ABSTRACT
The Deinococcus genus is renowned for its remarkable resilience against environmental stresses, including ionizing radiation, desiccation, and oxidative damage. This resilience is attributed to its sophisticated DNA repair mechanisms and robust defense systems, enabling it to recover from extensive damage and thrive under extreme conditions. Central to Deinococcus research, the D. radiodurans strains ATCC BAA-816 and ATCC 13939 facilitate extensive studies into this remarkably resilient genus. This study focused on delineating genetic discrepancies between these strains by sequencing our laboratory's ATCC 13939 specimen (ATCC 13939K) and juxtaposing it with ATCC BAA-816. We uncovered 436 DNA sequence differences within ATCC 13939K, including 100 single nucleotide variations, 278 insertions, and 58 deletions, which could induce frameshifts altering protein-coding genes. Gene annotation revisions accounting for gene fusions and the reconciliation of gene lengths uncovered novel protein-coding genes and refined the functional categorizations of established ones. Additionally, the analysis pointed out genome structural variations due to insertion sequence (IS) elements, underscoring the D. radiodurans genome's plasticity. Notably, ATCC 13939K exhibited a loss of six ISDra2 elements relative to BAA-816, restoring genes fragmented by ISDra2, such as those encoding for α/ß hydrolase and serine protease, and revealing new open reading frames, including genes imperative for acetoin decomposition. This comparative genomic study offers vital insights into the metabolic capabilities and resilience strategies of D. radiodurans.
ABSTRACT
The sophisticated ability of living organisms to sense and respond to external stimuli is critical for survival. This is particularly true for fungal pathogens, where the capacity to adapt and proliferate within a host is essential. To this end, signaling pathways, whether evolutionarily conserved or unique, have been refined through interactions with the host. Cryptococcus neoformans, an opportunistic fungal pathogen, is responsible for over 190,000 cases and an estimated 147,000 annual deaths globally. Extensive research over the past decades has shed light on the signaling pathways underpinning the pathogenicity of C. neoformans, as well as the host's responses during infection. In this context, we delineate the regulatory mechanisms employed by C. neoformans to detect and react to stresses derived from the host.
Subject(s)
Cryptococcus neoformans , Host-Pathogen Interactions , Signal Transduction , Stress, Physiological , Cryptococcus neoformans/pathogenicity , Cryptococcus neoformans/physiology , Cryptococcus neoformans/metabolism , Cryptococcus neoformans/genetics , Humans , Animals , Meningitis, Cryptococcal/microbiology , Gene Expression Regulation, Fungal , Fungal Proteins/metabolism , Fungal Proteins/geneticsABSTRACT
Dual-specificity LAMMER kinases are highly evolutionarily conserved in eukaryotes and play pivotal roles in diverse physiological processes, such as growth, differentiation, and stress responses. Although the functions of LAMMER kinase in fungal pathogens in pathogenicity and stress responses have been characterized, its role in Cryptococcus neoformans, a human fungal pathogen and a model yeast of basidiomycetes, remains elusive. In this study, we identified a LKH1 homologous gene and constructed a strain with a deleted LKH1 and a complemented strain. Similar to other fungi, the lkh1Δ mutant showed intrinsic growth defects. We observed that C. neoformans Lkh1 was involved in diverse stress responses, including oxidative stress and cell wall stress. Particularly, Lkh1 regulates DNA damage responses in Rad53-dependent and -independent manners. Furthermore, the absence of LKH1 reduced basidiospore formation. Our observations indicate that Lkh1 becomes hyperphosphorylated upon treatment with rapamycin, a TOR protein inhibitor. Notably, LKH1 deletion led to defects in melanin synthesis and capsule formation. Furthermore, we found that the deletion of LKH1 led to the avirulence of C. neoformans in a systemic cryptococcosis murine model. Taken together, Lkh1 is required for the stress response, sexual differentiation, and virulence of C. neoformans.
Subject(s)
Cryptococcosis , Cryptococcus neoformans , Fungal Proteins , Virulence , Animals , Female , Humans , Mice , Cell Wall/metabolism , Cryptococcosis/microbiology , Cryptococcus neoformans/pathogenicity , Cryptococcus neoformans/genetics , Cryptococcus neoformans/enzymology , Disease Models, Animal , DNA Damage , Fungal Capsules/metabolism , Fungal Capsules/genetics , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Deletion , Gene Expression Regulation, Fungal , Melanins/metabolism , Mice, Inbred BALB C , Oxidative Stress , Phosphorylation , Sirolimus/pharmacology , Spores, Fungal/growth & development , Stress, PhysiologicalABSTRACT
Reactive oxygen species induce DNA strand breaks and DNA oxidation. DNA oxidation leads to DNA mismatches, resulting in mutations in the genome if not properly repaired. Homologous recombination (HR) and non-homologous end-joining (NHEJ) are required for DNA strand breaks, whereas the base excision repair system mainly repairs oxidized DNAs, such as 8-oxoguanine and thymine glycol, by cleaving the glycosidic bond, inserting correct nucleotides, and sealing the gap. Our previous studies revealed that the Rad53-Bdr1 pathway mainly controls DNA strand breaks through the regulation of HR- and NHEJ-related genes. However, the functional roles of genes involved in the base excision repair system remain elusive in Cryptococcus neoformans. In the present study, we identified OGG1 and NTG1 genes in the base excision repair system of C. neoformans, which are involved in DNA oxidation repair. The expression of OGG1 was induced in a Hog1-dependent manner under oxidative stress. On the other hand, the expression of NTG1 was strongly induced by DNA damage stress in a Rad53-independent manner. We demonstrated that the deletion of NTG1, but not OGG1, resulted in elevated susceptibility to DNA damage agents and oxidative stress inducers. Notably, the ntg1Δ mutant showed growth defects upon antifungal drug treatment. Although deletion of OGG1 or NTG1 did not increase mutation rates, the mutation profile of each ogg1Δ and ntg1Δ mutant was different from that of the wild-type strain. Taken together, we found that DNA N-glycosylase Ntg1 is required for oxidative DNA damage stress and antifungal drug resistance in C. neoformans.
Subject(s)
Cryptococcus neoformans , Cryptococcus neoformans/genetics , DNA Damage , DNA Repair , Oxidative Stress , MutationABSTRACT
Oxidative stress, arising from excess reactive oxygen species (ROS) or insufficient antioxidant defenses, can damage cellular components, such as lipids, proteins, and nucleic acids, resulting in cellular dysfunction. The relationship between oxidative stress and various health disorders has prompted investigations into potent antioxidants that counteract ROS's detrimental impacts. In this context, antioxidant peptides, composed of two to twenty amino acids, have emerged as a unique group of antioxidants and have found applications in food, nutraceuticals, and pharmaceuticals. Antioxidant peptides are sourced from natural ingredients, mainly proteins derived from foods like milk, eggs, meat, fish, and plants. These peptides can be freed from their precursor proteins through enzymatic hydrolysis, fermentation, or gastrointestinal digestion. Previously published studies focused on the origin and production methods of antioxidant peptides, describing their structure-activity relationship and the mechanisms of food-derived antioxidant peptides. Yet, the role of microorganisms hasn't been sufficiently explored, even though the production of antioxidant peptides frequently employs a variety of microorganisms, such as bacteria, fungi, and yeasts, which are recognized for producing specific proteases. This review aims to provide a comprehensive overview of microorganisms and their proteases participating in enzymatic hydrolysis and microbial fermentation to produce antioxidant peptides. This review also covers endogenous peptides originating from microorganisms. The information obtained from this review might guide the discovery of novel organisms adept at generating antioxidant peptides.
Subject(s)
Antioxidants , Peptides , Animals , Antioxidants/metabolism , Reactive Oxygen Species , Peptides/chemistry , Dietary Supplements , Peptide HydrolasesABSTRACT
In this work, we proposed a new damage model for estimating radiation-induced direct damage to biomolecular systems and validated its the effectiveness for pBR322 plasmids. The proposed model estimates radiation-induced damage to biomolecular systems by: (1) simulation geometry modeling using the coarse-grained (CG) technique to replace the minimum repeating units of a molecule with a single bead, (2) approximation of the threshold energy for radiation damage through CG potential calculation, (3) calculation of cumulative absorption energy for each radiation event in microscopic regions of CG models using the Monte Carlo track structure (MCTS) code, and (4) estimation of direct radiation damage to biomolecular systems by comparing CG potentials and absorption energy. The proposed model replicated measured data with an average error of approximately 14.2% in the estimation of radiation damage to pBR322 plasmids using the common MCTS code Geant4-DNA. This is similar to the results of previous simulation studies. However, in existing damage models, parameters are adjusted based on experimental data to increase the reliability of simulation results, whereas in the proposed model, they can be determined without using empirical data. Because the proposed model proposed is applicable to DNA and various biomolecular systems with minimal experimental data, it provides a new method that is convenient and effective for predicting damage in living organisms caused by radiation exposure.
Subject(s)
DNA , Computer Simulation , DNA/chemistry , Monte Carlo Method , Plasmids/genetics , Reproducibility of ResultsABSTRACT
A balance in the deoxyribonucleotide (dNTPs) intracellular concentration is critical for the DNA replication and repair processes. In the model yeast Saccharomyces cerevisiae, the Mec1-Rad53-Dun1 kinase cascade mainly regulates the ribonucleotide reductase (RNR) gene expression during DNA replication and DNA damage stress. However, the RNR regulatory mechanisms in basidiomycete fungi during DNA replication and damage stress remain elusive. Here, we observed that in C. neoformans, RNR1 (large RNR subunit) and RNR21 (one small RNR subunit) were required for cell viability, but not RNR22 (another small RNR subunit). RNR22 overexpression compensated for the lethality of RNR21 suppression. In contrast to the regulatory mechanisms of RNRs in S. cerevisiae, Rad53 and Chk1 kinases cooperatively or divergently controlled RNR1 and RNR21 expression under DNA damage and DNA replication stress. In particular, this study revealed that Chk1 mainly regulated RNR1 expression during DNA replication stress, whereas Rad53, rather than Chk1, played a significant role in controlling the expression of RNR21 during DNA damage stress. Furthermore, the expression of RNR22, not but RNR1 and RNR21, was suppressed by the Ssn6-Tup1 complex during DNA replication stress. Notably, we observed that RNR1 expression was mainly regulated by Mbs1, whereas RNR21 expression was cooperatively controlled by Mbs1 and Bdr1 as downstream factors of Rad53 and Chk1 during DNA replication and damage stress. Collectively, the regulation of RNRs in C. neoformans has both evolutionarily conserved and divergent features in DNA replication and DNA damage stress, compared with other yeasts. IMPORTANCE Upon DNA replication or damage stresses, it is critical to provide proper levels of deoxynucleotide triphosphates (dNTPs) and activate DNA repair machinery. Ribonucleotide reductases (RNRs), which are composed of large and small subunits, are required for synthesizing dNTP. An imbalance in the intracellular concentration of dNTPs caused by the perturbation of RNR results in a reduction in DNA repair fidelity. Despite the importance of their roles, functions and regulations of RNR have not been elucidated in the basidiomycete fungi. In this study, we found that the roles of RNR1, RNR21, and RNR22 genes encoding RNR subunits in the viability of C. neoformans. Furthermore, their expression levels are divergently regulated by the Rad53-Chk1 pathway and the Ssn6-Tup1 complex in response to DNA replication and damage stresses. Therefore, this study provides insight into the regulatory mechanisms of RNR genes to DNA replication and damage stresses in basidiomycete fungi.
Subject(s)
Cryptococcus neoformans , DNA Damage , Ribonucleotide Reductases , Checkpoint Kinase 2/genetics , Checkpoint Kinase 2/metabolism , Cryptococcus neoformans/enzymology , Cryptococcus neoformans/genetics , DNA Replication , Ribonucleotide Reductases/genetics , Ribonucleotide Reductases/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolismABSTRACT
DNA double-strand breaks (DSBs) are the most deleterious type of DNA lesions because they cause loss of genetic information if not properly repaired. In eukaryotes, homologous recombination (HR) and non-homologous end joining (NHEJ) are required for DSB repair. However, the relationship of HR and NHEJ in DNA damage stress is unknown in the radiation-resistant fungus Cryptococcus neoformans. In this study, we found that the expression levels of HR- and NHEJ-related genes were highly induced in a Rad53-Bdr1 pathway-dependent manner under genotoxic stress. Deletion of RAD51, which is one of the main components in the HR, resulted in growth under diverse types of DNA damage stress, whereas perturbations of KU70 and KU80, which belong to the NHEJ system, did not affect the genotoxic stresses except when bleomycin was used for treatment. Furthermore, deletion of both RAD51 and KU70/80 renders cells susceptible to oxidative stress. Notably, we found that deletion of RAD51 induced a hypermutator phenotype in the fluctuation assay. In contrast to the fluctuation assay, perturbation of KU70 or KU80 induced rapid microevolution similar to that induced by the deletion of RAD51. Collectively, Rad51-mediated HR and Ku70/Ku80-mediated NHEJ regulate the DNA damage response and maintain genome stability.
ABSTRACT
Due to lifespan extension and changes in global climate, the increase in mycoses caused by primary and opportunistic fungal pathogens is now a global concern. Despite increasing attention, limited options are available for the treatment of systematic and invasive mycoses, owing to the evolutionary similarity between humans and fungi. Although plants produce a diversity of chemicals to protect themselves from pathogens, the molecular targets and modes of action of these plant-derived chemicals have not been well characterized. Using a reverse genetics approach, the present study revealed that thymol, a monoterpene alcohol from Thymus vulgaris L., (Lamiaceae), exhibits antifungal activity against Cryptococcus neoformans by regulating multiple signaling pathways including calcineurin, unfolded protein response, and HOG (high-osmolarity glycerol) MAPK (mitogen-activated protein kinase) pathways. Thymol treatment reduced the intracellular concentration of Ca2+ by controlling the expression levels of calcium transporter genes in a calcineurin-dependent manner. We demonstrated that thymol decreased N-glycosylation by regulating the expression levels of genes involved in glycan-mediated post-translational modifications. Furthermore, thymol treatment reduced endogenous ergosterol content by decreasing the expression of ergosterol biosynthesis genes in a HOG MAPK pathway-dependent manner. Collectively, this study sheds light on the antifungal mechanisms of thymol against C. neoformans.
Subject(s)
Antifungal Agents/pharmacology , Cryptococcosis/drug therapy , Cryptococcus neoformans/drug effects , Thymol/pharmacology , Calcineurin/metabolism , Cryptococcosis/metabolism , Cryptococcus neoformans/metabolism , Ergosterol/pharmacology , Fungal Proteins/metabolism , Humans , Mitogen-Activated Protein Kinases/metabolism , Monoterpenes/pharmacology , Signal Transduction/drug effects , Thymus Plant/chemistryABSTRACT
Signature-tagged mutagenesis (STM) is a high-throughput genetic technique that can be used to investigate the function of genes by constructing a large number of mutant strains with unique DNA identification tags, pooling them, and screening them for a particular phenotypic trait. STM was first designed for the identification of genes that contribute to the virulence or infectivity of a pathogen in its host. Recently, this method has also been applied for the identification of mutants with specific phenotypes, such as antifungal drug resistance and proliferation. In the present study, we describe an STM method for the identification of genes contributing to the infectivity of Cryptococcus neoformans using a mutant library, in which each strain was tagged with a unique DNA sequence.
Subject(s)
Cryptococcosis/pathology , Cryptococcus neoformans/genetics , Cryptococcus neoformans/pathogenicity , Genome, Fungal/genetics , Virulence/genetics , Animals , DNA, Fungal/genetics , Female , Gene Deletion , Genes, Fungal/genetics , Mice , Mice, Inbred A , PhenotypeABSTRACT
Phosphatases, together with kinases and transcription factors, are key components in cellular signalling networks. Here, we present a systematic functional analysis of the phosphatases in Cryptococcus neoformans, a fungal pathogen that causes life-threatening fungal meningoencephalitis. We analyse 230 signature-tagged mutant strains for 114 putative phosphatases under 30 distinct in vitro growth conditions, revealing at least one function for 60 of these proteins. Large-scale virulence and infectivity assays using insect and mouse models indicate roles in pathogenicity for 31 phosphatases involved in various processes such as thermotolerance, melanin and capsule production, stress responses, O-mannosylation, or retromer function. Notably, phosphatases Xpp1, Ssu72, Siw14, and Sit4 promote blood-brain barrier adhesion and crossing by C. neoformans. Together with our previous systematic studies of transcription factors and kinases, our results provide comprehensive insight into the pathobiological signalling circuitry of C. neoformans.
Subject(s)
Cryptococcus neoformans/genetics , Fungal Proteins/genetics , Gene Expression Profiling/methods , Genome, Fungal/genetics , Genome-Wide Association Study/methods , Phosphoric Monoester Hydrolases/genetics , Animals , Cluster Analysis , Cryptococcosis/microbiology , Cryptococcus neoformans/pathogenicity , Female , Fungal Proteins/classification , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Mice, Inbred Strains , Phosphoric Monoester Hydrolases/classification , Phosphoric Monoester Hydrolases/metabolism , Phosphotransferases/classification , Phosphotransferases/genetics , Phosphotransferases/metabolism , Signal Transduction/genetics , Thermotolerance/genetics , Transcription Factors/classification , Transcription Factors/genetics , Transcription Factors/metabolism , Virulence/geneticsABSTRACT
We describe the design and performance evaluation of a portable gas chromatograph suitable for the analysis of volatile organic and odorous compounds at trace levels. The system comprises a carbon nanotube sponge preconcentrator, an electronic pressure control (EPC) unit, a temperature-programmable column module, and a fast-response photoionization detector. A built-in tablet computer controls instrumental parameters and chromatogram display functions. The compact GC with dimensions of 35 cm (l) × 26 cm (w) × 15 cm (h) is self-contained, weighing less than 5 kg without a battery pack, and uses no auxiliary compressed gases. Our design has three main advantages over conventional portable GCs: recharging configuration of ambient air as the carrier gas using a miniature diaphragm pump, precise control of column flow by the built-in canister and EPC system, and rapid thermal desorption of the preconcentrator facilitated by intrinsic resistivity of the carbon nanotube sponge. A 30 m, 0.28 mm I.D. capillary column operated at a head pressure of 14 psi provided a peak capacity of 55 for a 10 min isothermal analysis. The temperature-programmability feature could decrease the analysis time of less than 5 min for vapor mixture of benzene, toluene, ethylbenzene, and o-xylene. More than a 100-fold increase in sensitivity by preconcentrating a sample adsorption volume of 90 mL resulted in improved detection limits of 0.13 (benzene), 0.20 (toluene), 0.23 (ethylbenzene), and 0.28 (o-xylene) ppb (v/v). Our instrument displayed good stability and reproducibility of retention times (< 0.14% RSD) and intensities (< 4.5% RSD) for continuous measurements using the preconcentrator over 10 h. Thus, continuous and on-site determinations of trace volatile organic compounds in air samples with this instrument appear feasible.
Subject(s)
Air Pollutants/analysis , Chromatography, Gas/methods , Computer Systems , Hydrocarbons, Aromatic/analysis , Volatile Organic Compounds/analysis , Calibration , Gases/analysis , Limit of Detection , Nanotubes, Carbon/chemistry , Odorants/analysis , Pressure , Reproducibility of Results , Temperature , Time FactorsABSTRACT
Cryptococcus neoformans causes fatal fungal meningoencephalitis. Here, we study the roles played by fungal kinases and transcription factors (TFs) in blood-brain barrier (BBB) crossing and brain infection in mice. We use a brain infectivity assay to screen signature-tagged mutagenesis (STM)-based libraries of mutants defective in kinases and TFs, generated in the C. neoformans H99 strain. We also monitor in vivo transcription profiles of kinases and TFs during host infection using NanoString technology. These analyses identify signalling components involved in BBB adhesion and crossing, or survival in the brain parenchyma. The TFs Pdr802, Hob1, and Sre1 are required for infection under all the conditions tested here. Hob1 controls the expression of several factors involved in brain infection, including inositol transporters, a metalloprotease, PDR802, and SRE1. However, Hob1 is dispensable for most cellular functions in Cryptococcus deuterogattii R265, a strain that does not target the brain during infection. Our results indicate that Hob1 is a master regulator of brain infectivity in C. neoformans.
Subject(s)
Blood-Brain Barrier/metabolism , Cryptococcus neoformans/pathogenicity , Homeodomain Proteins/metabolism , Meningitis, Cryptococcal/pathology , Meningoencephalitis/pathology , Transcription Factors/metabolism , Animals , Brain/microbiology , Brain/pathology , Cryptococcus gattii/genetics , Cryptococcus gattii/metabolism , Cryptococcus gattii/pathogenicity , Cryptococcus neoformans/genetics , Cryptococcus neoformans/metabolism , Disease Models, Animal , Female , Fungal Proteins , Gene Expression Profiling , Gene Expression Regulation, Fungal , Homeodomain Proteins/genetics , Humans , Meningitis, Cryptococcal/microbiology , Meningoencephalitis/microbiology , Mice , Mutagenesis , Mutation , Permeability , Phosphotransferases/genetics , Signal Transduction/genetics , Transcription Factors/geneticsABSTRACT
Sensitive methods are required for in situ monitoring of volatile organic compounds (VOCs). Herein, carbon nanotube (CNT) sponges were investigated as a new type of adsorbent for enriching trace aromatic VOCs. A square pillar configuration (3 mm × 3 mm × 45 mm, 5 mg) of a CNT sponge was enclosed in a glass tube (4 mm i.d.). After accumulating the sample vapor, a direct current pulse (26 V, 0.5-3.0 s) through the CNT sponge allowed narrow desorption bandwidths of 0.48-0.84 s (with a photoionization detector) and 1.2 s (with a flame ionization detector) and high desorption efficiency (>96.5%). Gas chromatographic analysis of a nine-component VOC mixture (100 mL adsorption volume) gave enrichment factors of 88 (benzene) to 323 (toluene and m-xylene) with detection limits in the range of 0.9-2.6 ppb (v/v). These results demonstrate that CNT sponges are a promising preconcentrator material for trace detection of VOCs. The adsorption breakthrough experiments exhibited good correlation with the kinetic adsorption and Langmuir isotherm models. The maximum adsorption capacities of the CNT sponge increased in the order benzene (0.13 mg/g) < toluene (2.45 mg/g) < ethylbenzene (13.90 mg/g) < o-xylene (14.31 mg/g), with R2 values of >0.95. The rollup phenomena observed during multicomponent adsorption were explained by the competitive displacement or adsorption affinities of aromatic VOCs. The feasibility of the CNT sponge preconcentrator in a real environment was tested for interfering species (NO2 and NH3), laboratory air, and a human breath sample and demonstrated similar performance as in the controlled nine-component tests.
Subject(s)
Nanotubes, Carbon/chemistry , Volatile Organic Compounds/analysis , Adsorption , Benzene/analysis , Benzene Derivatives/analysis , Chromatography, Gas , Kinetics , Toluene/analysis , Xylenes/analysisABSTRACT
In the article titled "Molecular Characterization of Adenylyl Cyclase Complex Proteins Using Versatile Protein-Tagging Plasmid Systems in Cryptococcus neoformans", the authors noticed that the B4028 primer sequence was given incorrectly in the Table. S1. The correct primer sequence is 5'-CGCAAGCTTGGAGCCATGAAGATCCTGA- 3. The correct 'Table S1' is now available online. Furthermore, we found typos in the supplementary data and revised them as follow. 'Fig. 2. Melanin and capsule analyses of tagging strains' should be changed to 'Fig. S2. Melanin and capsule analyses of tagging strains'. 'Table 2. Strains used in this study' should be changed to 'Table S2. Strains used in this study'. 'Table 2. Plasmid used in this study' should be changed to 'Table S3. Plasmids used in this study'.
ABSTRACT
A carbon nanotube (CNT) sponge was synthesized and examined as an adsorptive material for a thermally desorbed preconcentrator for volatile organic compounds (VOCs). The porous sponge-like material, retaining the intrinsic properties of individual multiwalled (MW) CNTs, was fabricated using spray pyrolysis chemical vapor deposition (CVD). The square pillar form of the CNT sponge was enclosed in a 1/4â³ glass tube with fittings for flow-through sampling. Flow of a direct current through the CNT sponge allowed rapid thermal heating to a surface temperature of 264.7 â at a rate of 481.5 â/s and a narrow desorption bandwidth of 0.74â¯s. The preconcentration concept was validated using gas chromatographic analysis of an aromatic VOC mixture, including benzene, toluene, ethylbenzene, and o-xylene (BTEX) vapors at concentrations of 100 parts per billion (ppb). With an adsorption volume of only 100â¯mL, the enrichment factor of each analyte was 300 (B), 240 (T), 210 (E), and 200 (X), enabling sensitive measurements with limits of detection at the parts per trillion level. Sequential desorption experiments confirmed that a single desorption process evaporates all the analytes inside the preconcentrator with >96% efficiency. There was no humidity effect and no sign of performance degradation after continuous operation for 45 repeated cycles. These results demonstrate that CNT sponges are a suitable material for the enrichment and sensitive determination of VOCs at trace levels. Thus, CNT sponge preconcentrators are advantageous in a variety of applications that permit fast and accurate real-time measurements, including ambient air and workplace air monitoring.
Subject(s)
Hydrocarbons, Aromatic/analysis , Nanotubes, Carbon/chemistry , Volatile Organic Compounds/analysis , Adsorption , Air Pollutants/analysis , Benzene/analysis , Chromatography, Gas , Humidity , Toluene/analysis , XylenesABSTRACT
Living organisms are constantly exposed to DNA damage stress caused by endogenous and exogenous events. Eukaryotic cells have evolutionarily conserved DNA damage checkpoint surveillance systems. We previously reported that a unique transcription factor, Bdr1, whose expression is strongly induced by the protein kinase Rad53 governs DNA damage responses by controlling the expression of DNA repair genes in the basidiomycetous fungus Cryptococcus neoformans However, the regulatory mechanism of the Rad53-dependent DNA damage signal cascade and its function in pathogenicity remain unclear. Here, we demonstrate that Rad53 is required for DNA damage response and is phosphorylated by two phosphatidylinositol 3-kinase (PI3K)-like kinases, Tel1 and Mec1, in response to DNA damage stress. Transcriptome analysis revealed that Rad53 regulates the expression of several DNA repair genes in response to gamma radiation. We found that expression of CHK1, another effector kinase involved in the DNA damage response, is regulated by Rad53 and that CHK1 deletion rendered cells highly susceptible to DNA damage stress. Nevertheless, BDR1 expression is regulated by Rad53, but not Chk1, indicating that DNA damage signal cascades mediated by Rad53 and Chk1 exhibit redundant and distinct functions. We found that perturbation of both RAD53 and CHK1 attenuated the virulence of C. neoformans, perhaps by promoting phagosome maturation within macrophage, reducing melanin production, and increasing susceptibility to oxidative stresses. Furthermore, deletion of both RAD53 and CHK1 increased susceptibility to certain antifungal drugs such as amphotericin B. This report provides insight into the regulatory mechanism of fungal DNA damage repair systems and their functional relationship with fungal virulence and antifungal drug susceptibility.IMPORTANCE Genome instability is detrimental for living things because it induces genetic disorder diseases and transfers incorrect genome information to descendants. Therefore, living organisms have evolutionarily conserved signaling networks to sense and repair DNA damage. However, how the DNA damage response pathway is regulated for maintaining the genome integrity of fungal pathogens and how this contributes to their pathogenicity remain elusive. In this study, we investigated the DNA damage response pathway in the basidiomycete pathogen Cryptococcus neoformans, which causes life-threatening meningoencephalitis in immunocompromised individuals, with an average of 223,100 infections leading to 181,100 deaths reported annually. Here, we found that perturbation of Rad53- and Chk1-dependent DNA damage response pathways attenuated the virulence of C. neoformans and increased its susceptibility to certain antifungal drugs, such as amphotericin B and flucytosine. Therefore, our work paves the way to understanding the important role of human fungal DNA damage networks in pathogenesis and antifungal drug susceptibility.
Subject(s)
Cell Cycle Proteins/metabolism , Cryptococcus neoformans/enzymology , Cryptococcus neoformans/growth & development , DNA Damage , DNA Repair , DNA, Fungal/metabolism , Gene Expression Regulation, Fungal , Antifungal Agents/pharmacology , Cryptococcus neoformans/drug effects , Cryptococcus neoformans/radiation effects , Gamma Rays , Gene Expression Profiling , Phosphorylation , Protein Processing, Post-TranslationalABSTRACT
Deinococcus radiodurans R1 is extremely resistant to ionizing radiation and oxidative stress. In this study, we characterized DR0846, a candidate peroxiredoxin in D. radiodurans. DR0846 is a peroxiredoxin Q containing two conserved cysteine residues. DR0846 exists mainly in monomeric form with an intramolecular disulfide bond between the two cysteine residues. We found that DR0846 functions as a molecular chaperone as well as a peroxidase. A mutational analysis indicates that the two cysteine residues are essential for enzymatic activity. A double-deletion mutant lacking DR0846 and catalase DR1998 exhibits decreased oxidative and heat shock stress tolerance with respect to the single mutants or the wild-type cells. These results suggest that DR0846 contributes to resistance against oxidative and heat stresses in D. radiodurans.
Subject(s)
Deinococcus/metabolism , Mutation , Peroxiredoxins/chemistry , Peroxiredoxins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cysteine/metabolism , Deinococcus/chemistry , Deinococcus/genetics , Gene Expression Regulation, Bacterial , Heat-Shock Response , Molecular Chaperones/chemistry , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Oxidative Stress , Peroxidase/chemistry , Peroxidase/genetics , Peroxidase/metabolism , Peroxiredoxins/geneticsABSTRACT
Cryptococcus neoformans is an opportunistic fungal pathogen, which causes life-threatening meningoencephalitis in immunocompromised individuals and is responsible for more than 1,000,000 infections and 600,000 deaths annually worldwide. Nevertheless, anti-cryptococcal therapeutic options are limited, mainly because of the similarity between fungal and human cellular structures. Owing to advances in genetic and molecular techniques and bioinformatics in the past decade, C. neoformans, belonging to the phylum basidiomycota, is now a major pathogenic fungal model system. In particular, genetic manipulation is the first step in the identification and characterization of the function of genes for understanding the mechanisms underlying the pathogenicity of C. neoformans. This unit describes protocols for constructing target gene deletion mutants using double-joint (DJ) PCR, constitutive overexpression strains using the histone H3 gene promoter, and epitope/fluorescence protein-tagged strains in C. neoformans. © 2018 by John Wiley & Sons, Inc.
Subject(s)
Blotting, Southern/methods , Cryptococcus neoformans/genetics , Genetic Complementation Test/methods , Genetic Engineering/methods , Polymerase Chain Reaction/methods , Cryptococcosis/microbiology , Cryptococcus neoformans/physiology , Humans , Transformation, BacterialABSTRACT
The unfolded protein response (UPR) pathway, consisting of the evolutionarily conserved Ire1 kinase/endonuclease and the bZIP transcription factor Hxl1, is critical for the pathogenicity of Cryptococcus neoformans; however, its role remains unknown in other pathogenic Cryptococcus species. Here, we investigated the role of the UPR pathway in C. deuterogattii, which causes pneumonia and systemic cryptococcosis, even in immunocompetent individuals. In response to ER stress, C. deuterogattii Ire1 triggers unconventional splicing of HXL1 to induce the expression of UPR target genes such as KAR2, DER1, ALG7, and ERG29. Furthermore, C. deuterogattii Ire1 is required for growth at mammalian body temperature, similar to C. neoformans Ire1. However, deletion of HXL1 does not significantly affect the growth of C. deuterogattii at 37 °C, which is in contrast to the indispensable role of HXL1 in the growth of C. neoformans at 37 °C. Nevertheless, both C. deuterogattii ire1Δ and hxl1Δ mutants are avirulent in a murine model of systemic cryptococcosis, suggesting that a non-thermotolerance phenotypic trait also contributes to the role of the UPR pathway in the virulence of pathogenic Cryptococcus species. In conclusion, the UPR pathway plays redundant and distinct roles in the virulence of members of the pathogenic Cryptococcus species complex.