Journal of Microbiology

Journal Article

Vaccine Development for Severe Fever with Thrombocytopenia Syndrome Virus in Dogs: Seok-Chan Park, Da-Eun Jeong, Sun-Woo Han, Joon-Seok Chae, Joo-Yong Lee, Hyun-Sook Kim, Bumseok Kim, Jun-Gu Kang; J. Microbiol. 2024;62(4):327-335. Published online April 18, 2024; DOI: https://doi.org/10.1007/s12275-024-00119-y

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Abstract: Severe fever with thrombocytopenia syndrome (SFTS) is a life-threatening viral zoonosis. The causative agent of this disease is the Dabie bandavirus, which is usually known as the SFTS virus (SFTSV). Although the role of vertebrates in SFTSV transmission to humans remains uncertain, some reports have suggested that dogs could potentially transmit SFTSV to humans. Consequently, preventive measures against SFTSV in dogs are urgently needed. In the present study, dogs were immunized three times at two-week intervals with formaldehyde-inactivated SFTSV with two types of adjuvants. SFTSV (KCD46) was injected into all dogs two weeks after the final immunization. Control dogs showed viremia from 2 to 4 days post infection (dpi), and displayed white pulp atrophy in the spleen, along with a high level of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay (TUNEL) positive area. However, the inactivated SFTSV vaccine groups exhibited rare pathological changes and significantly reduced TUNEL positive areas in the spleen. Furthermore, SFTSV viral loads were not detected at any of the tested dpi. Our results indicate that both adjuvants can be safely used in combination with an inactivated SFTSV formulation to induce strong neutralizing antibodies. Inactivated SFTSV vaccines effectively prevent pathogenicity and viremia in dogs infected with SFTSV. In conclusion, our study highlighted the potential of inactivated SFTSV vaccination for SFTSV control in dogs.

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

Negative regulation of the vacuole-mediated resistance to K+ stress by a novel C2H2 zinc finger transcription factor encoded by aslA in Aspergillus nidulans: Dong Soo Park , Yeong Man Yu , Yong Jin Kim , Pil Jae Maeng; J. Microbiol. 2015;53(2):100-110. Published online January 28, 2015; DOI: https://doi.org/10.1007/s12275-015-4701-8

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In fungi and plants, vacuoles function as a storage and sequestration vessel for a wide variety of ions and are responsible for cytosolic ion homeostasis and responses to ionic shock. In the filamentous fungus Aspergillus nidulans, however, little is known about the molecular genetic mechanisms of vacuolar biogenesis and function. In the present study, we analyzed the function of the aslA gene (AN5583) encoding a novel C2H2-type zinc finger transcription factor (TF) in relation to K+ stress resistance, vacuolar morphology, and vacuolar transporters. The mutant lacking aslA showed increased mycelial growth and decreased branching at high K+ concentrations. Deletion of aslA also caused elevated K+ stress-inducible expression of the genes, nhxA (AN2288), vnxA (AN6986), and vcxA (AN0471), encoding putative endosomal and vacuolar cation/H+ exchangers, as well as cpyA and vpsA genes encoding the proteins involved in vacuolar biogenesis. Interestingly, vacuolar fragmentation induced by K+ stress was alleviated by aslA deletion, resulting in persistence of unfragmented vacuoles. In the presence of bafilomycin, an inhibitor of vacuolar H+-ATPase, the mutant phenotype was suppressed in terms of growth rates and vacuolar morphology. These results together suggest that the C2H2- type zinc finger TF AslA attenuates the K+ stress-inducible expression of the genes encoding the ion pumps involved in vacuolar sequestration of K+ ions powered by vacuolar H+- ATPase, as well as the proteins that function in vacuolar biogenesis.

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The pleiotropic phenotype of FlbA of Aspergillus niger is explained in part by the activity of seven of its downstream-regulated transcription factors
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Survival Factor A (SvfA) Contributes to Aspergillus nidulans Pathogenicity
Joo-Yeon Lim, Ye-Eun Jung, Hye-Eun Hwang, Cheol-Hee Kim, Nese Basaran-Akgul, Sri Harshini Goli, Steven P. Templeton, Hee-Moon Park
Journal of Fungi.2023; 9(2): 143. CrossRef
A C2H2 Zinc Finger Protein PlCZF1 Is Necessary for Oospore Development and Virulence in Peronophythora litchii
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The Gβ-like Protein AfCpcB Affects Sexual Development, Response to Oxidative Stress and Phagocytosis by Alveolar Macrophages in Aspergillus fumigatus
Joo-Yeon Lim, Yeon-Ju Kim, Hee-Moon Park
Journal of Fungi.2022; 8(1): 56. CrossRef
Characterization of BbKlf1 as a novel transcription factor vital for asexual and infection cycles of Beauveria bassiana
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The LAMMER Kinase, LkhA, Affects Aspergillus fumigatus Pathogenicity by Modulating Reproduction and Biosynthesis of Cell Wall PAMPs
Joo-Yeon Lim, Yeon Ju Kim, Seul Ah Woo, Jae Wan Jeong, Yu-Ri Lee, Cheol-Hee Kim, Hee-Moon Park
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Mannitol-1-phosphate dehydrogenase, MpdA, is required for mannitol production in vegetative cells and involved in hyphal branching, heat resistance of conidia and sexual development in Aspergillus nidulans
Joo-Yeon Lim, Seung-Hyun Jang, Hee-Moon Park
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The putative C2H2 transcription factor RocA is a novel regulator of development and secondary metabolism in Aspergillus nidulans
Dong Chan Won, Yong Jin Kim, Da Hye Kim, Hee-Moon Park, Pil Jae Maeng
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Survival factor SvfA plays multiple roles in differentiation and is essential for completion of sexual development in Aspergillus nidulans
Joo-Yeon Lim, Eun-Hye Kang, Yun-Hee Park, Jun-Ho Kook, Hee-Moon Park
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Characterization of NpgA, a 4'-phosphopantetheinyl transferase of Aspergillus nidulans, and evidence of its involvement in fungal growth and formation of conidia and cleistothecia for development: Jung-Mi Kim , Ha-Yeon Song , Hyo-Jin Choi , Kum-Kang So , Dae-Hyuk Kim , Keon-Sang Chae , Dong-Min Han , Kwang-Yeop Jahng; J. Microbiol. 2015;53(1):21-31. Published online January 4, 2015; DOI: https://doi.org/10.1007/s12275-015-4657-8

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Abstract

The null pigmentation mutant (npgA1) in Aspergillus nidulans
results
in a phenotype with colorless organs, decreased branching growth, delayed of asexual spore development, and aberrant cell wall structure. The npgA gene was isolated from A. nidulans to investigate these pleiomorphic phenomena of npgA1 mutant. Sequencing analysis of the complementing gene indicated that it contained a 4􍿁-phosphopantetheinyl transferase (PPTase) superfamily domain. Enzymatic assay of the PPTase, encoded by the npgA gene, was implemented in vivo and in vitro. Loss-of-function of LYS5, which encoded a PPTase in Saccharomyces cerevisiae, was functionally complemented by NpgA, and Escherichia coli-derived NpgA revealed phosphopantetheinylation activity with the elaboration of 3􍿁5􍿁-ADP. Deletion of the npgA gene caused perfectly a lethal phenotype and the absence of asexual/sexual sporulation and secondary metabolites such as pigments in A. nidulans. However, a cross feeding effect with A. nidulans wild type allowed recovery from deletion defects, and phased-culture filtrate from the wild type were used to verify that the npgA gene was essential for formation of metabolites needed for development as well as growth. In addition, forced expression of npgA promoted the formation of conidia and cleistothecia as well as growth. These results indicate that the npgA gene is involved in the phosphopantetheinylation required for primary biological processes such as growth, asexual/sexual development, and the synthesis of secondary metabolites in A. nidulans.

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A Novel Rapid Fungal Promoter Analysis System Using the Phosphopantetheinyl Transferase Gene,npgA, inAspergillus nidulans
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Transcriptional Regulation of fksA, a β-1,3-Glucan Synthase Gene, by the APSES Protein StuA during Aspergillus nidulans Development: Bum-Chan Park , Yun-Hee Park , Soohyun Yi , Yu Kyung Choi , Eun-Hye Kang , Hee-Moon Park; J. Microbiol. 2014;52(11):940-947. Published online October 31, 2014; DOI: https://doi.org/10.1007/s12275-014-4517-y

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Abstract

The temporal and spatial regulation of β-1,3-glucan synthesis plays an important role in morphogenesis during fungal growth and development. Northern blot analysis showed that the transcription of fksA, the gene encoding β-1,3-glucan synthase in Aspergillus nidulans, was cell-cycle-dependent and increased steadily over the duration of the vegetative period, but its overall expression during the asexual and sexual stages was fairly constant up until the time of transcription cessation. In an A. nidulans strain mutated in the eukaryotic bHLH-like APSES transcription factor stuA1, the transcriptional level of fksA, and consequently the content of alkali-insoluble cell wall β-glucan, significantly increased at the conidial chain formation and maturation stage. Electrophoretic mobility shift assays revealed that StuA was bound to StREs (StuA Response Elements) on the fksA promoter region. Promoter analysis with sGFP-fusion constructs also indicated the negative regulation of fksA expression by StuA, especially during asexual development. Taken together, these data suggest that StuA plays an important role in cell wall biogenesis during the development of A. nidulans, by controlling the transcription level of fksA.

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Survival Factor A (SvfA) Contributes to Aspergillus nidulans Pathogenicity
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Applied and Environmental Microbiology.2019;[Epub] CrossRef
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Joo-Yeon Lim, Hee-Moon Park
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Journal Article

The Observation of PlcA Mutation and Localization in Aspergillus nidulans: Chun-Seob Ahn , Young Taek Oh , Jeong-Geun Kim , Kap-Hoon Han , Chang-Won Lee , Jae Won Kim; J. Microbiol. 2014;52(7):590-596. Published online June 28, 2014; DOI: https://doi.org/10.1007/s12275-014-3651-x

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Abstract

To know the function of the plcA gene, which encodes a putative phosphoinositide-specific phospholipase C, in a model filamentous fungus Aspergillus nidulans, it was disrupted thorough homologous recombination and examined. The germination rate of ΔplcA was reduced by approximately 65% and germination of ΔplcA at a lower temperature (25°C) was much slower than germination under normal conditions (37°C), suggesting the plcA is responsible for cold-sensitivity. The hyphal growth of ΔplcA was slightly reduced at 37°C and conspicuously reduced at 25°C. While germinating ΔplcA formed giant swollen spores, and generated short and thick hyphae. The results of the nuclear examination of ΔplcA showed nuclear division with missegregation, and the rate of nuclear division was lower than that of wild type at both 25°C and 37°C. The results of this study showed that plcA is localized to the nucleus through intracellular calcium signaling in A. nidulans. The abnormal nuclear division, resulting from plcA gene deletion, affects conidiation in asexual development. Taken together, these results suggested that plcA is required for normal vegetative growth, morphogenesis, conidiation, and nuclear division
in A. nidulans.

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Regulators of the Asexual Life Cycle of Aspergillus nidulans
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Research Support, Non-U.S. Gov'ts

A Putative APSES Transcription Factor Is Necessary for Normal Growth and Development of Aspergillus nidulans: Ji-Yeon Lee , Lee-Han Kim , Ha-Eun Kim , Jae-Sin Park , Kap-Hoon Han , Dong-Min Han; J. Microbiol. 2013;51(6):800-806. Published online December 19, 2013; DOI: https://doi.org/10.1007/s12275-013-3100-2

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Abstract

The nsdD gene encoding a GATA type transcription factor positively controls sexual development in Aspergillus nidulans. According to microarray data, 20 genes that were upregulated by deleting nsdD during various life cycle stages were randomly selected and deleted for functional analysis. None of the mutants showed apparent changes in growth or development compared with those of the wild-type except the AN3154 gene that encodes a putative APSES transcription factor and is an ortholog of Saccharomyces cerevisiae swi4. Deleting AN3154 resulted in retarded growth and development, and the gene was named rgdA (retared growth and development). The rgdA deletion mutant developed a reduced number of conidia even under favorable conditions for asexual development. The retarded growth and development was partially suppressed by the veA1 mutation. The conidial heads of the mutant aborted, showing reduced and irregular shaped phialides. Fruiting body development was delayed compared with that in the wild-type. The mutant did not respond to various nutritional or environmental factors that affected the development patterns. The rgdA gene was expressed at low levels throughout the life cycle and was not significantly affected by several regulators of sexual and asexual development such as nsdD, veA, stuA, or brlA. However, the rgdA gene affected brlA and abaA expression, which function as key regulators of asexual sporulation, suggesting that rgdA functions upstream of those genes.

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Transcriptional regulation of fksA, a β-1,3-glucan synthase gene, by the APSES protein StuA during Aspergillus nidulans development
Bum-Chan Park, Yun-Hee Park, Soohyun Yi, Yu Kyung Choi, Eun-Hye Kang, Hee-Moon Park
Journal of Microbiology.2014; 52(11): 940. CrossRef

NOTE] Isolation and Characterization of Self-Fertile Suppressors from the Sterile nsdD Deletion Mutant of Aspergillus nidulans: Dong-Beom Lee , Lee Han Kim , Jin-Pyo Kim , Kap-Hoon Han , Dong-Min Han; J. Microbiol. 2011;49(6):1054-1057. Published online December 28, 2011; DOI: https://doi.org/10.1007/s12275-011-1111-4

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Abstract: To identify downstream and/or interactive factors of the nsdD gene, which encodes a positive regulator of sexual development of Aspergillus nidulans, suppressor mutants displaying a self-fertile phenotype were isolated from a sterile nsdD deletion mutant. At least five different loci (sndA-E) were identified and genetically analyzed. In the nsdD+ background, most of the suppressors showed a marked increment of sexual development, even under the stress conditions that normally inhibited sexual development. The common phenotype of the suppressor mutants suggested the involvement of the snd genes in the negative regulation of sexual development in response to the environmental factors.

The MpkB MAP Kinase Plays a Role in Post-karyogamy Processes as well as in Hyphal Anastomosis During Sexual Development in Aspergillus nidulans: Sang-Cheol Jun , Sei-Jin Lee , Hyun-Joo Park , Ji-Young Kang , Young-Eun Leem , Tae-Ho Yang , Mi-Hee Chang , Jung-Mi Kim , Seung-Hwan Jang , Hwan-Gyu Kim , Dong-Min Han , Keon-Sang Chae , Kwang-Yeop Jahng; J. Microbiol. 2011;49(3):418-430. Published online June 30, 2011; DOI: https://doi.org/10.1007/s12275-011-0193-3

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Abstract: Two genes encoding MAP kinase homologs, designated as mpkB and mpkC, were isolated from Aspergillus nidulans by PCR with degenerate primers. Deletion and over-expression mutants of mpkC showed no detectable phenotypes under any external stress tested. Deletion of mpkB caused pleiotropic phenotypes including a failure in forming cleistothecia under any induction conditions for sexual development, increased Hülle cell production, slow hyphal growth and aberrant conidiophore morphology. Over-expression of mpkB led to increased cleistothecium production. While the transcripts of mpkB and mpkC were constitutively synthesized through the entire life cycle, their size and amount differed with developmental stages. An outcross test using fluorescent protein reporters showed that the mpkB deletion mutant could not form heterokaryons with wild type. Protoplast fusion experiments showed that the fusant of the mpkB mutant with wild type could undergo normal sexual development. However, heterokaryotic mycelia that were produced from a fusant between two mpkB deletion mutants could not form cleistothecia, although they did appear to form diploid nuclei. These results suggest that the MpkB MAP kinase is required for some post-karyogamy process as well as at the hyphal anastomosis stage to accomplish sexual development successfully.

NOTE] Simple Identification of veA1 Mutation in Aspergillus nidulans: Kap-Hoon Han , Jae-Sin Park , Keon Sang Chae , Dong-Min Han; J. Microbiol. 2010;48(6):885-887. Published online January 9, 2011; DOI: https://doi.org/10.1007/s12275-010-0506-y

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Abstract: The veA gene plays an important role in development of a homothallic filamentous fungus Aspergillus nidulans. The veA1 phenotype can be difficult to distinguish from the wild-type veA. Despite the importance of the veA allele, no efficient identification method has been reported besides DNA sequencing. Here, we present simple physiological and molecular biological ways to distinguish between the veA wild-type and veA1 allele. The novel approaches, which involve incubation in the presence of oxalic acid, polymerase chain reaction using double mismatched primers, and BstXI enzyme digestion, are simpler, faster and more cost-efficient than genome sequencing.

Screening of Growth- or Development-related Genes by Using Genomic Library with Inducible Promoter in Aspergillus nidulans: Bang-Yong Lee , Sang-Yong Han , Han Gil Choi , Jee Hyun Kim , Kap-Hoon Han , Dong-Min Han; J. Microbiol. 2005;43(6):523-528.; DOI: https://doi.org/2295 [pii]

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Abstract: Using the genomic library constructed at the downstream of the niiA promoter, which induces the over-expression of an inserted DNA fragment, we have attempted to screen the genes affecting growth or development by over-expression. The wild-type strain was transformed using the AMA-niiA(p) library and cultured on 1.2 M sorbitol media, in which asexual sporulation is induced, but sexual development is repressed. Over 100,000 strains transformed to pyrG+ were analyzed with regard to any changes in phenotype. Consequently, seven strains were isolated for further analyses. These strains were designated NOT [niiA(p) over-expression transformants] stains. Four of the strains were of the inducible type, and the remaining strains were of the multi-copy suppression type. Two of the inducible-type strains, NOT1 and NOT40, harbored genes which had been inserted in reverse direction, suggesting that the mutant phenotypes had been derived from an excess amount of anti-sense mRNA. Domain analyses of the deduced polypeptides from the DNA fragments rescued from the transformants revealed that NOT1, NOT40 and NOT6 harbored a LisH motif, a forkhead domain, and a Zn(II)2Cys6 binuclear zinc cluster, respectively.

Microscopic Examination of the Suppressive Action of Antifungal Substances from Pseudomonas aeruginosa on Asexual Sporulation of Fungi: Yoon, Kwon S. , Min, Bu Y. , Choi, Hyoung T. , Lee, Jong K. , Kim, Kun W.; J. Microbiol. 1999;37(1):27-34.

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Abstract: Two fractions with unusual antifungal activity that suppress asexual sporulation of several fungi were obtained from culture filtrate of Pseudomonas aeruginosa and were partially purified through the repeated silicagel flash column chromatographies. The sporulation-suppressive actions of these fractions in Aspergillus nidulans, Rhizopus stolonifer, and Coprinus cinereus, were analyzed by light and electron microscopes. The germination ability of the spores produced in the presence of these fractions were also checked to determine the persistent effects of these antifungal substances on the next generation. Light microscopic observation of developing sporangia of R. stolonifer grown in the presence of both fractions revealed that the significant number of sporangia failed to reach maturity, and frequently, uncontrolled growths of hyphae and rhizoids from the sporangiophores were found. In A. nidulans addition of these fractions appeared to cause different classes of morphological abnormality in conidia development, which included aborted formation of conidiogenous cells from the apex of conidiophores and enhanced hyphal growths either at the tip or middle of the conidiophores. Germination abilities of spores obtained from the cultures grown in the presence of antifungal fractions were 40∼60% in Aspergillus, 50∼80% in Coprinus (thallic spores), and 30∼40% in Rhizopus compared to those of normal spores.

Isolation of an Autonomously Replicating DNA Sequence from Aspergillus nidulans: Jang, Seung Hwan , Jahng, Kwang Yeop; J. Microbiol. 1999;37(2):51-58.

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Abstract: Using yeast, Saccharomyces cerevisiae, and the integrate vector system, we have isolated and characterized an autonomously replicating sequence (ARS) from Aspergillus nidulans. The DNA fragment, designated ANR1, is 5.0 kb in size and maintained free from the chromosome in S. cerevisiae. The YIplac211-ANR1 recombinant plasmid, which consists of sequences derived from the yeast integrative vector YIplac211 and 5.0 kb ANR1 fragment, showed a 10⁴-fold enhancement in transformation efficiency over that found for YIplac211, and was easily recovered from the transformed yeast. Genetic analysis of transformants showed that YIplac21-ANR1 could be over 96% cured when cultured over 20 generations in complete medium and thus suggests that this sequence is mitotically unstable. In A. nidulans, recombinant plasmid PILJ16-4.5 which carries the 4.5 kb EcoRI fragment of ANR1 showed a 170-fold enhancement in transformation efficiency compared to that of the integrative vector PILJ16.

Controlled Expression and Secretion of Aspergillus oryzae Alkaline Protease in Aspergillus nidulans: Eun Ah Kim , Jeong Goo Lee , Mi Kyung Whang , Hee Moon Park , Jeong Yoon Kim , Suhn Kee Chae , Pil Jae Maeng; J. Microbiol. 2001;39(2):95-101.

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Abstract: In an effort to develop an efficient expression and secretion system for heterologous proteins in Aspergillus nidulans, the PCR-amplified coding sequence for alkaline protease (AlpA) of A. oryzae was cloned into a fungal expression vector downstream of A. nidulans alcA (alcohol dehydrogenase) promoter to yield pRAAlp. Transformation of A. nidulans with pRAAlp gave stable transformants harboring various copy numbers (3 to 10) of integrated alpA gene, from among which 6 representatives were selected. On a medium containing 0.8% ammonium sulfate that represses the expression of the hosts own protease, the alcA promoter-controlled AlpA expression was strongly induced by threonine but repressed by glucose. The level of AlpA secretion was highest (approximately 666 mU/ml) in transformant ALP6 containing the largest copy number integrated alpA. However, the level of AlpA secretion was not necessarily proportional to the copy numbers of the integrated alpA genes. The N-terminal sequence of the secreted mature AlpA was determined to be Gly-Leu-Thr-Thr-Gln-Lys-Ser and its molecular mass to be approximately 34 kDa, indicating that AlpA is properly processed by the removal of 121 N-terminal amino acids.

A Novel UV-Sensitivity Mutation Induces Nucleotide Excision Repair Phenotype and Shows Epistatic Relationships with UvsF and UvsB Groups in Aspergillus nidulans: F. Baptista , M. A. A. Castro-Prado; J. Microbiol. 2001;39(2):102-108.

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Abstract: DNA damage response has a central role in the maintenance of genomic integrity while mutations in related genes may result in a range of disorders, including neoplasic formations. The uvsZ1 characterized in this report is a novel uvs mutation in Aspergillus nidulans, resulting in a nucleotide excision repair (NER) phenotype: UV-sensitivity before DNA synthesis (quiescent cells), high UV-induced mutation frequency and probable absence of involvement with mitotic and meiotic recombinations. The mutation is recessive and non-allelic to the previously characterized uvsA101 mutation, also located on the paba-y interval on chromosome I. uvsZ1 showed wild-type sensitivity to MMS, which suggests non-involvement of this mutation with BER. Epitasis tests showed that the uvsZ gene product is probably involved in the same repair pathways as UVSB or UVSH proteins. Although mutations in these proteins result in an NER phenotype, UVSB is related with cell cycle control and UVSH is associated with the post-replicational repair pathway. The epistatic interaction among uvsZ1 and uvsB413 and uvsH77 mutations indicates that different repair systems may be related with the common steps of DNA damage response in Aspergillus nidulans.

Cell Cycle-dependent Expression of Chitin Synthase Genes in Aspergillus nidulans: Bum-Chan Park , Pil-Jae Maeng , Hee-Moon Park; J. Microbiol. 2001;39(1):74-78.

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Abstract: The transcription of the chitin synthase genes (chss) was cell cycle-regulated in Aspergillus nidulans and the expression pattern was classified into two groups. Group one, containing chsA and chsC, showed decreasing transcription level upon entry into the S-phase and no further variation during the remainder of the cell cycle. However, group two, containing chsB, chsD, and csmA, showed a sharp decrease of mRNA level upon entry into the G2-phase and an increase during the M-phase. Our results suggested that the chss, belonging to same group with the similar expression pattern during the cell cycle are functionally linked and that chsD may play a role in hyphal growth and development in A. nidulans.

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