Competition of Candida glabrata against Lactobacillus is Hog1 dependent.

Candida glabrata is a common human fungal commensal and opportunistic pathogen. This fungus shows remarkable resilience as it can form recalcitrant biofilms on indwelling catheters, has intrinsic resistance against azole antifungals, and is causing vulvovaginal candidiasis. As a nosocomial pathogen, it can cause life-threatening bloodstream infections in immune-compromised patients. Here, we investigate the potential role of the high osmolarity glycerol response (HOG) MAP kinase pathway for C. glabrata virulence. The C. glabrata MAP kinase CgHog1 becomes activated by a variety of environmental stress conditions such as osmotic stress, low pH, and carboxylic acids and subsequently accumulates in the nucleus. We found that CgHog1 allows C. glabrata to persist within murine macrophages, but it is not required for systemic infection in a mouse model. C. glabrata and Lactobacilli co-colonise mucosal surfaces. Lactic acid at a concentration produced by vaginal Lactobacillus spp. causes CgHog1 phosphorylation and accumulation in the nucleus. In addition, CgHog1 enables C. glabrata to tolerate different Lactobacillus spp. and their metabolites when grown in co-culture. Using a phenotypic diverse set of clinical C. glabrata isolates, we find that the HOG pathway is likely the main quantitative determinant of lactic acid stress resistance. Taken together, our data indicate that CgHog1 has an important role in the confrontation of C. glabrata with the common vaginal flora.

Gentisaldehyde and Its Derivative 2,3-Dihydroxybenzaldehyde Show Antimicrobial Activities Against Bovine Mastitis Staphylococcus aureus.

Bovine mastitis is a worldwide disease of dairy cattle associated with significant economic losses for the dairy industry. One of the most common pathogens responsible for mastitis is Staphylococcus (S.) aureus. Due to the development and spreading of antibiotic resistance, the search for novel antimicrobial substances against S. aureus is of great importance. The aim of this study was to evaluate two dihydroxybenzaldehydes for the prevention of bovine mastitis. Therefore we determined the minimal inhibitory concentration (MICs) of gentisaldehyde (2,5-dihydroxybenzaldehyde) and 2,3-dihydroxybenzaldehyde of a diverse set of 172 bovine mastitis S. aureus isolates using an automated robot-based microdilution method. To characterize the bovine isolates we determined the genotype by spa-typing, the antimicrobial resistance to eight antibiotic classes using the disk diffusion method and the MICs of three commonly used antiseptics (benzalkonium chloride, chlorhexidine, and iodine). Further we investigated the cytotoxicity of gentisaldehyde and 2,3-dihydroxybenzaldehyde in bovine mammary epithelial MAC-T cells using the XTT assay. The S. aureus strains showed a high genetic diversity with 52 different spa-types, including five novel types. Antibiotic susceptibility testing revealed that 24% of isolates were resistant to one antimicrobial agent and 3% of isolates were multi-resistant. The occurrence of antibiotic resistance strongly correlated with the spa-type. Both dihydroxybenzaldehydes showed antimicrobial activities with a MIC50 of 500 mg/L. The MIC of gentisaldehyde significantly correlated with that of 2,3-dihydroxybenzaldehyde, whereas no correlation was observed with the MIC of the three antiseptics. Cytotoxicity testing using bovine mammary epithelial MAC-T cells revealed that gentisaldehyde and 2,3-dihydroxybenzaldehyde show low toxicity at MIC50 and MIC90 concentrations. In conclusion, gentisaldehyde and 2,3-dihydroxybenzaldehyde exhibited antimicrobial activities against a diverse range of bovine mastitis S. aureus strains at low-cytotoxic concentrations. Therefore, both compounds are potential candidates as antiseptics to prevent bovine mastitis and to reduce the use of antibiotics in dairy cows.

A robust high-throughput fungal biosensor assay for the detection of estrogen activity.

Estrogenic active compounds are present in a variety of sources and may alter biological functions in vertebrates. Therefore, it is crucial to develop innovative analytical systems that allow us to screen a broad spectrum of matrices and deliver fast and reliable results. We present the adaptation and validation of a fungal biosensor for the detection of estrogen activity in cow derived samples and tested the clinical applicability for pregnancy diagnosis in 140 mares and 120 cows. As biosensor we used a previously engineered genetically modified strain of the filamentous fungus Aspergillus nidulans, which contains the human estrogen receptor alpha and a reporter construct, in which ß-galactosidase gene expression is controlled by an estrogen-responsive-element. The estrogen response of the fungal biosensor was validated with blood, urine, feces, milk and saliva. All matrices were screened for estrogenic activity prior to and after chemical extraction and the results were compared to an enzyme immunoassay (EIA). The biosensor showed consistent results in milk, urine and feces, which were comparable to those of the EIA. In contrast to the EIA, no sample pre-treatment by chemical extraction was needed. For 17ß-estradiol, the biosensor showed a limit of detection of 1ng/L. The validation of the biosensor for pregnancy diagnosis revealed a specificity of 100% and a sensitivity of more than 97%. In conclusion, we developed and validated a highly robust fungal biosensor for detection of estrogen activity, which is highly sensitive and economic as it allows analyzing in high-throughput formats without the necessity for organic solvents.

Butyrate influences intracellular levels of adenine and adenine derivatives in the fungus Penicillium restrictum.

Butyrate, a small fatty acid, has an important role in the colon of ruminants and mammalians including the inhibition of inflammation and the regulation of cell proliferation. There is also growing evidence that butyrate is influencing the histone structure in mammalian cells by inhibition of histone deacetylation. Butyrate shows furthermore an antimicrobial activity against fungi, yeast and bacteria, which is linked to its toxicity at a high concentration. In fungi there are indications that butyrate induces the production of secondary metabolites potentially via inhibition of histone deacetylases. However, information about the influence of butyrate on growth, primary metabolite production and metabolism, besides lipid catabolism, in fungi is scarce. We have identified the filamentous fungus Penicillium (P.) restrictum as a susceptible target for butyrate treatment in an antimicrobial activity screen. The antimicrobial activity was detected only in the mycelium of the butyrate treated culture. We investigated the effect of butyrate ranging from low (0.001mM) to high (30mM), potentially toxic, concentrations on biomass and antimicrobial activity. Butyrate at high concentrations (3 and 30mM) significantly reduced the fungal biomass. In contrast P. restrictum treated with 0.03mM of butyrate showed the highest antimicrobial activity. We isolated three antimicrobial active compounds, active against Staphylococcus aureus, from P. restrictum cellular extracts treated with butyrate: adenine, its derivate hypoxanthine and the nucleoside derivate adenosine. Production of all three compounds was increased at low butyrate concentrations. Furthermore we found that butyrate influences the intracellular level of the adenine nucleoside derivate cAMP, an important signalling molecule in fungi and various organisms. In conclusion butyrate treatment increases the intracellular levels of adenine and its respective derivatives.

Valproic Acid Induces Antimicrobial Compound Production in Doratomyces microspores.

One of the biggest challenges in public health is the rising number of antibiotic resistant pathogens and the lack of novel antibiotics. In recent years there is a rising focus on fungi as sources of antimicrobial compounds due to their ability to produce a large variety of bioactive compounds and the observation that virtually every fungus may still contain yet unknown so called "cryptic," often silenced, compounds. These putative metabolites could include novel bioactive compounds. Considerable effort is spent on methods to induce production of these "cryptic" metabolites. One approach is the use of small molecule effectors, potentially influencing chromatin landscape in fungi. We observed that the supernatant of the fungus Doratomyces (D.) microsporus treated with valproic acid (VPA) displayed antimicrobial activity against Staphylococcus (S.) aureus and two methicillin resistant clinical S. aureus isolates. VPA treatment resulted in enhanced production of seven antimicrobial compounds: cyclo-(L-proline-L-methionine) (cPM), p-hydroxybenzaldehyde, cyclo-(phenylalanine-proline) (cFP), indole-3-carboxylic acid, phenylacetic acid (PAA) and indole-3-acetic acid. The production of the antimicrobial compound phenyllactic acid was exclusively detectable after VPA treatment. Furthermore three compounds, cPM, cFP, and PAA, were able to boost the antimicrobial activity of other antimicrobial compounds. cPM, for the first time isolated from fungi, and to a lesser extent PAA, are even able to decrease the minimal inhibitory concentration of ampicillin in MRSA strains. In conclusion we could show in this study that VPA treatment is a potent tool for induction of "cryptic" antimicrobial compound production in fungi, and that the induced compounds are not exclusively linked to the secondary metabolism. Furthermore this is the first discovery of the rare diketopiperazine cPM in fungi. Additionally we could demonstrate that cPM and PAA boost antibiotic activity against antibiotic resistant strains, suggesting a possible application in combinatorial antibiotic treatment against resistant pathogens.

Development of Genetic Dereplication Strains in Aspergillus nidulans Results in the Discovery of Aspercryptin.

To reduce the secondary metabolite background in Aspergillus nidulans and minimize the rediscovery of compounds and pathway intermediates, we created a "genetic dereplication" strain in which we deleted eight of the most highly expressed secondary metabolite gene clusters (more than 244,000 base pairs deleted in total). This strain allowed us to discover a novel compound that we designate aspercryptin and to propose a biosynthetic pathway for the compound. Interestingly, aspercryptin is formed from compounds produced by two separate gene clusters, one of which makes the well-known product cichorine. This raises the exciting possibility that fungi use differential regulation of expression of secondary metabolite gene clusters to increase the diversity of metabolites they produce.

Fungi treated with small chemicals exhibit increased antimicrobial activity against facultative bacterial and yeast pathogens.

For decades, fungi have been the main source for the discovery of novel antimicrobial drugs. Recent sequencing efforts revealed a still high number of so far unknown "cryptic" secondary metabolites. The production of these metabolites is presumably epigenetically silenced under standard laboratory conditions. In this study, we investigated the effect of six small mass chemicals, of which some are known to act as epigenetic modulators, on the production of antimicrobial compounds in 54 spore forming fungi. The antimicrobial effect of fungal samples was tested against clinically facultative pathogens and multiresistant clinical isolates. In total, 30 samples of treated fungi belonging to six different genera reduced significantly growth of different test organisms compared to the untreated fungal sample (growth log reduction 0.3-4.3). For instance, the pellet of Penicillium restrictum grown in the presence of butyrate revealed significant higher antimicrobial activity against Staphylococcus (S.) aureus and multiresistant S. aureus strains and displayed no cytotoxicity against human cells, thus making it an ideal candidate for antimicrobial compound discovery. Our study shows that every presumable fungus, even well described fungi, has the potential to produce novel antimicrobial compounds and that our approach is capable of rapidly filling the pipeline for yet undiscovered antimicrobial substances.

Small chemical chromatin effectors alter secondary metabolite production in Aspergillus clavatus.

The filamentous fungus Aspergillus clavatus is known to produce a variety of secondary metabolites (SM) such as patulin, pseurotin A, and cytochalasin E. In fungi, the production of most SM is strongly influenced by environmental factors and nutrients. Furthermore, it has been shown that the regulation of SM gene clusters is largely based on modulation of a chromatin structure. Communication between fungi and bacteria also triggers chromatin-based induction of silent SM gene clusters. Consequently, chemical chromatin effectors known to inhibit histone deacetylases (HDACs) and DNA-methyltransferases (DNMTs) influence the SM profile of several fungi. In this study, we tested the effect of five different chemicals, which are known to affect chromatin structure, on SM production in A. clavatus using two growth media with a different organic nitrogen source. We found that production of patulin was completely inhibited and cytochalasin E levels strongly reduced, whereas growing A. clavatus in media containing soya-derived peptone led to substantially higher pseurotin A levels. The HDAC inhibitors valproic acid, trichostatin A and butyrate, as well as the DNMT inhibitor 5-azacytidine (AZA) and N-acetyl-D-glucosamine, which was used as a proxy for bacterial fungal co-cultivation, had profound influence on SM accumulation and transcription of the corresponding biosynthetic genes. However, the repressing effect of the soya-based nitrogen source on patulin production could not be bypassed by any of the small chemical chromatin effectors. Interestingly, AZA influenced some SM cluster genes and SM production although no Aspergillus species has yet been shown to carry detectable DNA methylation.