Journal of Microbiology

Journal Articles

Identification and Characterization of HEPN‑MNT Type II TA System from Methanothermobacter thermautotrophicus ΔH: Wonho Choi , Anoth Maharjan , Hae Gang Im , Ji-Young Park , Jong-Tae Park , Jung-Ho Park; J. Microbiol. 2023;61(4):411-421. Published online April 18, 2023; DOI: https://doi.org/10.1007/s12275-023-00041-9

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Abstract: Toxin-antitoxin (TA) systems are widespread in bacteria and archaea plasmids and genomes to regulate DNA replication, gene transcr!ption, or protein translation. Higher eukaryotic and prokaryotic nucleotide-binding (HEPN) and minimal nucleotidyltransferase (MNT) domains are prevalent in prokaryotic genomes and constitute TA pairs. However, three gene pairs (MTH304/305, 408/409, and 463/464) of Methanothermobacter thermautotropicus ΔH HEPN-MNT family have not been studied as TA systems. Among these candidates, our study characterizes the MTH463/MTH464 TA system. MTH463 expression inhibited Escherichia coli growth, whereas MTH464 did not and blocked MTH463 instead. Using site-directed MTH463 mutagenesis, we determined that amino acids R99G, H104A, and Y106A from the R[ɸX]4-6H motif are involved with MTH463 cell toxicity. Furthermore, we established that purified MTH463 could degrade MS2 phage RNA, whereas purified MTH464 neutralized MTH463 activity in vitro. Our results indicate that the endonuclease toxin MTH463 (encoding a HEPN domain) and its cognate antitoxin MTH464 (encoding the MNT domain) may act as a type II TA system in M. thermautotropicus ΔH. This study provides initial and essential information studying TA system functions, primarily archaea HEPN-MNT family.

Vibrio vulnificus PlpA facilitates necrotic host cell death induced by the pore forming MARTX toxin: Changyi Cho , Sanghyeon Choi , Myung Hee Kim , Byoung Sik Kim; J. Microbiol. 2022;60(2):224-233. Published online February 1, 2022; DOI: https://doi.org/10.1007/s12275-022-1448-x

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Abstract: Opportunistic pathogen Vibrio vulnificus causes severe systemic infection in humans with high mortality. Although multiple exotoxins have been characterized in V. vulnificus, their interactions and potential synergistic roles in pathogen-induced host cell death have not been investigated previously. By employing a series of multiple exotoxin deletion mutants, we investigated whether specific exotoxins of the pathogen functioned together to achieve severe and rapid necrotic cell death. Human epithelial cells treated with V. vulnificus with a plpA deletion background exhibited an unusually prolonged cell blebbing, suggesting the importance of PlpA, a phospholipase A2, in rapid necrotic cell death by this pathogen. Additional deletion of the rtxA gene encoding the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin did not result in necrotic cell blebs. However, if the rtxA gene was engineered to produce an effector-free MARTX toxin, the cell blebbing was observed, indicating that the pore forming activity of the MARTX toxin is sufficient, but the MARTX toxin effector domains are not necessary, for the blebbing. When a recombinant PlpA was treated on the blebbed cells, the blebs were completely disrupted. Consistent with this, MARTX toxin-pendent rapid release of cytosolic lactate dehydrogenase was significantly delayed in the plpA deletion background. Mutations in other exotoxins such as elastase, cytolysin/hemolysin, and/or extracellular metalloprotease did not affect the bleb formation or disruption. Together, these findings indicate that the pore forming MARTX toxin and the phospholipase A2, PlpA, cooperate sequentially to achieve rapid necrotic cell death by inducing cell blebbing and disrupting the blebs, respectively.

Analyses of DNA double-strand break repair pathways in tandem arrays of HXT genes of Saccharomyces cerevisiae: Ju-Hee Choi , Ye-Seul Lim , Min-Ku Kim , Sung-Ho Bae; J. Microbiol. 2020;58(11):957-966. Published online October 30, 2020; DOI: https://doi.org/10.1007/s12275-020-0461-1

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5 Citations

Abstract: Eukaryotic genomes contain numerous homologous repeat sequences including redundant genes with divergent homology that can be potential recombination targets. Recombination between divergent sequences is rare but poses a substantial threat to genome stability. The hexose transporter (HXT) gene family shares high sequence similarities at both protein and DNA levels, and some members are placed close together in tandem arrays. In this study, we show that spontaneous interstitial deletions occur at significantly high rates in HXT gene clusters, resulting in chimeric HXT sequences that contain a single junction point. We also observed that DNA double-strand breaks created between HXT genes produce primarily interstitial deletions, whereas internal cleavage of the HXT gene resulted in gene conversions as well as deletion products. Interestingly, interstitial deletions were less constrained by sequence divergence than gene conversion. Moreover, recombination-defective mutations differentially affected the survival frequency. Mutations that impair single-strand annealing (SSA) pathway greatly reduced the survival frequency by 10–1,000-fold, whereas disruption of Rad51-dependent homologous recombination exhibited only modest reduction. Our results indicate that recombination in the tandemly repeated HXT genes occurs primarily via SSA pathway.

IgG and IgM responses to human papillomavirus L1 virus-like particle as a function of dosing schedule and vaccine formulation: Min-Hye Park , Ji Won You , Hyoung Jin Kim , Hong-Jin Kim; J. Microbiol. 2019;57(9):821-827. Published online August 27, 2019; DOI: https://doi.org/10.1007/s12275-019-9308-z

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4 Citations

Abstract: Most commercialized virus-like particle (VLP) vaccines use aluminum salt as adjuvant, even though VLPs provoke adequate antibody responses without adjuvant. We do not have detailed knowledge of how adjuvant affects the profile of anti- VLP antibodies. Meanwhile, there is evidence that differences between vaccination protocols influence the glycosylation of antibodies, which may alter their effector functions. In the present study a murine model was used to investigate the effects of dosing schedule and adjuvant on the antibody profiles and glycosylation levels of antigen-specific antibody responses to human papillomavirus type 16 L1 (HPV16 L1) VLPs. Mice received subcutaneously 2,000 ng of antigen divided into 4 or 7 doses. The HPV16 L1 VLPs elicited > 4 log10 anti-HPV16 L1 IgG titers without adjuvant, and aluminum hydroxide as adjuvant increased IgG titers 1.3- to 4-fold and reduced the anti-HPV16 L1 IgG2a / anti-HPV16 L1 IgG1 ratio value (use of aluminum hydroxide reduced the ratio of the IgG2a). Immunization with HPV16 L1 VLPs in combination with Freund’s adjuvant enhanced IgG titers 5- to 12- fold. Seven-dose immunization markedly increased anti- HPV16 L1 IgM titers compared to four-dose immunization, as well as increasing the proportion of glycosylated antibodies. Our results suggest that antibody glycosylation can be controlled immunologically, and IgG and IgM profiles and glycosylation profiles of the vaccine-induced antibodies can be used as indicators reflecting the vaccine characteristics. These
results
indicate that the HPV16 L1 VLP dosing schedule can affect the quality of antigen-specific antibody responses. We suggest that dosing schedules should be noted in vaccination protocols for VLP-based vaccines.

Review

[Minireview] Antibiotic resistance of pathogenic Acinetobacter species and emerging combination therapy: Bora Shin , Woojun Park; J. Microbiol. 2017;55(11):837-849. Published online October 27, 2017; DOI: https://doi.org/10.1007/s12275-017-7288-4

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40 Citations

Abstract: The increasing antibiotic resistance of Acinetobacter species in both natural and hospital environments has become a serious problem worldwide in recent decades. Because of both intrinsic and acquired antimicrobial resistance (AMR) against last-resort antibiotics such as carbapenems, novel therapeutics are urgently required to treat Acinetobacter-associated infectious diseases. Among the many pathogenic Acinetobacter species, A. baumannii has been reported to be resistant to all classes of antibiotics and contains many AMR genes, such as blaADC (Acinetobacter-derived cephalosporinase). The AMR of pathogenic Acinetobacter species is the result of several different mechanisms, including active efflux pumps, mutations in antibiotic targets, antibiotic modification, and low antibiotic membrane permeability. To overcome the limitations of existing drugs, combination theraphy that can increase the activity of antibiotics should be considered in the treatment of Acinetobacter infections. Understanding the molecular mechanisms behind Acinetobacter AMR resistance will provide vital information for drug development and therapeutic strategies using combination treatment. Here, we summarize the classic mechanisms of Acinetobacter AMR, along with newly-discovered genetic AMR factors and currently available antimicrobial adjuvants that can enhance drug efficacy in the treatment of A. baumannii infections.

Research Support, Non-U.S. Gov't

Adjuvant Efficacy of mOMV against Avian Influenza Virus Infection in Mice: Byeong-Jae Lee , Sang-Ho Lee , Min-Suk Song , Philippe Noriel Q. Pascua , Hyeok-il Kwon , Su-Jin Park , Eun-Ha Kim , Arun Decano , Se Mi Kim , Gyo Jin Lim , Doo-Jin Kim , Kyu-Tae Chang , Sang-Hyun Kim , Young Ki Choi; J. Microbiol. 2013;51(5):682-688. Published online October 31, 2013; DOI: https://doi.org/10.1007/s12275-013-3411-3

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Abstract: Highly pathogenic avian influenza H5N1 viruses are found chiefly in birds and have caused severe disease and death in infected humans. Development of influenza vaccines capable of inducing heterosubtypic immunity against a broad range of influenza viruses is the best option for the preparedness, since vaccination remains the principal method in controlling influenza viral infections. Here, a mOMV-adjuvanted recombinant H5N2 (rH5N2) whole virus antigen vaccine with A/Environment/Korea/W149/06(H5N1)-derived H5 HA and A/Chicken/Korea/ma116/04(H9N2)-derived N2 NA in the backbone of A/Puerto Rico/8/34(H1N1) was prepared and generated by reverse genetics. Groups of mice were vaccinated by a prime-boost regime with the rH5N2 vaccine (1.75 μg of HA with/without 10 μg mOMV or aluminum hydroxide adjuvant for comparison). At two weeks post-immunizations, vaccinated mice were challenged with lethal doses of 103.5 EID50/ml of H5N1 or H9N2 avian influenza viruses, and were monitored for 15 days. Both mOMV- and alum-adjuvant vaccine groups had high survival rates after H5N1 infection and low levels of body weight changes compared to control groups. Interestingly, the mOMV-adjuvanted group induced better cross-reactive antibody responses serologically and promoted cross-protectivity against H5N1 and H9N2 virus challenges. Our results suggest that mOMV could be used as a vaccine adjuvant in the development of effective vaccines used to control influenza A virus transmission.

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