX-resistant and BRI+IBX can inhibit their growth. The combination of BRI+IBX plays a fungicidal role, increases the fungal cell permeability and decreases the fungal survival in the presence of A549 epithelial cells.

Brilacidin, a host defense peptide mimetic, potentiates ibrexafungerp antifungal activity against the human pathogenic fungus Aspergillus fumigatus.

Aspergillus fumigatus is the primary etiological agent of aspergillosis. Here, we show that the host defense peptide mimetic brilacidin (BRI) can potentiate ibrexafungerp (IBX) against clinical isolates of A. fumigatus. BRI + IBX can inhibit the growth of A. fumigatus voriconazole- and caspofungin-resistant clinical isolates. BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against viruses, bacteria, and fungi. In vitro, combination of BRI + IBX plays a fungicidal role, increases the fungal cell permeability, decreases the fungal survival in the presence of A549 epithelial cells, and appears as a promising antifungal therapeutic alternative against A. fumigatus. IMPORTANCE: Invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. Aspergillus fumigatus causes a series of distinct invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. A. fumigatus causes a spectrum of distinct clinical entities named aspergillosis, which the most severe form is the invasive pulmonary aspergillosis. There are few therapeutic options for treating aspergillosis and searching for new antifungal agents against this disease is very important. Here, we present brilacidin (BRI) as a synergizer o fibrexafungerp (IBX) against A. fumigatus. BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against bacteria and viruses. We propose the combination of BRI and IBX as a new antifungal combinatorial treatment against aspergillosis.

Milteforan, a promising veterinary commercial product against feline sporotrichosis.

Sporotrichosis, the cutaneous mycosis most commonly reported in Latin America, is caused by the Sporothrix clinical clade species, including Sporothrix brasiliensis and Sporothrix schenckii sensu stricto. In Brazil, S. brasiliensis represents a vital health threat to humans and domestic animals due to its zoonotic transmission. Itraconazole, terbinafine, and amphotericin B are the most used antifungals for treating sporotrichosis. However, many strains of S. brasiliensis and S. schenckii have shown resistance to these agents, highlighting the importance of finding new therapeutic options. Here, we demonstrate that milteforan, a commercial veterinary product against dog leishmaniasis whose active principle is miltefosine, is a possible therapeutic alternative for the treatment of sporotrichosis, as observed by its fungicidal activity in vitro against different strains of S. brasiliensis and S. schenckii, and by its antifungal activity when used to treat infected epithelial cells and macrophages. Our results suggest milteforan as a possible alternative to treat feline sporotrichosis.

Strain heterogeneity in a non-pathogenic fungus highlights factors contributing to virulence.

Fungal pathogens exhibit extensive strain heterogeneity, including variation in virulence. Whether closely related non-pathogenic species also exhibit strain heterogeneity remains unknown. Here, we comprehensively characterized the pathogenic potentials (i.e., the ability to cause morbidity and mortality) of 16 diverse strains of Aspergillus fischeri, a non-pathogenic close relative of the major pathogen Aspergillus fumigatus. In vitro immune response assays and in vivo virulence assays using a mouse model of pulmonary aspergillosis showed that A. fischeri strains varied widely in their pathogenic potential. Furthermore, pangenome analyses suggest that A. fischeri genomic and phenotypic diversity is even greater. Genomic, transcriptomic, and metabolomic profiling identified several pathways and secondary metabolites associated with variation in virulence. Notably, strain virulence was associated with the simultaneous presence of the secondary metabolites hexadehydroastechrome and gliotoxin. We submit that examining the pathogenic potentials of non-pathogenic close relatives is key for understanding the origins of fungal pathogenicity.

Aspergillus fumigatus mitogen-activated protein kinase MpkA is involved in gliotoxin production and self-protection.

Aspergillus fumigatus is a saprophytic fungus that can cause a variety of human diseases known as aspergillosis. Mycotoxin gliotoxin (GT) production is important for its virulence and must be tightly regulated to avoid excess production and toxicity to the fungus. GT self-protection by GliT oxidoreductase and GtmA methyltransferase activities is related to the subcellular localization of these enzymes and how GT can be sequestered from the cytoplasm to avoid increased cell damage. Here, we show that GliT:GFP and GtmA:GFP are localized in the cytoplasm and in vacuoles during GT production. The Mitogen-Activated Protein kinase MpkA is essential for GT production and self-protection, interacts physically with GliT and GtmA and it is necessary for their regulation and subsequent presence in the vacuoles. The sensor histidine kinase SlnASln1 is important for modulation of MpkA phosphorylation. Our work emphasizes the importance of MpkA and compartmentalization of cellular events for GT production and self-defense.

Sirtuin E deacetylase is required for full virulence of Aspergillus fumigatus.

Aspergillus fumigatus represents a public health problem due to the high mortality rate in immunosuppressed patients and the emergence of antifungal-resistant isolates. Protein acetylation is a crucial post-translational modification that controls gene expression and biological processes. The strategic manipulation of enzymes involved in protein acetylation has emerged as a promising therapeutic approach for addressing fungal infections. Sirtuins, NAD+-dependent lysine deacetylases, regulate protein acetylation and gene expression in eukaryotes. However, their role in the human pathogenic fungus A. fumigatus remains unclear. This study constructs six single knockout strains of A. fumigatus and a strain lacking all predicted sirtuins (SIRTKO). The mutant strains are viable under laboratory conditions, indicating that sirtuins are not essential genes. Phenotypic assays suggest sirtuins' involvement in cell wall integrity, secondary metabolite production, thermotolerance, and virulence. Deletion of sirE attenuates virulence in murine and Galleria mellonella infection models. The absence of SirE alters the acetylation status of proteins, including histones and non-histones, and triggers significant changes in the expression of genes associated with secondary metabolism, cell wall biosynthesis, and virulence factors. These findings encourage testing sirtuin inhibitors as potential therapeutic strategies to combat A. fumigatus infections or in combination therapy with available antifungals.

Brilacidin, a novel antifungal agent against Cryptococcus neoformans.

Cryptococcus neoformans causes cryptococcosis, one of the most prevalent fungal diseases, generally characterized by meningitis. There is a limited and not very effective number of drugs available to combat this disease. In this manuscript, we show the host defense peptide mimetic brilacidin (BRI) as a promising antifungal drug against C. neoformans. BRI can affect the organization of the cell membrane, increasing the fungal cell permeability. We also investigated the effects of BRI against the model system Saccharomyces cerevisiae by analyzing libraries of mutants grown in the presence of BRI. In S. cerevisiae, BRI also affects the cell membrane organization, but in addition the cell wall integrity pathway and calcium metabolism. In vivo experiments show BRI significantly reduces C. neoformans survival inside macrophages and partially clears C. neoformans lung infection in an immunocompetent murine model of invasive pulmonary cryptococcosis. We also observed that BRI interacts with caspofungin (CAS) and amphotericin (AmB), potentiating their mechanism of action against C. neoformans. BRI + CAS affects endocytic movement, calcineurin, and mitogen-activated protein kinases. Our results indicate that BRI is a novel antifungal drug against cryptococcosis. IMPORTANCE: Invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. Cryptococcosis, one of the most prevalent fungal diseases, is generally characterized by meningitis and is mainly caused by two closely related species of basidiomycetous yeasts, Cryptococcus neoformans and Cryptococcus gattii. There are few therapeutic options for treating cryptococcosis, and searching for new antifungal agents against this disease is very important. Here, we present brilacidin (BRI) as a potential antifungal agent against C. neoformans. BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against bacteria and viruses. BRI alone was shown to inhibit the growth of C. neoformans, acting as a fungicidal drug, but surprisingly also potentiated the activity of caspofungin (CAS) against this species. We investigated the mechanism of action of BRI and BRI + CAS against C. neoformans. We propose BRI as a new antifungal agent against cryptococcosis.

Biochemical, physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves.

BACKGROUND: Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H2O2 and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx). METHODS: Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography. RESULTS: Vu-2-Cys-Prx reduces H2O2 using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56­4.72; its NH2-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% α-helix, 39% ß-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys52 residue and the amino acids Pro45, Thr49 and Arg128 are conserved as in other 2-Cys-Prx. GENERAL SIGNIFICANCE: The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins.

Identification of the cell targets important for propolis-induced cell death in Candida albicans.

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 result in invasive, systemic infections that are associated with a high incidence of mortality. Propolis is a complex mixture of several resinous substances which are collected from plants by bees. Here, 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. 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.