Transcriptome analysis reveals gene expression changes of the basidiomycetous yeast Apiotrichum mycotoxinivorans in response to ochratoxin A exposure.

Ochratoxin A (OTA) is one of the most common and deleterious mycotoxins found in food and feedstuffs worldwide; however, Apiotrichum mycotoxinivorans can detoxify OTA. Our results show that A. mycotoxinivorans GUM1709 efficiently degraded OTA, but it caused the accumulation of intracellular reactive oxygen species. The main aim of this study was to identify potential OTA-detoxifying enzymes and to explore the effects of OTA on A. mycotoxinivorans GMU1709. RNA-seq data revealed that 1643 and 1980 genes were significantly upregulated and downregulated, respectively, after OTA exposure. Functional enrichment analyses indicated that OTA exposure enhanced defense capability, protein transport, endocytosis, and energy metabolism; caused ribosomal stress; suppressed DNA replication and transcription; inhibited cell growth and division; and promoted cell death. The integration of secretome, gene expression, and molecular docking analyses revealed that two carboxypeptidase homologues (members of the metallocarboxypeptidase family) were most likely responsible for the detoxification of both extracellular and intracellular OTA. Superoxide dismutase and catalase were the main genes activated in response to oxidative stress. In addition, analysis of key genes associated with cell division and apoptosis showed that OTA exposure inhibited mitosis and promoted cell death. This study revealed the possible OTA response and detoxification mechanisms in A. mycotoxinivorans.

Bacterial DNA promotes Tau aggregation.

A hallmark feature of Alzheimer's disease (AD) and other tauopathies is the misfolding, aggregation and cerebral accumulation of tau deposits. Compelling evidence indicates that misfolded tau aggregates are neurotoxic, producing synaptic loss and neuronal damage. Misfolded tau aggregates are able to spread the pathology from cell-to-cell by a prion like seeding mechanism. The factors implicated in the initiation and progression of tau misfolding and aggregation are largely unclear. In this study, we evaluated the effect of DNA extracted from diverse prokaryotic and eukaryotic cells in tau misfolding and aggregation. Our results show that DNA from various, unrelated gram-positive and gram-negative bacteria results in a more pronounced tau misfolding compared to eukaryotic DNA. Interestingly, a higher effect in promoting tau aggregation was observed for DNA extracted from certain bacterial species previously detected in the brain, CSF or oral cavity of patients with AD. Our findings indicate that microbial DNA may play a previously overlooked role in the propagation of tau protein misfolding and AD pathogenesis, providing a new conceptual framework that positions the compromised blood-brain and intestinal barriers as important sources of microbial DNA in the CNS, opening novel opportunities for therapeutic interventions.

Activation of myeloid dendritic cells by deoxynucleic acids from Cordyceps sinensis via a Toll-like receptor 9-dependent pathway.

The mechanism by which host cells recognize Cordyceps sinensis, a Chinese herbal medicine that is known to exhibit immunomodulating activity, remains poorly understood. In this study, we investigated whether the DNA of this fungus could activate mouse bone marrow-derived dendritic cells (BM-DCs). Upon stimulation with C. sinensis DNA, BM-DCs released IL-12p40 and TNF-alpha and expressed CD40. Cytokine production and CD40 expression were attenuated by chloroquin and bafilomycin A. Activation of BM-DCs by C. sinensis DNA was almost completely abrogated in TLR9KO mice. According to a luciferase reporter assay, C. sinensis DNA activated NF-kappaB in HEK293T cells transfected with the TLR9 gene. Finally, a confocal microscopic analysis showed that C. sinensis DNA was co-localized with CpG-ODN and partly with TLR9 and LAMP-1, a late endosomal marker, in BM-DCs. Our results demonstrated that C. sinensis DNA caused activation of BM-DCs in a TLR9-dependent manner.

Presence of extracellular DNA in the Candida albicans biofilm matrix and its contribution to biofilms.

DNA has been described as a structural component of the extracellular matrix (ECM) in bacterial biofilms. In Candida albicans, there is a scarce knowledge concerning the contribution of extracellular DNA (eDNA) to biofilm matrix and overall structure. This work examined the presence and quantified the amount of eDNA in C. albicans biofilm ECM and the effect of DNase treatment and the addition of exogenous DNA on C. albicans biofilm development as indicators of a role for eDNA in biofilm development. We were able to detect the accumulation of eDNA in biofilm ECM extracted from C. albicans biofilms formed under conditions of flow, although the quantity of eDNA detected differed according to growth conditions, in particular with regards to the medium used to grow the biofilms. Experiments with C. albicans biofilms formed statically using a microtiter plate model indicated that the addition of exogenous DNA (>160 ng/ml) increases biofilm biomass and, conversely, DNase treatment (>0.03 mg/ml) decreases biofilm biomass at later time points of biofilm development. We present evidence for the role of eDNA in C. albicans biofilm structure and formation, consistent with eDNA being a key element of the ECM in mature C. albicans biofilms and playing a predominant role in biofilm structural integrity and maintenance.

Effect of Candida albicans dsDNA in gastrointestinal Candida infection.

Neonates are highly sensitive to infections because they are biased to develop Th2 immune responses. When exposed to certain agents, such as DNA vaccines or CpG DNA motifs, neonates are capable to mount adult-like Th1 protective responses. This study investigates the capacity of Candida albicans (C. albicans) dsDNA to induce host resistance in newborn mice against gastrointestinal C. albicans infection. The protective properties of dsDNA are related to an increased number of spleen CD4+ T cells secreting IFN-gamma. In infected DNA-treated mice, an enhanced production of IFN-gamma by Peyer's patch cells was observed together with reduced colonization and histopathological changes in the stomach. Our results indicated that C. albicans dsDNA administration in neonates elicited the protective immune response against gastrointestinal Candida infection.

Candida albicans double-stranded DNA can participate in the host defense against disseminated candidiasis.

In the present work, we studied the in vitro immunomodulatory properties of double-stranded Candida albicans DNA and its protective effect in murine disseminated candidiasis. DNA induced the production of TNF-alpha by peritoneal macrophages and splenocytes in vitro through a chloroquine-dependent mechanism. Yeast DNA acted synergistically with IFN-gamma in triggering the secretion of nitric oxide by macrophages and enabled them to stimulate the proliferation of T cells in response to soluble anti-CD3. The effect of DNA on splenocytes is associated with an enhanced synthesis of IFN-gamma, IL-2 and IL-10. In vivo, DNA decreased the mortality and lowered the kidney contamination in mice intraperitoneally inoculated with C. albicans simultaneously with an increase in the specific proliferative response and cytokine production. The present results indicate that C. albicans DNA can provide protection against disseminated infection.

G-protein beta-subunit specificity in the fast membrane-delimited inhibition of Ca2+ channels.

We investigated which subtypes of G-protein beta subunits participate in voltage-dependent modulation of N-type calcium channels. Calcium currents were recorded from cultured rat superior cervical ganglion neurons injected intranuclearly with DNA encoding five different G-protein beta subunits. Gbeta1 and Gbeta2 strongly mimicked the fast voltage-dependent inhibition of calcium channels produced by many G-protein-coupled receptors. The Gbeta5 subunit produced much weaker effects than Gbeta1 and Gbeta2, whereas Gbeta3 and Gbeta4 were nearly inactive in these electrophysiological studies. The specificity implied by these results was confirmed and extended using the yeast two-hybrid system to test for protein-protein interactions. Here, Gbeta1 or Gbeta2 coupled to the GAL4-activation domain interacted strongly with a channel sequence corresponding to the intracellular loop connecting domains I and II of a alpha1 subunit of the class B calcium channel fused to the GAL4 DNA-binding domain. In this assay, the Gbeta5 subunit interacted weakly, and Gbeta3 and Gbeta4 failed to interact. Together, these results suggest that Gbeta1 and/or Gbeta2 subunits account for most of the voltage-dependent inhibition of N-type calcium channels and that the linker between domains I and II of the calcium channel alpha1 subunit is a principal receptor for this inhibition.

[The interferonogenic properties of yeast RNA-tilorone complexes in a cell culture]. / Izuchenie interferonogennykh svoistv kompleksov drozhzhevaia DNK--tiloron v kul'ture kletok.

The use of molecular complexes of yeast RNA with thyloron as inductors of type I interferons (alpha/beta-interferons) in the culture of cells L929 is described. It was shown that the complexes induced the interferon synthesis in the cells at the level comparable to that of the standard inductors of the polyribonucleotide nature i.e. larifan and poly(I)-poly(C). In the experimental doses the complexes proved to be nontoxic. It was concluded that the use of the yeast RNA and thyloron complexes as inductors in large-scale production of type I interferons was promising.

Polyphosphate present in DNA preparations from filamentous fungal species of Colletotrichum inhibits restriction endonucleases and other enzymes.

During the development of a procedure for the isolation of total genomic DNA from filamentous fungi (Rodriguez, R. J., and Yoder, O.C., Exp. Mycol. 15, 232-242, 1991) a cell fraction was isolated which inhibited the digestion of DNA by restriction enzymes. After elimination of DNA, RNA, proteins, and lipids, the active compound was purified by gel filtration to yield a single fraction capable of complete inhibition of restriction enzyme activity. The inhibitor did not absorb uv light above 220 nm, and was resistant to alkali and acid at 25 degrees C and to temperatures as high as 100 degrees C. More extensive analyses demonstrated that the inhibitor was also capable of inhibiting T4 DNA ligase and TaqI DNA polymerase, but not DNase or RNase. Chemical analyses indicated that the inhibitor was devoid of carbohydrates, proteins, lipids, and nucleic acids but rich in phosphorus. A combination of nuclear magnetic resonance, metachromatic shift of toluidine blue, and gel filtration indicated that the inhibitor was a polyphosphate (polyP) containing approximately 60 phosphate molecules. The mechanism of inhibition appeared to involve complexing of polyP to the enzymatic proteins. All species of Colletotrichum analyzed produced polyP equivalent in chain length and concentration. A modification to the original DNA extraction procedure is described which eliminates polyP and reduces the time necessary to obtain DNA of sufficient purity for restriction enzyme digestion and TaqI polymerase amplification.