Journal of Microbiology

Volume 59(2); February 2021

Reviews

Rediscovery of antimicrobial peptides as therapeutic agents: Minkyung Ryu , Jaeyeong Park , Ji-Hyun Yeom , Minju Joo , Kangseok Lee; J. Microbiol. 2021;59(2):113-123. Published online February 1, 2021; DOI: https://doi.org/10.1007/s12275-021-0649-z

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Abstract: In recent years, the occurrence of antibiotic-resistant pathogens is increasing rapidly. There is growing concern as the development of antibiotics is slower than the increase in the resistance of pathogenic bacteria. Antimicrobial peptides (AMPs) are promising alternatives to antibiotics. Despite their name, which implies their antimicrobial activity, AMPs have recently been rediscovered as compounds having antifungal, antiviral, anticancer, antioxidant, and insecticidal effects. Moreover, many AMPs are relatively safe from toxic side effects and the generation of resistant microorganisms due to their target specificity and complexity of the mechanisms underlying their action. In this review, we summarize the history, classification, and mechanisms of action of AMPs, and provide descriptions of AMPs undergoing clinical trials. We also discuss the obstacles associated with the development of AMPs as therapeutic agents and recent strategies formulated to circumvent these obstacles.

Raloxifene as a treatment option for viral infections: Subin Hong , JuOae Chang , Kwiwan Jeong , Wonsik Lee; J. Microbiol. 2021;59(2):124-131. Published online February 1, 2021; DOI: https://doi.org/10.1007/s12275-021-0617-7

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32 Citations

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARSCoV- 2) has caused corona virus disease 2019 (COVID-19) pandemic and led to mass casualty. Even though much effort has been put into development of vaccine and treatment
methods
to combat COVID-19, no safe and efficient cure has been discovered. Drug repurposing or drug repositioning which is a process of investigating pre-existing drug candidates for novel applications outside their original medical indication can speed up the drug development process. Raloxifene is a selective estrogen receptor modulator (SERM) that has been approved by FDA in 1997 for treatment and prevention of postmenopausal osteoporosis and cancer. Recently, raloxifene demonstrates efficacy in treating viral infections by Ebola, influenza A, and hepatitis C viruses and shows potential for drug repurposing for the treatment of SARS-CoV-2 infection. This review will provide an overview of raloxifene’s mechanism of action as a SERM and present proposed mechanisms of action in treatment of viral infections.

Journal Articles

Description of Vagococcus coleopterorum sp. nov., isolated from the intestine of the diving beetle, Cybister lewisianus, and Vagococcus hydrophili sp. nov., isolated from the intestine of the dark diving beetle, Hydrophilus acuminatus, and emended description of the genus Vagococcus: Dong-Wook Hyun , Euon Jung Tak , Pil Soo Kim , Jin-Woo Bae; J. Microbiol. 2021;59(2):132-141. Published online December 23, 2020; DOI: https://doi.org/10.1007/s12275-021-0485-1

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6 Citations

Abstract: A polyphasic taxonomic approach was used to characterize two novel bacterial strains, HDW17AT and HDW17BT, isolated from the intestine of the diving beetle Cybister lewisianus, and the dark diving beetle Hydrophilus acuminatus, respectively. Both strains were Gram-positive and facultative anaerobic cocci forming cream-colored colonies. The isolates grew optimally at 25°C, pH 7, in the presence of 0.3% (wt/vol) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences and genome sequences showed that the isolates were members of the genus Vagococcus, and strain HDW17AT was closely related to Vagococcus fessus CCUG 41755T (98.9% of 16S rRNA gene sequence similarity and 74.3% of average nucleotide identity [ANI]), whereas strain HDW17BT was closely related to Vagococcus fluvialis NCFB 2497T (98.9% of 16S rRNA gene sequence similarity and 76.6% of ANI). Both strains contained C16:0, and C18:1 ω9c as the major cellular fatty acids, but C16:1 ω9c was also observed only in strain HDW17BT as the major cellular fatty acid. The respiratory quinone of the isolates was MK-7. The major polar lipid components were phosphatidylglycerol, phosphatidylethanolamine, and diphosphatidylglycerol. The genomic DNA G + C content of strains HDW17AT and HDW17BT were 36.6 and 34.4%, respectively. Both strains had cell wall peptidoglycan composed of the amino acids L-alanine, glycine, D-glutamic acid, L-tryptophan, L-lysine, and L-aspartic acid, and the sugars ribose, glucose, and galactose. Based on phylogenetic, phenotypic, chemotaxonomic, and genotypic analyses, strains HDW17AT and HDW17BT represent two novel species in the genus Vagococcus. We propose the name Vagococcus coleopterorum sp. nov. for strain HDW17AT (= KACC 21348T = KCTC 49324T = JCM 33674T) and the name Vagococcus hydrophili sp. nov. for strain HDW17BT (= KACC 21349T = KCTC 49325T = JCM 33675T).

iTRAQ-facilitated proteomic analysis of Bacillus cereus via degradation of malachite green: Bobo Wang , Jing Lu , Junfang Zheng , Zhisheng Yu; J. Microbiol. 2021;59(2):142-150. Published online February 1, 2021; DOI: https://doi.org/10.1007/s12275-021-0441-0

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Abstract: The wide use of malachite green (MG) as a dye has caused substantial concern owing to its toxicity. Bacillus cereus can against the toxic effect of MG and efficiently decolourise it. However, detailed information regarding its underlying adaptation and degradation mechanisms based on proteomic data is scarce. In this study, the isobaric tags for relative and absolute quantitation (iTRAQ)-facilitated quantitative method was applied to analyse the molecular mechanisms by which B. cereus degrades MG. Based on this analysis, 209 upregulated proteins and 198 downregulated proteins were identified with a false discovery rate of 1% or less during MG biodegradation. Gene ontology and KEGG analysis determined that the differentially expressed proteins were enriched in metabolic processes, catalytic activity, antioxidant activity, and responses to stimuli. Furthermore, real-time qPCR was utilised to further confirm the regulated proteins involved in benzoate degradation. The proteins BCE_4076 (Acetyl-CoA acetyltransferase), BCE_5143 (Acetyl-CoA acetyltransferase), BCE_5144 (3-hydroxyacyl-CoA dehydrogenase), BCE_4651 (Enoyl-CoA hydratase), and BCE_5474 (3-hydroxyacyl-CoA dehydrogenase) involved in the benzoate degradation pathway may play an important role in the biodegradation of MG by B. cereus. The results of this study not only provide a comprehensive view of proteomic changes in B. cereus upon MG loading but also shed light on the mechanism underlying MG biodegradation by B. cereus.

Molecular characterization of Hsf1 as a master regulator of heat shock response in the thermotolerant methylotrophic yeast Ogataea parapolymorpha: Jin Ho Choo , Su-Bin Lee , Hye Yun Moon , Kun Hwa Lee , Su Jin Yoo , Keun Pil Kim , Hyun Ah Kang; J. Microbiol. 2021;59(2):151-163. Published online February 1, 2021; DOI: https://doi.org/10.1007/s12275-021-0646-2

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Abstract: Ogataea parapolymorpha (Hansenula polymorpha DL-1) is a thermotolerant methylotrophic yeast with biotechnological applications. Here, O. parapolymorpha genes whose expression is induced in response to heat shock were identified by transcriptome analysis and shown to possess heat shock elements (HSEs) in their promoters. The function of O. parapolymorpha HSF1 encoding a putative heat shock transcription factor 1 (OpHsf1) was characterized in the context of heat stress response. Despite exhibiting low sequence identity (26%) to its Saccharomyces cerevisiae homolog, OpHsf1 harbors conserved domains including a DNA binding domain (DBD), domains involved in trimerization (TRI), transcriptional activation (AR1, AR2), transcriptional repression (CE2), and a C-terminal modulator (CTM) domain. OpHSF1 could complement the temperature sensitive (Ts) phenotype of a S. cerevisiae hsf1 mutant. An O. parapolymorpha strain with an H221R mutation in the DBD domain of OpHsf1 exhibited significantly retarded growth and a Ts phenotype. Intriguingly, the expression of heat-shock-protein‒coding genes harboring HSEs was significantly decreased in the H221R mutant strain, even under non-stress conditions, indicating the importance of the DBD for the basal growth of O. parapolymorpha. Notably, even though the deletion of C-terminal domains (ΔCE2, ΔAR2, ΔCTM) of OpHsf1 destroyed complementation of the growth defect of the S. cerevisiae hsf1 strain, the C-terminal domains were shown to be dispensable in O. parapolymorpha. Overexpression of OpHsf1 in S. cerevisiae increased resistance to transient heat shock, supporting the idea that OpHsf1 could be useful in the development of heatshock‒ resistant yeast host strains.

Biophysical characterization of antibacterial compounds derived from pathogenic fungi Ganoderma boninense: Syahriel Abdullah , Yoon Sin Oh , Min-Kyu Kwak , KhimPhin Chong; J. Microbiol. 2021;59(2):164-174. Published online December 23, 2020; DOI: https://doi.org/10.1007/s12275-021-0551-8

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Abstract: There have been relatively few studies which support a link between Ganoderma boninense, a phytopathogenic fungus that is particularly cytotoxic and pathogenic to plant tissues and roots, and antimicrobial compounds. We previously observed that liquid-liquid extraction (LLE) using chloroformmethanol- water at a ratio (1:1:1) was superior at detecting antibacterial activities and significant quantities of antibacterial compounds. Herein, we demonstrate that antibacterial secondary metabolites are produced from G. boninense mycelia. Antibacterial compounds were monitored in concurrent biochemical and biophysical experiments. The combined
methods
included high performance thin-layer chromatography (HPTLC), gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), fourier transform infrared (FTIR), and nuclear magnetic resonance (NMR) spectroscopy. The antibacterial compounds derived from mycelia with chloroform-methanol extraction through LLE were isolated via a gradient solvent elution system using HPTLC. The antibacterial activity of the isolated compounds was observed to be the most potent against Staphylococcus aureus ATCC 25923 and multidrug-resistant S. aureus NCTC 11939. GC-MS, HPLC, and FTIR analysis confirmed two antibacterial compounds, which were identified as 4,4,14α-trimethylcholestane (m/z = 414.75; lanostane, C30H54) and ergosta-5,7,22-trien-3β-ol (m/z = 396.65; ergosterol, C28H44O). With the aid of spectroscopic evaluations, ganoboninketal (m/z = 498.66, C30H42O6), which belongs to the 3,4-seco-27-norlanostane triterpene family, was additionally characterized by 2D-NMR analysis. Despite the lack of antibacterial potential exhibited by lanostane; both ergosterol and ganoboninketal displayed significant antibacterial activities against bacterial pathogens. Results provide evidence for the existence of bioactive compounds in the mycelia of the relatively unexplored phytopathogenic G. boninense, together with a robust method for estimating the corresponding potent antibacterial secondary metabolites.

Spot 42 RNA regulates putrescine catabolism in Escherichia coli by controlling the expression of puuE at the post-transcription level: Xin Sun , Ruyan Li , Guochen Wan , Wanli Peng , Shuangjun Lin , Zixin Deng , Rubing Liang; J. Microbiol. 2021;59(2):175-185. Published online February 1, 2021; DOI: https://doi.org/10.1007/s12275-021-0421-4

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Abstract: Putrescine, a typical polyamine compound important for cell growth and stress resistance, can be utilized as an energy source. However, the regulation of its catabolism is unclear. Here the small RNA (sRNA) Spot 42, an essential regulator of carbon catabolite repression (CCR), was confirmed to participate in the post-transcriptional regulation of putrescine catabolism in Escherichia coli. Its encoding gene spf exclusively exists in the γ-proteobacteria and contains specific binding sites to the 5􍿁-untranslated regions of the puuE gene, which encodes transaminase in the glutamylated putrescine pathway of putrescine catabolism converting γ-aminobutyrate (GABA) into succinate semialdehyde (SSA). The transcription of the spf gene was induced by glucose, inhibited by putrescine, and unaffected by PuuR, the repressor of puu genes. Excess Spot 42 repressed the expression of PuuE significantly in an antisense mechanism through the direct and specific base-pairing between the 51–57 nt of Spot 42 and the 5􍿁- UTR of puuE. Interestingly, Spot 42 mainly influenced the stability of the puuCBE transcript. This work revealed the regulatory role of Spot 42 in putrescine catabolism, in the switch between favorable and non-favorable carbon source utilization, and in the balance of metabolism of carbon and nitrogen sources.

Functional and structural characterization of Deinococcus radiodurans R1 MazEF toxin-antitoxin system, Dr0416-Dr0417: Immanuel Dhanasingh , Eunsil Choi , Jeongeun Lee , Sung Haeng Lee , Jihwan Hwang; J. Microbiol. 2021;59(2):186-201. Published online February 1, 2021; DOI: https://doi.org/10.1007/s12275-021-0523-z

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Abstract: In prokaryotes, toxin-antitoxin (TA) systems are commonly found. They likely reflect the adaptation of pathogenic bacteria or extremophiles to various unfavorable environments by slowing their growth rate. Genomic analysis of the extremophile Deinococcus radiodurans R1 revealed the presence of eight type II TA systems, including the genes dr0417, dr0660, dr1530, dr0690, and dr1807. Expression of these toxin genes led to inhibition of Escherichia coli growth, whereas their antidote antitoxins were able to recover the growth defect. Remarkably, Dr0417 (DrMazF) showed endoribonuclease activity toward rRNAs as well as mRNAs, as determined by in vivo and in vitro RNA cleavage assays, and this activity was inhibited by Dr0416 (DrMazE). It was also found that the expression of dr0416-0417 module is directly regulated by the DrMazE-MazF complex. Furthermore, this TA module was induced under stress conditions and plays an important role in survival. To understand the regulatory mechanism at the molecular level, we determined the first high-resolution structures of DrMazF alone and of the DrMazE-MazF complex. In contrast with the hetero-hexameric state of typical MazEMazF complexes found in other species, DrMazE-MazF crystal structure consists of a hetero-trimer, with the DNA-binding domain of DrMazE undergoing self-cleavage at the flexible linker loop. Our structure revealed that the unique residue R54 provides an additional positive charge to the substratebinding pocket of DrMazF, its mutation significantly affects the endonuclease activity. Thus, our work reports the unique structural and biochemical features of the DrMazE-MazF system.

GABA-producing Lactobacillus plantarum inhibits metastatic properties and induces apoptosis of 5-FU-resistant colorectal cancer cells via GABAB receptor signaling: JaeJin An , Heon Seok , Eun-Mi Ha; J. Microbiol. 2021;59(2):202-216. Published online February 1, 2021; DOI: https://doi.org/10.1007/s12275-021-0562-5

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29 Citations

Abstract: 5-Fluorouracil (5-FU) is an essential drug in systemic chemotherapy treatments for colorectal cancer (CRC). Despite the development of several treatment strategies over the past decades, the patient benefits of 5-FU-based therapies have been compromised by the development of chemoresistance. Differences in treatment responses among CRC patients may be due to genetic and epigenetic factors unique to individuals. Therefore, important factors for realizing personalized medicine are to accurately understand the causes and mechanisms of drug resistance to 5-FU-based therapies and to identify and validate prognostic biomarkers. Gut microbes that interact directly with the host contribute to human health and cancer control. Lactobacillus plantarum, in particular, has the potential to be a therapeutic agent by producing bioactive compounds that may benefit the host. Here, we investigated the gamma-aminobutyric acid (GABA) and GABAB receptor (GABABR)-dependent signaling pathway as a treatment option for 5-FU-resistant HT-29 cells. GABA-producing L. plantarum activates anti-proliferative, anti-migration, and anti-invasion effects against 5-FU-resistant HT-29 cells. The inhibitory effects of GABA-producing L. plantarum are mediated via GABABR. Activated GABABR induces apoptosis through the inhibition of cAMP-dependent signaling pathways and cellular inhibitor of apoptosis protein 2 (cIAP2) expression. Thus, the GABAergic system has potential in 5- FU-resistant HT-29 cells as a predictive biomarker. In addition, GABA-producing L. plantarum is promising as an adjuvant treatment for 5-FU-resistant CRC, and its intervention in neurobiological signaling imply new possibilities for chemoprevention and the treatment of colon cancer-related diseases.

Effects of digested Cheonggukjang on human microbiota assessed by in vitro fecal fermentation: Vineet Singh , Nakwon Hwang , Gwangpyo Ko , Unno Tatsuya; J. Microbiol. 2021;59(2):217-227. Published online February 1, 2021; DOI: https://doi.org/10.1007/s12275-021-0525-x

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Abstract: In vitro fecal fermentation is an assay that uses fecal microbes to ferment foods, the results of which can be used to evaluate the potential of prebiotic candidates. To date, there have been various protocols used for in vitro fecal fermentation- based assessments of food substances. In this study, we investigated how personal gut microbiota differences and external factors affect the results of in vitro fecal fermentation assays. We used Cheonggukjang (CGJ), a Korean traditional fermented soybean soup that is acknowledged as healthy functional diet. CGJ was digested in vitro using acids and enzymes, and then fermented with human feces anaerobically. After fecal fermentation, the microbiota was analyzed using MiSeq, and the amount of short chain fatty acids (SCFAs) were measured using GC-MS. Our results suggest that CGJ was effectively metabolized by fecal bacteria to produce SCFAs, and this process resulted in an increase in the abundance of Coprococcus, Ruminococcus, and Bifidobacterium and a reduction in the growth of Sutterella, an opportunistic pathogen. The metabolic activities predicted from the microbiota shifts indicated enhanced metabolism linked to methionine biosynthesis and depleted chondroitin sulfate degradation. Moreover, the amount of SCFAs and microbiota shifts varied depending on personal microbiota differences. Our findings also suggest that in vitro fecal fermentation of CGJ for longer durations may partially affect certain fecal microbes. Overall, the study discusses the usability of in vitro gastrointestinal digestion and fecal fermentation (GIDFF) to imitate the effects of diet-induced microbiome modulation and its impact on the host.

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