Journal of Microbiology

Volume 56(8); August 2018

Review

[MINIREVIEW] Phylogenetic comparison of Epstein-Barr virus genomes: Su Jin Choi , Seok Won Jung , Sora Huh , Hyosun Cho , Hyojeung Kang; J. Microbiol. 2018;56(8):525-533. Published online June 14, 2018; DOI: https://doi.org/10.1007/s12275-018-8039-x

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Abstract: Technologies used for genome analysis and whole genome sequencing are useful for us to understand genomic characterization and divergence. The Epstein-Barr virus (EBV) is an oncogenic virus that causes diverse diseases such as Burkitt’s lymphoma (BL), nasopharyngeal carcinoma (NPC), Hodgkin’s lymphoma (HL), and gastric carcinoma (GC). EBV genomes found in these diseases can be classified either by phases of EBV latency (type-I, -II, and -III latency) or types of EBNA2 sequence difference (type-I EBV, type-II EBV or EBV-1, EBV-2). EBV from EBV-transformed lymphoblastoid cell line (LCL) establishes type-III latency, EBV from NPC establishes type-II latency, and EBV from GC establishes type-I latency. However, other important factors play key roles in classifying numerous EBV strains because EBV genomes are highly diverse and not phylogenetically related to types of EBV-associated diseases. Herein, we first reviewed previous studies to describe molecular characteristics of EBV genomes. Then, using comparative and phylogenetic analyses, we phylogenetically analyzed molecular variations of EBV genomes and proteins. The review of previous studies and our phylogenetic analysis showed that EBV genomes and proteins were highly diverse regardless of types of EBV-associated diseases. Other factors should be considered in determining EBV taxonomy. This review will be helpful to understand complicated phylogenetic relationships of EBV genomes.

Journal Articles

[PROTOCOL] Drosophila melanogaster as a polymicrobial infection model for Pseudomonas aeruginosa and Staphylococcus aureus: Young-Joon Lee , Hye-Jeong Jang , In-Young Chung , You-Hee Cho; J. Microbiol. 2018;56(8):534-541. Published online July 25, 2018; DOI: https://doi.org/10.1007/s12275-018-8331-9

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20 Citations

Abstract: Non-mammalian infection models have been developed over the last two decades, which is a historic milestone to understand the molecular basis of bacterial pathogenesis. They also provide small-scale research platforms for identification of virulence factors, screening for antibacterial hits, and evaluation of antibacterial efficacy. The fruit fly, Drosophila melanogaster is one of the model hosts for a variety of bacterial pathogens, in that the innate immunity pathways and tissue physiology are highly similar to those in mammals. We here present a relatively simple protocol to assess the key aspects of the polymicrobial interaction in vivo between the human opportunistic pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, which is based on the systemic infection by needle pricking at the dorsal thorax of the flies. After infection, fly survival and bacteremia over time for both P. aeruginosa and S. aureus within the infected flies can be monitored as a measure of polymicrobial virulence potential. The infection takes ~24 h including bacterial cultivation. Fly survival and bacteremia are assessed using the infected flies that are monitored up to ~60 h post-infection. These methods can be used to identify presumable as well as unexpected phenotypes during polymicrobial interaction between P. aeruginosa and S. aureus mutants, regarding bacterial pathogenesis and host immunity.

Flavobacterium parvum sp. nov., isolated from soil polluted by sewer water: Hyun Seo Lee , Woon Mo Hwang , Keunsoo Kang , Tae-Young Ahn; J. Microbiol. 2018;56(8):542-548. Published online July 25, 2018; DOI: https://doi.org/10.1007/s12275-018-8145-9

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Abstract: A novel Gram-stain-negative, motile by means of gliding, and short rod-shaped bacterium, designated HS916T, was isolated from soil polluted by sewer water in Cheonan-si, South Korea. Growth occurred at 10–35°C (optimum 30°C), pH 6.0–8.0 (optimum pH 7.0), and 0–1% sodium chloride (NaCl, w/v). Based on similarities of 16S rRNA gene sequences, strain HS916T was closely related to members of the genus Flavobacterium, exhibiting the highest sequence similarities with Flavobacterium glycines Gm-149T (96.4%), followed by F. granuli Kw05T (96.3%), F. fluminis 3R17T (96.3%), F. aquicola TMd3a3T (96.2%), and F. nitratireducens N1T (96.2%). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain HS916T was placed in a monophyletic cluster with F. nitratireducens N1T and F. fluminis 3R17T. The predominant fatty acids (> 5% of the total) of strain HS916T were iso-C15:0, anteiso-C15:0, iso-C15:0 3-OH, C17:1 ω6с, C16:0 3-OH, iso-C17:0 3-OH, and summed feature 3 (C16:1 ω7с and/or C16:1 ω6с). The major polar lipids of the strain comprised phosphatidylethanolamine, unidentified aminolipids, and five unidentified lipids. The predominant respiratory quinone and the major polyamine were menaquinone-6 (MK-6) and symhomospermidine, respectively. The DNA G + C content of strain HS916T was 34.9 mol%. Based on polyphasic analyses, strain HS916T represents a novel species belonging to the genus Flavobacterium, for which the name Flavobacterium parvum sp. nov. is proposed. The type strain is HS916T (= KACC 19448T = JCM 32368T).

Leifsonia flava sp. nov., a novel actinobacterium isolated from the rhizosphere of Aquilegia viridiflora: Ying Cai , Wen-Zhen Tao , Yu-Jun Ma , Juan Cheng , Meng-Yue Zhang , Yi-Xuan Zhang; J. Microbiol. 2018;56(8):549-555. Published online July 25, 2018; DOI: https://doi.org/10.1007/s12275-018-8061-z

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Abstract: SYP-B2174T is a yellow-pigmented, Gram-positive, non-motile, and rod-shaped actinobacterium isolated from the rhizospheric soil of Aquilegia viridiflora Pall. collected from the Xinjiang uygur autonomous region of China. The strain’s growth temperature ranges from 1 to 35°C, with an optimal growth being observed at 28°C. Growth occurs from 0 to 5% NaCl and at pH 6–8, with optimal growth being observed in 1% NaCl at pH 7. Comparative 16S rRNA gene sequencebased phylogenetic analysis placed the strain in a clade with the species Leifsonia kafniensis JCM 17021T and Leifsonia psychrotolerans DSM 22824T with similarities of 97.8 and 97.6%, respectively. The DNA-DNA hybridization values of the strain SYP-B2174T to its closest phylogenetic neighbors were significantly lower than 35.7%. The strain was identified as a novel species of the genus Leifsonia judging by the coryneform morphology, peptidoglycans based upon 2,4-diaminobutyric acid, principal phospholipids phosphatidylglycerol and diphosphatidylglycerol, major menaquinone MK-11, predominant fatty acids of anteiso-C15:0, anteiso-C17:0, and iso-C16:0, and a DNA G + C base composition of 68.7 mol%, for which the name Leifsonia flava sp. nov. is proposed. The type strain is SYP-B2174T (= CGMCC 1.15856T = DSM 105144T = KCTC 39963T).

Evidence of the genetic diversity and clonal population structure of Oenococcus oeni strains isolated from different wine-making regions of China: Dongliang Yu , Kan Shi , Xiangyuan Wen , Fangshu Xie , Tao Wang , Shuwen Liu , Ling He; J. Microbiol. 2018;56(8):556-564. Published online July 25, 2018; DOI: https://doi.org/10.1007/s12275-018-7568-7

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5 Citations

Abstract: Studies of the genetic diversity and population structure of Oenococcus oeni (O. oeni) strains from China are lacking compared to other countries and regions. In this study, amplified fragment length polymorphism (AFLP) and multilocus sequence typing (MLST) methods were used to investigate the genetic diversity and regional evolutionary patterns of 38 O. oeni strains isolated from different wine-making regions in China. The results indicated that AFLP was markedly more efficient than MLST for typing O. oeni strains. AFLP distinguished 37 DNA patterns compared to 7 sequence types identified using MLST, corresponding to discriminatory indices of 0.999 and 0.602, respectively. The AFLP results revealed a high level of genetic diversity among the O. oeni strains from different regions of China, since two subpopulations and an intraspecific homology higher than 60% were observed. Phylogenetic analysis of the O. oeni strains using the MLST method also identified two major phylogroups, which were differentiated into two distinct clonal complexes by minimum spanning tree analysis. Neither intragenic nor intergenic recombination verified the existence of the clonal population structure of the O. oeni strains.

A rule governing the FtsH-mediated proteolysis of the MgtC virulence protein from Salmonella enterica serovar Typhimurium: Jonghyun Baek , Eunna Choi , Eun-Jin Lee; J. Microbiol. 2018;56(8):565-570. Published online July 25, 2018; DOI: https://doi.org/10.1007/s12275-018-8245-6

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Abstract: A tightly controlled turnover of membrane proteins is required for lipid bilayer stability, cell metabolism, and cell viability. Among the energy-dependent AAA+ proteases in Salmonella, FtsH is the only membrane-bound protease that contributes to the quality control of membrane proteins. FtsH preferentially degrades the C-terminus or N-terminus of misfolded, misassembled, or damaged proteins to maintain physiological functions. We found that FtsH hydrolyzes the Salmonella MgtC virulence protein when we substitute the MgtC 226th Trp, which is well conserved in other intracellular pathogens and normally protects MgtC from the FtsH-mediated proteolysis. Here we investigate a rule determining the FtsHmediated proteolysis of the MgtC protein at Trp226 residue. Substitution of MgtC tryptophan 226th residue to alanine, glycine, or tyrosine leads to MgtC proteolysis in a manner dependent on the FtsH protease whereas substitution to phenylalanine, methionine, isoleucine, leucine, or valine resists MgtC degradation by FtsH. These data indicate that a large and hydrophobic side chain at 226th residue is required for protection from the FtsH-mediated MgtC proteolysis.

Biosynthesis of 2-amino-3-hydroxycyclopent-2-enone moiety of bafilomycin in Kitasatospora cheerisanensis KCTC2395: Nguyen Phan Kieu Hanh , Jae Yoon Hwang , Hye Ryeung Oh , Geum Jin Kim , Hyukjae Choi , Doo Hyun Nam; J. Microbiol. 2018;56(8):571-578. Published online July 25, 2018; DOI: https://doi.org/10.1007/s12275-018-8267-0

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Abstract: Bafilomycins produced by Kitasatospora cheerisanensis KCTC- 2395 belong to the 16-membered macrolactone family plecomacrolide antibiotics. Bafilomycin B1 contains 2-amino- 3-hydroxycyclopent-2-enone (C5N), a five membered ring, which gets condensed via an amide linkage to bafilomycin polyketide. To study the biosynthetic pathway of C5N during bafilomycin biosynthesis in K. cheerisanensis KCTC2395, we attempted the functional analysis of two putative genes, encoding 5-aminolevulinic acid synthase (ALAS) and acyl- CoA ligase (ACL). The amplified putative genes for ALAS and ACL were cloned into the E. coli expression vector pET- 32a(+) plasmid, following which the soluble recombinant ALAS and ACL proteins were purified through nickel-affinity column chromatography. Through HPLC analysis of the enzyme reaction mixture, we confirmed the products of putative ALAS and ACL reaction as 5-aminolevulinic acid (5- ALA) and 5-ALA-CoA, respectively. The optimal pH for the putative ALAS reaction was 7.5, and for putative ACL reaction was 7.0, as confirmed by the colorimetric assay. Furthermore, pyridoxal 5􍿁-phosphate (PLP) was found to be an essential cofactor in the putative ALAS reaction, and ATP was a cofactor for the putative ACL catalysis. Finally, we also confirmed that the simultaneous treatment of putative ACL and putative ALAS enzymes resulted in the production of C5N compound from 5-ALA.

Gamma-irradiation of Streptococcus pneumoniae for the use as an immunogenic whole cell vaccine: Min Yong Jwa , Soyoung Jeong , Eun Byeol Ko , A Reum Kim , Hyun Young Kim , Sun Kyung Kim , Ho Seong Seo , Cheol-Heui Yun , Seung Hyun Han; J. Microbiol. 2018;56(8):579-585. Published online July 25, 2018; DOI: https://doi.org/10.1007/s12275-018-8347-1

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19 Citations

Abstract: Streptococcus pneumoniae is a major respiratory pathogen that causes millions of deaths worldwide. Although subunit vaccines formulated with the capsular polysaccharides or their protein conjugates are currently-available, low-cost vaccines with wide serotype coverage still remain to be developed, especially for developing countries. Recently, gamma- irradiation has been considered as an effective inactivation
method
to prepare S. pneumoniae vaccine candidate. In this study, we investigated the immunogenicity and protective immunity of gamma-irradiated S. pneumoniae (r-SP), by comparing with heat-inactivated S. pneumoniae (h-SP) and formalin-inactivated S. pneumoniae (f-SP), both of which were made by traditional inactivation methods. Intranasal immunization of C57BL/6 mice with r-SP in combination with cholera toxin as an adjuvant enhanced S. pneumoniaespecific antibodies on the airway mucosal surface and in sera more potently than that with h-SP or f-SP under the same conditions. In addition, sera from mice immunized with r- SP potently induced opsonophagocytic killing activity more effectively than those of h-SP or f-SP, implying that r-SP could induce protective antibodies. Above all, immunization with r-SP effectively protected mice against S. pneumoniae infection. Collectively, these results suggest that gamma- irradiation is an effective method for the development of a killed whole cell pneumococcal vaccine that elicits robust mucosal and systemic immune responses.

Antiviral activity of Poncirus trifoliata seed extract against oseltamivirresistant influenza virus: Yoonki Heo , Yeondong Cho , Kwon sung Ju , Hansam Cho , Ki Hoon Park , Hanul Choi , Jong Kwang Yoon , Chiung Moon , Young Bong Kim; J. Microbiol. 2018;56(8):586-592. Published online July 25, 2018; DOI: https://doi.org/10.1007/s12275-018-8222-0

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Abstract: The emergence of oseltamivir-resistant variants of influenza virus has highlighted the necessity for the development of more effective novel antiviral drugs. To date, numerous researchers have focused on developing antiviral drugs using natural resources, such as traditional herbal medicines. Poncirus trifoliata is widely used in oriental medicine as a remedy for gastritis, dysentery, inflammation and digestive ulcers. In this study, we investigated the potential antiviral effect of the Poncirus trifoliata orange seed extract against influenza virus. An ethanol extract of Poncirus trifoliata seeds (PTex) inhibited the activity of influenza viruses, in particular, oseltamivir- resistant strains, in Madin-Darby canine kidney cells. In contrast to oseltamivir, PTex exerted a significant inhibitory effect on the cellular penetration pathway of the virus rather than HA receptor binding. The potent antiviral effect and novel working mechanism of PTex support its further development as an effective natural antiviral drug with a wide spectrum of activity against influenza and oseltamivir-resistant viruses.

Development and validation of multiplex real-time PCR assays for rapid detection of cytomegalovirus, Epstein-Barr virus, and polyomavirus BK in whole blood from transplant candidates: Kyung-Ah Hwang , Ji Hoon Ahn , Jae-Hwan Nam; J. Microbiol. 2018;56(8):593-599. Published online July 25, 2018; DOI: https://doi.org/10.1007/s12275-018-8273-2

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Abstract: Transplant recipients are more susceptible to bacterial and viral infections. Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and polyomavirus BK (BK) are risk factors for graft dysfunction. All three of them are latent viruses that can cause serious disease in immunocompromised patients. Mainly qualitative PCR tests are required for diagnosis and quantitative monitoring, which are used to follow the response to transplantation. We developed a multiplex real-time PCR (qPCR)
method
to detect these viruses during blood screenings of transplant recipients. We also validated analytical and clinical performance tests using the developed multiplex qPCR. The limit of detection (LOD) was 100, 125, and 183 copies/ml for CMV, EBV, and BK, respectively. These results had high linearity (R2 = 0.997) and reproducibility (CV range, 0.95– 2.38%, 0.52–3.32%, and 0.31–2.45%, respectively). Among 183 samples, we detected 8 samples that were positive for CMV, while only 6 were positive for EBV, and 3 were positive for BK. Therefore, the viral infection prevalence in transplant candidates was 4.40% for CMV, 3.29% for EBV, and 1.64% for BK. This multiplex qPCR method should be used widely for diagnosing and monitoring latent viral infections in transplant recipients.

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