Journal of Microbiology

Journal Articles

CXCL12/CXCR4 Axis is Involved in the Recruitment of NK Cells by HMGB1 Contributing to Persistent Airway Inflammation and AHR During the Late Stage of RSV Infection: Sisi Chen , Wei Tang , Guangyuan Yu , Zhengzhen Tang , Enmei Liu; J. Microbiol. 2023;61(4):461-469. Published online February 13, 2023; DOI: https://doi.org/10.1007/s12275-023-00018-8

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Abstract: We previously showed that both high-mobility group box-1 (HMGB1) and natural killer (NK) cells contribute to respiratory syncytial virus (RSV)-induced persistent airway inflammation and airway hyperresponsiveness (AHR). Meanwhile, Chemokine (C-X-C motif) ligand 12 (CXCL12) and its specific receptor (chemokine receptor 4, CXCR4) play important roles in recruitment of immune cells. CXCL12 has been reported to form a complex with HMGB1 that binds to CXCR4 and increases inflammatory cell migration. The relationship between HMGB1, NK cells and chemokines in RSV-infected model remains unclear. An anti-HMGB1 neutralizing antibody and inhibitor of CXCR4 (AMD3100) was administered to observe changes of NK cells and airway disorders in nude mice and BALB/c mice. Results showed that the mRNA expression and protein levels of HMGB1 were elevated in late stage of RSV infection and persistent airway inflammation and AHR were diminished after administration of anti-HMGB1 antibodies, with an associated significant decrease in CXCR4+ NK cells. In addition, CXCL12 and CXCR4 were reduced after HMGB1 blockade. Treatment with AMD3100 significantly suppressed the recruitment of NK cells and alleviated the airway disorders. Thus, CXCL12/CXCR4 axis is involved in the recruitment of NK cells by HMGB1, contributing to persistent airway inflammation and AHR during the late stage of RSV infection.

Transcriptome‑based Mining of the Constitutive Promoters for Tuning Gene Expression in Aspergillus oryzae: Kobkul Laoteng , Jutamas Anantayanon , Chanikul Chutrakul , Sarocha Panchanawaporn , Sukanya Jeennor; J. Microbiol. 2023;61(2):199-210. Published online February 6, 2023; DOI: https://doi.org/10.1007/s12275-023-00020-0

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Abstract: Transcriptional regulation has been adopted for developing metabolic engineering tools. The regulatory promoter is a crucial genetic element for strain optimization. In this study, a gene set of Aspergillus oryzae with highly constitutive expression across different growth stages was identified through transcriptome data analysis. The candidate promoters were functionally characterized in A. oryzae by transcriptional control of β-glucuronidase (GUS) as a reporter. The results showed that the glyceraldehyde triphosphate dehydrogenase promoter (PgpdA1) of A. oryzae with a unique structure displayed the most robust strength in constitutively controlling the expression compared to the PgpdA2 and other putative promoters tested. In addition, the ubiquitin promoter (Pubi) of A. oryzae exhibited a moderate expression strength. The deletion analysis revealed that the 5' untranslated regions of gpdA1 and ubi with the length of 1028 and 811 nucleotides, counted from the putative translation start site (ATG), respectively, could efficiently drive the GUS expression. Interestingly, both promoters could function on various carbon sources for cell growth. Glucose was the best fermentable carbon source for allocating high constitutive expressions during cell growth, and the high concentrations (6–8% glucose, w/v) did not repress their functions. It was also demonstrated that the secondary metabolite gene coding for indigoidine could express under the control of PgpdA1 or Pubi promoter. These strong and moderate promoters of A. oryzae provided beneficial options in tuning the transcriptional expression for leveraging the metabolic control towards the targeted products.

[Protocol]Rapid method for chromatin immunoprecipitation (ChIP) assay in a dimorphic fungus, Candida albicans: Jueun Kim , Jung-Shin Lee; J. Microbiol. 2020;58(1):11-16. Published online June 11, 2019; DOI: https://doi.org/10.1007/s12275-020-9143-2

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Abstract: A chromatin immunoprecipitation (ChIP) assay is a method to identify how much a protein of interest binds to the DNA region. This method is indispensable to study the mechanisms of how the transcription factors or chromatin modifications regulate the gene expression. Candida albicans is a dimorphic pathogenic fungus, which can change its morphology very rapidly from yeast to hypha in response to the environmental signal. The morphological change of C. albicans is one of the critical factors for its virulence. Therefore, it is necessary to understand how to regulate the expression of genes for C. albicans to change its morphology. One of the essential methods for us to understand this regulation is a ChIP assay. There have been many efforts to optimize the protocol to lower the background signal and to analyze the results accurately because a ChIP assay can provide very different results even with slight differences in the experimental procedure. We have optimized the rapid and efficient ChIP protocol so that it could be applied equally for both yeast and hyphal forms of C. albicans. Our method in this protocol is also comparatively rapid to the method widely used. In this protocol, we described our rapid method for the ChIP assay in C. albicans in detail.

Review

REVIEW] Revisiting old friends: Developments in understanding Histoplasma capsulatum pathogenesis: Jon P. Woods; J. Microbiol. 2016;54(3):265-276. Published online February 27, 2016; DOI: https://doi.org/10.1007/s12275-016-6044-5

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39 Citations

Abstract: Histoplasma capsulatum is a dimorphic pathogenic fungus and causative agent of histoplasmosis, which is a respiratory and systemic infection that is particularly severe in immunocompromised hosts and represents the fungal homolog of tuberculosis. In highly endemic regions, the majority of individuals have been infected and carry the organism in a persistent latent form that is a danger for reactivation if host defenses are suppressed. H. capsulatum has been a model organism for intracellular pathogenesis and fungal morphogenesis for decades. New genomic information and application of approaches for molecular genetic manipulation are shedding new light on virulence mechanisms.

Research Support, Non-U.S. Gov'ts

Azole-synergistic Anti-Candidal Activity of Altenusin, a Biphenyl Metabolite of the Endophytic Fungus Alternaria alternata Isolated from Terminalia chebula Retz.: Jatuporn Phaopongthai , Suthep Wiyakrutta , Duangdeun Meksuriyen , Nongluksna Sriubolmas , Khanit Suwanborirux; J. Microbiol. 2013;51(6):821-828. Published online December 19, 2013; DOI: https://doi.org/10.1007/s12275-013-3189-3

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28 Citations

Abstract: In this study, a tropical endophytic fungus, Alternaria alternata Tche-153 was isolated from a Thai medicinal plant Terminalia chebula Rezt. The ethyl acetate extract prepared from the fermentation broth exhibited significant ketoconazole- synergistic activity against Candida albicans. Bioassaydirected fractionation of the ethyl acetate extract led to the isolation of altenusin (1), isoochracinic acid (2), and altenuic acid (3) together with 2,5-dimethyl-7-hydroxychromone (4). Using the disc diffusion method and the microdilution chequerboard technique, only altenusin (1) in combination with each of three azole drugs, ketoconazole, fluconazole or itraconazole at their low sub-inhibitory concentrations exhibited potent synergistic activity against C. albicans with the fractional inhibitory concentration index range of 0.078 to 0.188. This first discovery of altenusin (1) as a new azole-synergistic prototype possessing a biphenyl structure is of significance for further development of new azole-synergists to treat invasive candidiasis.

The Role of a Dark Septate Endophytic Fungus, Veronaeopsis simplex Y34, in Fusarium Disease Suppression in Chinese Cabbage: Rida O. Khastini , Hiroyuki Ohta , Kazuhiko Narisawa; J. Microbiol. 2012;50(4):618-624. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-2105-6

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35 Citations

Abstract: The soil-inhabiting fungal pathogen Fusarium oxysporum has been an increasing threat to Chinese cabbage (Brassica campestris L.). A dark septate endophytic fungus, Veronaeopsis simplex Y34, isolated from Yaku Island, Japan, was evaluated in vitro for the ability to suppress Fusarium disease. Seedlings grown in the presence of the endophyte showed a 71% reduction in Fusarium wilt disease and still had good growth. The disease control was achieved through a synergetic effect involving a mechanical resistance created by a dense network of V. simplex Y34 hyphae, which colonized the host root, and siderophore production acting indirectly to induce a resistance mechanism in the plant. Changes in the relative abundance of the fungal communities in the soil as determined by fluorescently labelled T-RFs (terminal restriction fragments), appeared 3 weeks after application of the fungus. Results showed the dominance of V. simplex Y34, which became established in the rhizosphere and out-competed F. oxysporum.

Identification and Functional Analysis of a Gene Encoding β-Glucosidase from the Brown-Rot Basidiomycete Fomitopsis palustris: Hwang-Woo Ji , Chang-Jun Cha; J. Microbiol. 2010;48(6):808-813. Published online January 9, 2011; DOI: https://doi.org/10.1007/s12275-010-0482-2

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Abstract: The brown-rot basidiomycete Fomitopsis palustris is known to degrade crystalline cellulose (Avicel) and produce three major cellulases, exoglucanases, endoglucanases, and β-glucosidases. A novel β-glucosidase designated as Cel3A was identified from F. palustris grown at the expense of Avicel. The deduced amino acid sequence of Cel3A showed high homology with those of other fungal β-glucosidases that belong to glycosyl hydrolase (GH) family 3. The sequence analysis also indicated that Cel3A contains the N- and C-terminal domains of GH family 3 and Asp-209 was conserved as a catalytic nucleophile. The cloned gene was successfully expressed in the yeast Pichia pastoris and the recombinant protein exhibited β-glucosidase activity with cellobiose and some degree of thermostability. Considering the size and sequence of the protein, the β-glucosidase identified in this study is different from the protein purified directly from F. palustris in the previous study. Our results suggest that the fungus possesses at least two β-glucosidase genes.

Production, Partial Characterization, and Immobilization in Alginate Beads of an Alkaline Protease from a New Thermophilic Fungus Myceliophthora sp.: Letícia Maria Zanphorlin , Fernanda Dell Antonio Facchini , Filipe Vasconcelos , Rafaella Costa Bonugli-Santos , André Rodrigues , Lara Durães Sette , Eleni Gomes , Gustavo Orlando Bonilla-Rodriguez; J. Microbiol. 2010;48(3):331-336. Published online June 23, 2010; DOI: https://doi.org/10.1007/s12275-010-9269-8

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Abstract: Thermophilic fungi produce thermostable enzymes which have a number of applications, mainly in biotechnological processes. In this work, we describe the characterization of a protease produced in solidstate (SSF) and submerged (SmF) fermentations by a newly isolated thermophilic fungus identified as a putative new species in the genus Myceliophthora. Enzyme-production rate was evaluated for both fermentation processes, and in SSF, using a medium composed of a mixture of wheat bran and casein, the proteolytic output was 4.5-fold larger than that obtained in SmF. Additionally, the peak of proteolytic activity was obtained after 3 days for SSF whereas for SmF it was after 4 days. The crude enzyme obtained by both SSF and SmF displayed similar optimum temperature at 50°C, but the optimum pH shifted from 7 (SmF) to 9 (SSF). The alkaline protease produced through solid-state fermentation (SSF), was immobilized on beads of calcium alginate, allowing comparative analyses of free and immobilized proteases to be carried out. It was observed that both optimum temperature and thermal stability of the immobilized enzyme were higher than for the free enzyme. Moreover, the immobilized enzyme showed considerable stability for up to 7 reuses.

Effect of Fungal Pellet Morphology on Enzyme Activities Involved in Phthalate Degradation: Young-Mi Kim , Hong-Gyu Song; J. Microbiol. 2009;47(4):420-424. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-009-0051-8

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Abstract: Pellet size of white rot fungus, Pleurotus ostreatus may affect the secretion of its degradative enzymes and accompanying biodegrading capability, but could be controlled by several physical culture conditions in liquid culture. The pellet size of P. ostreatus was affected by the volume of inoculum, flask, and medium, but the agitation speed was the most important control factor. At the lower agitation speed of 100 rpm, the large pellets were formed and the laccase activity was higher than that of small pelleted culture at 150 rpm, which might be due to loose intrapellet structure. However, the biodegradation rates of benzylbutylphthalate and dimethylphthalate were higher in the small pelleted culture, which indicated the involvement of other degradative enzyme rather than laccase. The activity of esterase which catalyzes the nonphenolic compounds before the reaction of ligninolytic enzymes was higher in the small pelleted culture, and coincided with the degradation pattern of phthalates. This study suggests the optimization of pellet morphology and subsequent secretion of degradative enzymes is necessary for the efficient removal of recalcitrants by white rot fungi.

Isolation and Identification of an Anticancer Drug, Taxol from Phyllosticta tabernaemontanae, a Leaf Spot Fungus of an Angiosperm, Wrightia tinctoria: Rangarajulu Senthil Kumaran , Johnpaul Muthumary , Byung-Ki Hur; J. Microbiol. 2009;47(1):40-49. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0127-x

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Abstract: Phyllosticta tabernaemontanae, a leaf spot fungus isolated from the diseased leaves of Wrightia tinctoria, showed the production of taxol, an anticancer drug, on modified liquid medium (M1D) and potato dextrose broth (PDB) medium in culture for the first time. The presence of taxol was confirmed by spectroscopic and chromatographic methods of analysis. The amount of taxol produced by this fungus was quantified using high performance liquid chromatography (HPLC). The maximum amount of taxol production was recorded in the fungus grown on M1D medium (461 ug/L) followed by PDB medium (150 ug/L). The production rate was increased to 9.2x103 fold than that found in the culture broth of earlier reported fungus, Taxomyces andreanae. The results designate that P. tabernaemontanae is an excellent candidate for taxol production. The fungal taxol extracted also showed a strong cytotoxic activity in the in vitro culture of tested human cancer cells by apoptotic assay.

Purification and Characterization of Laccase from the White Rot Fungus Trametes versicolor: Moon-Jeong Han , Hyoung-Tae Choi , Hong-Gyu Song; J. Microbiol. 2005;43(6):555-560.; DOI: https://doi.org/2290 [pii]

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Abstract: Laccase is one of the ligninolytic enzymes of white rot fungus Trametes versicolor 951022, a strain first isolated in Korea. This laccase was purified 209-fold from culture fluid with a yield of 6.2% using ethanol precipitation, DEAE-Sepharose, Phenyl-Sepharose, and Sephadex G-100 chromatography. T. versicolor 951022 excretes a single monomeric laccase showing a high specific activity of 91,443 U/mg for 2,2''-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as a substrate. The enzyme has a molecular mass of approximately 97 kDa as determined by SDS-PAGE, which is larger than those of other laccases reported. It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 3.0 and a temperature of 50oC. The Km value of the enzyme for substrate ABTS is 12.8 M and its corresponding Vmax value is 8125.4 U/mg. The specific activity and substrate affinity of this laccase are higher than those of other white rot fungi, therefore, it may be potentially useful for industrial purposes.

Transformation and Mutagenesis of the Nematode-trapping Fungus Monacrosporium sphaeroides by Restriction Enzyme-mediated Integration (REMI): Xu Jin , Ming-He Mo , Zhou Wei , Xiao-Wei Huang , Ke-Qin Zhang; J. Microbiol. 2005;43(5):417-423.; DOI: https://doi.org/2281 [pii]

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Abstract: In this study, the nematode-trapping fungus, Monacrosporium sphaeroides, was transformed with a plasmid harboring the hygromycin B phosphotransferase gene, via restriction enzyme-mediated integration (REMI). Frequencies of up to 94 transformants g-1 per linearized plasmid DNA were obtained by optimizing the PEG concentration, as well as the category and quantity of the added restriction enzyme. 90% of the transformants were determined to be stable for drug resistance when 20 randomly selected transformants were tested. Southern analyses revealed that the transforming DNA was integrated into the M. sphaeroides genome either with or without rearrangement. Five mitotic stable mutant strains were obtained using this approach, all of which had been altered with regard to sporulation capacity and pathogenicity toward nematodes. Southern blot analyses of the five mutants revealed that foreign plasmid DNA had integrated into the genome. Three of the mutants, Tms2316, Tms3583 and Tms1536, exhibited integration at a single location, whereas the remaining two, Tms32 and Tms1913, manifested integration at double or multiple locations. Our results suggest that the transformation of M. sphaeroides via REMI will facilitate insertional mutagenesis, the functional analysis of a variety of genes, and the tagging or cloning of genes of interest.

Establishment of a Micro-Particle Bombardment Transformation System for Dunaliella salina: Congping Tan , Song Qin , Qun Zhang , Peng Jiang , Fangqing Zhao; J. Microbiol. 2005;43(4):361-365.; DOI: https://doi.org/2253 [pii]

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Abstract: In this study, we chronicle the establishment of a novel transformation system for the unicellular marine green alga, Dunaliella salina. We introduced the CaMV35S promoter-GUS construct into D. salina with a PDS1000/He micro-particle bombardment system. Forty eight h after transformation, via histochemical staining, we observed the transient expression of GUS in D. salina cells which had been bombarded under rupture-disc pressures of 450 psi and 900 psi. We observed no GUS activity in either the negative or the blank controls. Our findings indicated that the micro-particle bombardment method constituted a feasible approach to the genetic transformation of D. salina. We also conducted tests of the cells'' sensitivity to seven antibiotics and one herbicide, and our results suggested that 20 ug/ml of Basta could inhibit cell growth completely. The bar gene, which encodes for phosphinothricin acetyltransferase and confers herbicide tolerance, was introduced into the cells via the above established method. The results of PCR and PCR-Southern blot analyses indicated that the gene was successfully integrated into the genome of the transformants.

Purification and Characterization of Thermostable β-Glucosidase from the Brown-Rot Basidiomycete Fomitopsis palustris Grown on Microcrystalline Cellulose: Jeong-Jun Yoon , Ki-Yeon Kim , Chang-Jun Cha; J. Microbiol. 2008;46(1):51-55.; DOI: https://doi.org/10.1007/s12275-007-0230-4

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51 Citations

Abstract: An extracellular β-glucosidase was purified 154-fold to electrophoretic homogeneity from the brown-rot basidiomycete Fomitopsis palustris grown on 2.0% microcrystalline cellulose. SDS-polyacrylamide gel electrophoresis gel gave a single protein band and the molecular mass of purified enzyme was estimated to be approximately 138 kDa. The amino acid sequences of the proteolytic fragments determined by nano-LC- MS/MS suggested that the protein has high homology with fungal β-glucosidases that belong to glycosyl hydrolase family 3. The Kms for p-nitorophenyl-β-D-glucoside (p-NPG) and cellobiose hydrolyses were 0.117 and 4.81 mM, and the Kcat values were 721 and 101.8 per sec, respectively. The enzyme was competitively inhibited by both glucose (Ki= 0.35 mM) and gluconolactone (Ki= 0.008 mM), when p-NPG was used as substrate. The optimal activity of the purified β-glucosidase was observed at pH 4.5 and 70°C. The F. palustris protein exhibited half-lives of 97 h at 55°C and 15 h at 65°C, indicating some degree of thermostability. The enzyme has high activity against p-NPG and cellobiose but has very little or no activity against p-nitrophenyl-β-lactoside, p-nitrophenyl-β-xyloside, p-nitrophenyl-α-arabinofuranoside, xylan, and carboxymethyl cellulose. Thus, our results revealed that the β-glucosidase from F. palustris can be classified as an aryl-β-glucosidase with cellobiase activity.

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