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Current Protocols in Microbiology最新文献

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Flow Cytometry Analysis of Fungal Ploidy 真菌倍性的流式细胞术分析
Current Protocols in Microbiology Pub Date : 2018-07-20 DOI: 10.1002/cpmc.58
Robert T. Todd, Ann L. Braverman, Anna Selmecki
{"title":"Flow Cytometry Analysis of Fungal Ploidy","authors":"Robert T. Todd,&nbsp;Ann L. Braverman,&nbsp;Anna Selmecki","doi":"10.1002/cpmc.58","DOIUrl":"10.1002/cpmc.58","url":null,"abstract":"<p>Ploidy, the number of sets of homologous chromosomes in a cell, can alter cellular physiology, gene regulation, and the spectrum of acquired mutations. Advances in single-cell flow cytometry have greatly improved the understanding of how genome size contributes to diverse biological processes including speciation, adaptation, pathogenesis, and tumorigenesis. For example, fungal pathogens can undergo whole genome duplications during infection of the human host and during acquisition of antifungal drug resistance. Quantification of ploidy is dramatically affected by the nucleic acid staining technique and the flow cytometry analysis of single cells. Ploidy in fungi is also impacted by samples that are heterogeneous for both ploidy and morphology, and control strains with known ploidy must be included in every flow cytometry experiment. To detect ploidy changes within fungal strains, the following protocol was developed to accurately and dependably interrogate single-cell ploidy. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"50 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-07-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.58","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36331245","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 15
Genetic Manipulation of Cryptococcus neoformans 新型隐球菌的遗传操作
Current Protocols in Microbiology Pub Date : 2018-07-17 DOI: 10.1002/cpmc.59
Kwang-Woo Jung, Kyung-Tae Lee, Yee-Seul So, Yong-Sun Bahn
{"title":"Genetic Manipulation of Cryptococcus neoformans","authors":"Kwang-Woo Jung,&nbsp;Kyung-Tae Lee,&nbsp;Yee-Seul So,&nbsp;Yong-Sun Bahn","doi":"10.1002/cpmc.59","DOIUrl":"10.1002/cpmc.59","url":null,"abstract":"<p><i>Cryptococcus neoformans</i> is an opportunistic fungal pathogen, which causes life-threatening meningoencephalitis in immunocompromised individuals and is responsible for more than 1,000,000 infections and 600,000 deaths annually worldwide. Nevertheless, anti-cryptococcal therapeutic options are limited, mainly because of the similarity between fungal and human cellular structures. Owing to advances in genetic and molecular techniques and bioinformatics in the past decade, <i>C. neoformans</i>, belonging to the phylum basidiomycota, is now a major pathogenic fungal model system. In particular, genetic manipulation is the first step in the identification and characterization of the function of genes for understanding the mechanisms underlying the pathogenicity of <i>C. neoformans</i>. This unit describes protocols for constructing target gene deletion mutants using double-joint (DJ) PCR, constitutive overexpression strains using the histone H3 gene promoter, and epitope/fluorescence protein-tagged strains in <i>C. neoformans</i>. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"50 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-07-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.59","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36318583","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 17
In Vitro Culturing and Screening of Candida albicans Biofilms 白色念珠菌生物膜的体外培养与筛选
Current Protocols in Microbiology Pub Date : 2018-07-11 DOI: 10.1002/cpmc.60
Megha Gulati, Matthew B. Lohse, Craig L. Ennis, Ruth E. Gonzalez, Austin M. Perry, Priyanka Bapat, Ashley Valle Arevalo, Diana L. Rodriguez, Clarissa J. Nobile
{"title":"In Vitro Culturing and Screening of Candida albicans Biofilms","authors":"Megha Gulati,&nbsp;Matthew B. Lohse,&nbsp;Craig L. Ennis,&nbsp;Ruth E. Gonzalez,&nbsp;Austin M. Perry,&nbsp;Priyanka Bapat,&nbsp;Ashley Valle Arevalo,&nbsp;Diana L. Rodriguez,&nbsp;Clarissa J. Nobile","doi":"10.1002/cpmc.60","DOIUrl":"10.1002/cpmc.60","url":null,"abstract":"<p><i>Candida albicans</i> is a normal member of the human microbiota that asymptomatically colonizes healthy individuals, however it is also an opportunistic pathogen that can cause severe infections, especially in immunocompromised individuals. The medical impact of <i>C. albicans</i> depends, in part, on its ability to form biofilms, communities of adhered cells encased in an extracellular matrix. Biofilms can form on both biotic and abiotic surfaces, such as tissues and implanted medical devices. Once formed, biofilms are highly resistant to antifungal agents and the host immune system, and can act as a protected reservoir to seed disseminated infections. Here, we present several <i>in vitro</i> biofilm protocols, including protocols that are optimized for high-throughput screening of mutant libraries and antifungal compounds. We also present protocols to examine specific stages of biofilm development and protocols to evaluate interspecies biofilms that <i>C. albicans</i> forms with interacting microbial partners. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"50 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.60","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36303215","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 61
Shigella Pathogenesis Modeling with Tissue Culture Assays 志贺氏菌发病机制的组织培养模型
Current Protocols in Microbiology Pub Date : 2018-05-24 DOI: 10.1002/cpmc.57
Benjamin J. Koestler, Cara M. Ward, Shelley M. Payne
{"title":"Shigella Pathogenesis Modeling with Tissue Culture Assays","authors":"Benjamin J. Koestler,&nbsp;Cara M. Ward,&nbsp;Shelley M. Payne","doi":"10.1002/cpmc.57","DOIUrl":"10.1002/cpmc.57","url":null,"abstract":"<p><i>Shigella</i> is an enteroinvasive human pathogen that infects the colonic epithelium and causes Shigellosis, an infectious diarrheal disease. There is no vaccine for the prevention or treatment of Shigellosis and antibiotic-resistant strains of <i>Shigella</i> are increasing, emphasizing the need for a deeper understanding of <i>Shigella</i> pathogenesis in order to design effective antimicrobial therapies. Small animal models do not recapitulate Shigellosis, therefore tissue-cultured cells have served as model systems to study <i>Shigella</i> pathogenesis. Here, protocols to enumerate <i>Shigella</i> invasion, cell-cell spread, and plaque formation in the tissue-cultured cell lines Henle-407 and CoN-841 are described. Additionally, a new method to study <i>Shigella</i> invasion in primary intestinal enteroids is described. These protocols can be used to examine different aspects of <i>Shigella</i> virulence. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"50 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.57","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36244157","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 11
Use of Axenic Culture Tools to Study Coxiella burnetii 利用无菌培养工具研究伯氏杆菌
Current Protocols in Microbiology Pub Date : 2018-05-18 DOI: 10.1002/cpmc.52
Savannah E. Sanchez, Eduardo Vallejo-Esquerra, Anders Omsland
{"title":"Use of Axenic Culture Tools to Study Coxiella burnetii","authors":"Savannah E. Sanchez,&nbsp;Eduardo Vallejo-Esquerra,&nbsp;Anders Omsland","doi":"10.1002/cpmc.52","DOIUrl":"10.1002/cpmc.52","url":null,"abstract":"<p><i>Coxiella burnetii</i> is a highly infectious obligate intracellular bacterium and the etiological agent of the zoonosis Query (Q) fever. This Gram-negative gamma-proteobacterium has adapted to replicate within a specialized compartment in mammalian phagocytic cells, known as the <i>Coxiella</i>-containing vacuole (CCV). Knowledge of critical characteristics of the CCV microenvironment (e.g., luminal pH), analysis of the <i>C. burnetii</i> genome sequence, and strategic metabolic profiling have provided the basis for determining the physicochemical and nutritional conditions necessary to support axenic replication of <i>C. burnetii</i>. In this unit, the media currently utilized for axenic culture of <i>C. burnetii</i> are described, with emphasis on application. To aid in experimental reproducibility and interpretation of results, considerations and limitations are discussed. Lastly, expected results for <i>C. burnetii</i> axenic growth under control conditions are provided as a reference. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"50 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-05-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.52","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36242689","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 25
Salmonella Typhimurium Infection of Human Monocyte-Derived Macrophages 鼠伤寒沙门菌感染人单核细胞源性巨噬细胞
Current Protocols in Microbiology Pub Date : 2018-05-18 DOI: 10.1002/cpmc.56
Stephanie K. Lathrop, Kendal G. Cooper, Kelsey A. Binder, Tregei Starr, Veena Mampilli, Corrella S. Detweiler, Olivia Steele-Mortimer
{"title":"Salmonella Typhimurium Infection of Human Monocyte-Derived Macrophages","authors":"Stephanie K. Lathrop,&nbsp;Kendal G. Cooper,&nbsp;Kelsey A. Binder,&nbsp;Tregei Starr,&nbsp;Veena Mampilli,&nbsp;Corrella S. Detweiler,&nbsp;Olivia Steele-Mortimer","doi":"10.1002/cpmc.56","DOIUrl":"10.1002/cpmc.56","url":null,"abstract":"<p>The successful infection of macrophages by non-typhoidal serovars of <i>Salmonella enterica</i> is likely essential to the establishment of the systemic disease they sometimes cause in susceptible human populations. However, the interactions between <i>Salmonella</i> and human macrophages are not widely studied, with mouse macrophages being a much more common model system. Fundamental differences between mouse and human macrophages make this less than ideal. Additionally, the inability of human macrophage-like cell lines to replicate some properties of primary macrophages makes the use of primary cells desirable. Here we present protocols to study the infection of human monocyte-derived macrophages with <i>Salmonella</i> Typhimurium. These include a method for differentiating monocyte-derived macrophages <i>in vitro</i> and protocols for infecting them with <i>Salmonella</i> Typhimurium, as well as assays to measure the extent of infection, replication, and death. These protocols are useful for the investigation of both bacterial and host factors that determine the outcome of infection. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"50 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-05-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.56","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36244228","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 12
Human Intestinal Enteroids for the Study of Bacterial Adherence, Invasion, and Translocation 人类肠道细菌粘附、侵袭和易位的研究
Current Protocols in Microbiology Pub Date : 2018-05-17 DOI: 10.1002/cpmc.55
Nina M. Poole, Anubama Rajan, Anthony W. Maresso
{"title":"Human Intestinal Enteroids for the Study of Bacterial Adherence, Invasion, and Translocation","authors":"Nina M. Poole,&nbsp;Anubama Rajan,&nbsp;Anthony W. Maresso","doi":"10.1002/cpmc.55","DOIUrl":"10.1002/cpmc.55","url":null,"abstract":"<p>Adherence, invasion, and translocation to and through the intestinal epithelium are important drivers of disease for many enteric bacteria. However, most work has been limited to transformed intestinal cell lines or murine models that often do not faithfully recapitulate key elements associated with human disease. The recent technological advances in organotypic tissue and cell culture are providing unparalleled access to systems with human physiology and complexity. Human intestinal enteroids (HIEs), derived from patient biopsy or surgical specimens of intestinal tissues, are organotypic cultures now being adapted to the study of enteric infections. HIEs are comprised of the dominant cell types of the human gastrointestinal epithelium, can be grown in two- or three-dimensional structures, form a crypt–villus axis with defined apical and basolateral compartments, and undergo physiologic responses to many different stimuli. Here, we describe a series of protocols that encompass the use of human enteroids for the measurement of the adherence, invasion, and translocation of <i>E. coli</i> to and through the intestinal epithelium. We also outline the steps needed to grow and prepare enteroids for this purpose and highlight some common problems to troubleshoot. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"50 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.55","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36244231","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 15
In Situ Detection of Adenovirus DNA and mRNA in Individual Cells 单个细胞中腺病毒DNA和mRNA的原位检测
Current Protocols in Microbiology Pub Date : 2018-04-27 DOI: 10.1002/cpmc.54
Tanel Punga, Sibel Ciftci, Mats Nilsson, Tomasz Krzywkowski
{"title":"In Situ Detection of Adenovirus DNA and mRNA in Individual Cells","authors":"Tanel Punga,&nbsp;Sibel Ciftci,&nbsp;Mats Nilsson,&nbsp;Tomasz Krzywkowski","doi":"10.1002/cpmc.54","DOIUrl":"10.1002/cpmc.54","url":null,"abstract":"<p>Infection by DNA viruses such as human adenoviruses (HAdVs) causes a high-level accumulation of viral DNA and mRNA in the cell population. However, the average viral DNA and mRNA content in a heterogeneous cell population does not inevitably reflect the abundance in individual cells. As the vast majority of virus infection studies is carried out using standard experimental procedures with heterogeneous cell populations, there is a need for a method allowing simultaneous detection and quantitative analysis of viral genome accumulation and gene expression in individual infected cells within a population. This article describes a padlock probe–based rolling-circle amplification protocol that allows simultaneous detection of HAdV type 5 (HAdV-5) DNA and various virus-encoded mRNAs, as well as quantitative analysis of HAdV-5 DNA copies and mRNA species, in individual cells within a heterogeneous population. This versatile method can be used to detect the extent of pathogenic DNA virus infection in different cell types over prolonged infection times. Furthermore, simultaneous viral DNA and mRNA quantification in individual cells allows identification of cells in which persistent infections may be established. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"49 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.54","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36340321","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 2
Mucor circinelloides: Growth, Maintenance, and Genetic Manipulation 环形毛霉:生长、维持和基因操作
Current Protocols in Microbiology Pub Date : 2018-04-27 DOI: 10.1002/cpmc.53
Sandeep Vellanki, Maria Isabel Navarro-Mendoza, Alexis Garcia, Laura Murcia, Carlos Perez-Arques, Victoriano Garre, Francisco E. Nicolas, Soo Chan Lee
{"title":"Mucor circinelloides: Growth, Maintenance, and Genetic Manipulation","authors":"Sandeep Vellanki,&nbsp;Maria Isabel Navarro-Mendoza,&nbsp;Alexis Garcia,&nbsp;Laura Murcia,&nbsp;Carlos Perez-Arques,&nbsp;Victoriano Garre,&nbsp;Francisco E. Nicolas,&nbsp;Soo Chan Lee","doi":"10.1002/cpmc.53","DOIUrl":"10.1002/cpmc.53","url":null,"abstract":"<p><i>Mucor circinelloides</i> is a fungus that belongs to the order Mucorales. It grows as mold in the environment and can cause mucormycosis, a potentially fatal infection in immunocompromised patients. <i>M. circinelloides</i> is a biodiesel producer and serves as a model organism for studying several biological processes, such as light responses and RNA interference–mediated gene silencing. Over the past decade, the increasing number of molecular tools has also allowed us to manipulate the genome of this fungus. This article outlines the fundamental protocols for the <i>in vitro</i> growth, maintenance, and genetic manipulation of <i>M. circinelloides</i> in the laboratory. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"49 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-04-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.53","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"36338202","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 38
Molecular Genetic Manipulation of Sarcocystis neurona 神经元肌囊虫的分子遗传操作
Current Protocols in Microbiology Pub Date : 2018-02-22 DOI: 10.1002/cpmc.48
Daniel K. Howe, Michelle Yeargan, Landon Simpson, Sriveny Dangoudoubiyam
{"title":"Molecular Genetic Manipulation of Sarcocystis neurona","authors":"Daniel K. Howe,&nbsp;Michelle Yeargan,&nbsp;Landon Simpson,&nbsp;Sriveny Dangoudoubiyam","doi":"10.1002/cpmc.48","DOIUrl":"10.1002/cpmc.48","url":null,"abstract":"<p><i>Sarcocystis neurona</i> is a member of the important phylum Apicomplexa and the primary cause of equine protozoal myeloencephalitis (EPM). Moreover, <i>S. neurona</i> is the best-studied species in the genus <i>Sarcocystis</i>, one of the most successful parasite taxa, as virtually all vertebrate animals may be infected by at least one species. Consequently, scientific investigation of <i>S. neurona</i> will aid in the control of EPM and neurologic disease in sea mammals, while also improving our understanding of a prominent branch on the apicomplexan phylogenetic tree. These protocols describe methods that expand the capabilities to study this prominent member of the Apicomplexa. © 2018 by John Wiley &amp; Sons, Inc.</p>","PeriodicalId":39967,"journal":{"name":"Current Protocols in Microbiology","volume":"48 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2018-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpmc.48","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"35889247","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 3
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