A reverse genetics system for the respiratory syncytial virus (RSV), which causes acute respiratory illness, is an effective tool for understanding the pathogenicity of RSV. To date, a method dependent on T7 RNA polymerase is commonly used for RSV. Although this method is well established and recombinant RSV is well rescued from transfected cells, the requirement for artificial supply of T7 RNA polymerase limits its application. To overcome this, we established a reverse genetics system dependent on RNA polymerase II, which is more convenient for the recovery of recombinant viruses from various cell lines. First, we identified human cell lines with high transfection efficiency in which RSV can replicate effectively. Two human cell lines, Huh-7 and 293T, permitted the propagation of recombinant green fluorescent protein–expressing RSV. Our minigenome system revealed that efficient transcription and replication of RSV occurred in both Huh-7 and 293T cells. We then confirmed that recombinant green fluorescent protein–expressing RSV was rescued in both Huh-7 and 293T cells. Furthermore, the growth capability of viruses rescued from Huh-7 and 293T cells was similar to that of recombinant RSV rescued using the conventional method. Thus, we succeeded in establishing a new reverse genetics system for RSV that is dependent on RNA polymerase II.
{"title":"Establishment of a new reverse genetics system for respiratory syncytial virus under the control of RNA polymerase II","authors":"Tatsuki Takahashi, Shiori Ueno, Yoshiro Sugiura, Kenta Shimizu, Wataru Kamitani","doi":"10.1111/1348-0421.13088","DOIUrl":"10.1111/1348-0421.13088","url":null,"abstract":"<p>A reverse genetics system for the respiratory syncytial virus (RSV), which causes acute respiratory illness, is an effective tool for understanding the pathogenicity of RSV. To date, a method dependent on T7 RNA polymerase is commonly used for RSV. Although this method is well established and recombinant RSV is well rescued from transfected cells, the requirement for artificial supply of T7 RNA polymerase limits its application. To overcome this, we established a reverse genetics system dependent on RNA polymerase II, which is more convenient for the recovery of recombinant viruses from various cell lines. First, we identified human cell lines with high transfection efficiency in which RSV can replicate effectively. Two human cell lines, Huh-7 and 293T, permitted the propagation of recombinant green fluorescent protein–expressing RSV. Our minigenome system revealed that efficient transcription and replication of RSV occurred in both Huh-7 and 293T cells. We then confirmed that recombinant green fluorescent protein–expressing RSV was rescued in both Huh-7 and 293T cells. Furthermore, the growth capability of viruses rescued from Huh-7 and 293T cells was similar to that of recombinant RSV rescued using the conventional method. Thus, we succeeded in establishing a new reverse genetics system for RSV that is dependent on RNA polymerase II.</p>","PeriodicalId":18679,"journal":{"name":"Microbiology and Immunology","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2023-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10521913","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Anke Neidig, Nikola Strempel, Nadine Bianca Waeber, Waleska Stephanie da Cruz Nizer, Joerg Overhage
Multidrug efflux systems of the resistance-nodulation-cell division family play a crucial role in resistance of Pseudomonas aeruginosa to a large variety of antibiotics. Here, we investigated the role of clinically relevant efflux pumps MexAB−OprM, MexCD−OprJ, and MexXY−OprM in resistance against different cationic antimicrobial peptides (AMPs). Our results indicate that a knock-out in efflux pump MexXY-OprM increased susceptibility to some AMPs by two- to eightfold. Our data suggest a contribution of MexXY-OprM in resistance to certain AMPs in P. aeruginosa, which should be considered in the future development of new and highly active antimicrobial peptides to fight multidrug resistant infections.
{"title":"Knock-out of multidrug efflux pump MexXY-OprM results in increased susceptibility to antimicrobial peptides in Pseudomonas aeruginosa","authors":"Anke Neidig, Nikola Strempel, Nadine Bianca Waeber, Waleska Stephanie da Cruz Nizer, Joerg Overhage","doi":"10.1111/1348-0421.13089","DOIUrl":"10.1111/1348-0421.13089","url":null,"abstract":"<p>Multidrug efflux systems of the resistance-nodulation-cell division family play a crucial role in resistance of <i>Pseudomonas aeruginosa</i> to a large variety of antibiotics. Here, we investigated the role of clinically relevant efflux pumps MexAB<sup>−</sup>OprM, MexCD<sup>−</sup>OprJ, and MexXY<sup>−</sup>OprM in resistance against different cationic antimicrobial peptides (AMPs). Our results indicate that a knock-out in efflux pump MexXY-OprM increased susceptibility to some AMPs by two- to eightfold. Our data suggest a contribution of MexXY-OprM in resistance to certain AMPs in <i>P. aeruginosa</i>, which should be considered in the future development of new and highly active antimicrobial peptides to fight multidrug resistant infections.</p>","PeriodicalId":18679,"journal":{"name":"Microbiology and Immunology","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2023-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1111/1348-0421.13089","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10521912","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jiajing Li, Yining Wang, Sajjan Rajpoot, Marla Lavrijsen, Qiuwei Pan, Pengfei Li, Mirza S. Baig
Coronaviruses (CoVs) have long been known to infect humans, mainly alpha-CoV and beta-CoV. The vaccines developed for SARS-CoV-2 are likely not effective against other coronavirus species, whereas the risk of the emergence of new strains that may cause the next epidemic/pandemic is high. The development of antiviral drugs that are effective across different CoVs represents a viable strategy for improving pandemic preparedness. In this study, we aim to identify pan-coronaviral agents by targeting the conserved main protease (Mpro). For drug screening, the catalytic dyad of four human CoVs (HCoVs: SARS-CoV-2, and seasonal CoV NL63, OC43, and 229E) was targeted by molecular docking. The identified leading candidate theobromine, a xanthine derivative, was further tested in cell culture models of coronavirus infection. Theobromine binds strongly with the catalytic dyad (His41 and Cys144/145) of SARS-CoV-2 and HCoV-NL63 Mpro, mildly with HCoV-OC43, but not with HCoV-229E. However, theobromine only shows dose-dependent inhibition in Calu3 cells inoculated with SARS-CoV-2, but not in cells inoculated with seasonal CoVs. Theobromine exerts antiviral activity against coronavirus infections potentially through targeting Mpro. However, the antiviral potency is distinct among different CoVs.
{"title":"Investigating theobromine as a potential anti-human coronaviral agent","authors":"Jiajing Li, Yining Wang, Sajjan Rajpoot, Marla Lavrijsen, Qiuwei Pan, Pengfei Li, Mirza S. Baig","doi":"10.1111/1348-0421.13086","DOIUrl":"10.1111/1348-0421.13086","url":null,"abstract":"<p>Coronaviruses (CoVs) have long been known to infect humans, mainly alpha-CoV and beta-CoV. The vaccines developed for SARS-CoV-2 are likely not effective against other coronavirus species, whereas the risk of the emergence of new strains that may cause the next epidemic/pandemic is high. The development of antiviral drugs that are effective across different CoVs represents a viable strategy for improving pandemic preparedness. In this study, we aim to identify pan-coronaviral agents by targeting the conserved main protease (Mpro). For drug screening, the catalytic dyad of four human CoVs (HCoVs: SARS-CoV-2, and seasonal CoV NL63, OC43, and 229E) was targeted by molecular docking. The identified leading candidate theobromine, a xanthine derivative, was further tested in cell culture models of coronavirus infection. Theobromine binds strongly with the catalytic dyad (His41 and Cys144/145) of SARS-CoV-2 and HCoV-NL63 Mpro, mildly with HCoV-OC43, but not with HCoV-229E. However, theobromine only shows dose-dependent inhibition in Calu3 cells inoculated with SARS-CoV-2, but not in cells inoculated with seasonal CoVs. Theobromine exerts antiviral activity against coronavirus infections potentially through targeting Mpro. However, the antiviral potency is distinct among different CoVs.</p>","PeriodicalId":18679,"journal":{"name":"Microbiology and Immunology","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2023-07-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10205028","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Xia Chuai, Yaya Zhou, Junhua Feng, Menghan Yu, Yan Wu, Lujuan Han, Yan Zhao, Hongxiu Qiao, Zhiyun Gao, Jian Li, Lixin Xie, Wenting Zhao, Changle Wang
Acinetobacter baumannii is a multidrug-resistant coccobacillus responsible for severe nosocomial infectious diseases. This study mainly focuses on investigating the antimicrobial resistance features of a clinically isolated strain (A. baumannii CYZ) using the PacBio Sequel II sequencing platform. The chromosomal size of A. baumannii CYZ is 3,960,760 bp, which contains a total of 3803 genes with a G + C content of 39.06%. Functional analysis performed using the Clusters of Orthologous Groups of Proteins (COGs), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, as well as the Comprehensive Antibiotic Resistance Database (CARD) revealed a complicated set of antimicrobial resistance determinants in the genome of A. baumannii CYZ, which were mainly classified into multidrug efflux pumps and transport systems, β-lactamase relative and penicillin-binding proteins, aminoglycoside modification enzymes, alternation of antibiotic target sites, lipopolysaccharide relative, and other mechanisms. A total of 35 antibiotics were tested for the antimicrobial susceptibility of A. baumannii CYZ, and the organism exhibited a stronger antimicrobial resistance ability. The phylogenetic relationship indicated that A. baumannii CYZ has high homology with A. baumannii ATCC 17978; however, the former also exhibited its specific genome characteristics. Our research results give insight into the genetic antimicrobial-resistant features of A. baumannii CYZ as well as provide a genetic basis for the further study of the phenotype.
鲍曼不动杆菌是一种多重耐药球芽孢杆菌,可导致严重的医院传染病。本研究主要利用PacBio Sequel II测序平台研究临床分离菌株(鲍曼不动杆菌CYZ)的耐药特征。鲍曼不动杆菌CYZ染色体大小为3960760 bp,共包含3803个基因,G + C含量为39.06%。利用同源蛋白群(Clusters of Orthologous Groups of Proteins, COGs)、基因本体(Gene Ontology, GO)、京都基因与基因组百科全书(KEGG)数据库以及抗生素耐药性综合数据库(Comprehensive Antibiotic Resistance Database, CARD)进行的功能分析显示,鲍曼不动杆菌CYZ基因组中存在一组复杂的耐药决定因素,主要分为多药外排泵和转运系统、β-内酰胺酶相关蛋白和青霉素结合蛋白。氨基糖苷修饰酶,抗生素靶位点的改变,脂多糖相关,和其他机制。鲍曼不动杆菌CYZ共对35种抗生素进行了药敏试验,结果表明该菌具有较强的耐药能力。系统发育关系表明,鲍曼不动杆菌CYZ与鲍曼不动杆菌ATCC 17978具有高度同源性;然而,前者也表现出其特定的基因组特征。我们的研究结果揭示了鲍曼不动杆菌CYZ的遗传耐药特征,并为进一步研究其表型提供了遗传基础。
{"title":"Analysis of multidrug-resistant determinants of clinically isolated Acinetobacter baumannii CYZ via whole genome sequencing","authors":"Xia Chuai, Yaya Zhou, Junhua Feng, Menghan Yu, Yan Wu, Lujuan Han, Yan Zhao, Hongxiu Qiao, Zhiyun Gao, Jian Li, Lixin Xie, Wenting Zhao, Changle Wang","doi":"10.1111/1348-0421.13087","DOIUrl":"10.1111/1348-0421.13087","url":null,"abstract":"<p><i>Acinetobacter baumannii</i> is a multidrug-resistant coccobacillus responsible for severe nosocomial infectious diseases. This study mainly focuses on investigating the antimicrobial resistance features of a clinically isolated strain (<i>A. baumannii</i> CYZ) using the PacBio Sequel II sequencing platform. The chromosomal size of <i>A. baumannii</i> CYZ is 3,960,760 bp, which contains a total of 3803 genes with a G + C content of 39.06%. Functional analysis performed using the Clusters of Orthologous Groups of Proteins (COGs), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, as well as the Comprehensive Antibiotic Resistance Database (CARD) revealed a complicated set of antimicrobial resistance determinants in the genome of <i>A. baumannii</i> CYZ, which were mainly classified into multidrug efflux pumps and transport systems, β-lactamase relative and penicillin-binding proteins, aminoglycoside modification enzymes, alternation of antibiotic target sites, lipopolysaccharide relative, and other mechanisms. A total of 35 antibiotics were tested for the antimicrobial susceptibility of <i>A. baumannii</i> CYZ, and the organism exhibited a stronger antimicrobial resistance ability. The phylogenetic relationship indicated that <i>A. baumannii</i> CYZ has high homology with <i>A. baumannii</i> ATCC 17978; however, the former also exhibited its specific genome characteristics. Our research results give insight into the genetic antimicrobial-resistant features of <i>A. baumannii</i> CYZ as well as provide a genetic basis for the further study of the phenotype.</p>","PeriodicalId":18679,"journal":{"name":"Microbiology and Immunology","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2023-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10205016","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Cover photograph: Electron microscopy of lectin-treated SARS-CoV-2. SARS-CoV-2 was concentrated by using a porous membrane-filter unit and incubated with saline or 9 μM OAA, ESA-2, or HypninA-2 for 10 min. The sample was negative-stained and observed by transmission electron microscopy. The left panel for each lectin is a 2000x view, and the right panel is a magnified view of the designated region. The area of n viruses on the electron micrographs was measured and divided into groups corresponding to less than 2 viral particles (~2), 2-5 (2~5), and 5 or more (5~), and the percentages were plotted on a pie chart. For HypninA-2, all virus particles made up about 10 huge clumps, and nothing that appeared to be free virus particles was found. Therefore, no pie chart was drawn. Microbiol Immunol: 67:334–344. Article link here