Curr Protoc Microbiol [JOURNAL]
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King KM, Van Doorslaer K
Curr Protoc Microbiol
· 2018 Nov · PMID 30265446
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Phylogenetic analyses allow for inferring a hypothesis about the evolutionary history of a set of homologous molecular sequences. This hypothesis can be used as the basis for further molecular and computational studies....
Phylogenetic analyses allow for inferring a hypothesis about the evolutionary history of a set of homologous molecular sequences. This hypothesis can be used as the basis for further molecular and computational studies. In this unit, we offer one specific method to construct a Maximum Likelihood phylogenetic tree. We outline how to identify homologous sequences and construct a multiple sequence alignment. Following alignment, sequences are screened for potentially confounding factors such as recombination and genetic saturation. Finally, a Maximum Likelihood phylogenetic tree can be constructed implementing a rigorously tested model of evolution. The workflow outlined in this unit provides sufficient background for inferring a robust phylogenetic tree starting from a particular gene of interest. © 2018 by John Wiley & Sons, Inc.
Anderson EM, Maldarelli F
Curr Protoc Microbiol
· 2018 Nov · PMID 30253074
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HIV persists, despite effective antiretroviral therapy, in long-lived cells, posing a major barrier toward a cure. A key step in the HIV replication cycle and a hallmark of the Retroviridae family is the integration of t...
HIV persists, despite effective antiretroviral therapy, in long-lived cells, posing a major barrier toward a cure. A key step in the HIV replication cycle and a hallmark of the Retroviridae family is the integration of the viral DNA into the host genome. Once integrated, HIV expression is regulated by host machinery and the provirus persists until the cell dies. A reservoir of cells harboring replication-competent proviruses can survive for years, and mechanisms that maintain that reservoir are under investigation. The majority of integrated proviruses, however, are defective or have large deletions, and the composition of the proviral landscape during therapy remains unknown. Methods to quantify HIV proviruses are useful in investigating HIV persistence. Presented in this unit is a method for total HIV DNA quantification of various HIV genome targets that utilizes the next-generation PCR platform, digital PCR. The abundance of various HIV gene targets reflects the overall proviral composition. In this protocol, total genomic DNA is isolated from patient-derived cells and then used as a template for droplet digital PCR, in which the PCR reaction is partitioned into approximately 20,000 individual droplets, PCR amplified to an end point, and subjected to absolute quantification by counting the number of positive and negative droplets. Copy number is directly calculated using straightforward Poisson correction. Additionally, this methodological approach can be used to obtain absolute quantification of other DNA targets. © 2018 by John Wiley & Sons, Inc.
Redmond CJ, Fu H, Aladjem MI
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, McBride AA
Curr Protoc Microbiol
· 2018 Nov · PMID 30129235
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Human papillomaviruses (HPVs) are frequently integrated in HPV-associated cancers. HPV genomes can be integrated in three patterns: A single integrated HPV genome (type I), multiple, tandemly integrated HPV genomes (type...
Human papillomaviruses (HPVs) are frequently integrated in HPV-associated cancers. HPV genomes can be integrated in three patterns: A single integrated HPV genome (type I), multiple, tandemly integrated HPV genomes (type II), and multiple, tandemly integrated HPV genomes interspersed with host DNA (type III). Analysis of the organization of type II and type III integration sites is complicated by their repetitive nature, as sequences of individual repeats are difficult to distinguish from each other. This article presents a method for directly visualizing HPV integration sites using molecular combing combined with fluorescent in situ hybridization, also known as fiber-FISH. In this technique, genomic DNA is stretched across a glass coverslip and individual integrated HPV sequences are detected and directly visualized by in situ hybridization with a resolution of ∼1 kb. Fiber-FISH allows comprehensive characterization of the genomic organization of HPV integration sites containing type II and type III integration. © 2018 by John Wiley & Sons, Inc.
Vellanki S, Navarro-Mendoza MI, Garcia A
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, Murcia L, Perez-Arques C, Garre V, Nicolas FE, Lee SC
Curr Protoc Microbiol
· 2018 May · PMID 30040216
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Mucor circinelloides 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. M. circinelloides is a bi...
Mucor circinelloides 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. M. circinelloides 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 in vitro growth, maintenance, and genetic manipulation of M. circinelloides in the laboratory. © 2018 by John Wiley & Sons, Inc.
Punga T, Ciftci S, Nilsson M
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, Krzywkowski T
Curr Protoc Microbiol
· 2018 May · PMID 30040197
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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 populatio...
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 & Sons, Inc.
Todd RT, Braverman AL, Selmecki A
Curr Protoc Microbiol
· 2018 Aug · PMID 30028911
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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 und...
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 & Sons, Inc.
Jung KW, Lee KT, So YS
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, Bahn YS
Curr Protoc Microbiol
· 2018 Aug · PMID 30016567
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Cryptococcus neoformans 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 ann...
Cryptococcus neoformans 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, C. neoformans, 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 C. neoformans. 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 C. neoformans. © 2018 by John Wiley & Sons, Inc.
Gulati M, Lohse MB, Ennis CL
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, Gonzalez RE, Perry AM, Bapat P, Arevalo AV, Rodriguez DL, Nobile CJ
Curr Protoc Microbiol
· 2018 Aug · PMID 29995344
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Candida albicans 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 immunocompromi...
Candida albicans 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 C. albicans 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 in vitro 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 C. albicans forms with interacting microbial partners. © 2018 by John Wiley & Sons, Inc.
Koestler BJ, Ward CM, Payne SM
Curr Protoc Microbiol
· 2018 Aug · PMID 29927109
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Shigella 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-...
Shigella 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 Shigella are increasing, emphasizing the need for a deeper understanding of Shigella 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 Shigella pathogenesis. Here, protocols to enumerate Shigella 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 Shigella invasion in primary intestinal enteroids is described. These protocols can be used to examine different aspects of Shigella virulence. © 2018 by John Wiley & Sons, Inc.
Sanchez SE, Vallejo-Esquerra E, Omsland A
Curr Protoc Microbiol
· 2018 Aug · PMID 29927105
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Coxiella burnetii 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...
Coxiella burnetii 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 Coxiella-containing vacuole (CCV). Knowledge of critical characteristics of the CCV microenvironment (e.g., luminal pH), analysis of the C. burnetii genome sequence, and strategic metabolic profiling have provided the basis for determining the physicochemical and nutritional conditions necessary to support axenic replication of C. burnetii. In this unit, the media currently utilized for axenic culture of C. burnetii are described, with emphasis on application. To aid in experimental reproducibility and interpretation of results, considerations and limitations are discussed. Lastly, expected results for C. burnetii axenic growth under control conditions are provided as a reference. © 2018 by John Wiley & Sons, Inc.
Poole NM, Rajan A, Maresso AW
Curr Protoc Microbiol
· 2018 Aug · PMID 29927096
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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 muri...
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 E. coli 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 & Sons, Inc.
Lathrop SK, Cooper KG, Binder KA
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, Starr T, Mampilli V, Detweiler CS, Steele-Mortimer O
Curr Protoc Microbiol
· 2018 Aug · PMID 29927091
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The successful infection of macrophages by non-typhoidal serovars of Salmonella enterica is likely essential to the establishment of the systemic disease they sometimes cause in susceptible human populations. However, th...
The successful infection of macrophages by non-typhoidal serovars of Salmonella enterica is likely essential to the establishment of the systemic disease they sometimes cause in susceptible human populations. However, the interactions between Salmonella 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 Salmonella Typhimurium. These include a method for differentiating monocyte-derived macrophages in vitro and protocols for infecting them with Salmonella 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 & Sons, Inc.
Kim SH, Samal SK
Curr Protoc Microbiol
· 2018 Feb · PMID 29512119
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Newcastle disease virus (NDV) is an economically important pathogen in the poultry industry worldwide. Recovery of infectious NDV from cDNA using reverse genetics has made it possible to manipulate the genome of NDV. Thi...
Newcastle disease virus (NDV) is an economically important pathogen in the poultry industry worldwide. Recovery of infectious NDV from cDNA using reverse genetics has made it possible to manipulate the genome of NDV. This has greatly contributed to our understanding of the molecular biology and pathogenesis of NDV. Furthermore, NDV has modular genome and accommodates insertion of a foreign gene as a transcriptional unit, thus enabling NDV as a vaccine vector against diseases of humans and animals. Avirulent NDV strains (e.g., LaSota and B1) have been commonly used as vaccine vectors. In this protocol, we have described reverse genetics of NDV to be used as a vaccine vector by exemplifying the recovery of NDV vectored avian influenza virus vaccine. Specifically, cloning and recovery of NDV expressing the hemagglutinin protein of highly pathogenic influenza virus were explained. © 2018 by John Wiley & Sons, Inc.
O'Neal L, Mukherjee T, Alexandre G
Curr Protoc Microbiol
· 2018 Feb · PMID 29512118
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Bacteria of the genus A. brasilense are motile and capable of chemotaxis and aerotaxis (taxis in gradient of oxygen) using a single polar flagellum that propels the cells in aqueous environments. Responses to attractants...
Bacteria of the genus A. brasilense are motile and capable of chemotaxis and aerotaxis (taxis in gradient of oxygen) using a single polar flagellum that propels the cells in aqueous environments. Responses to attractants and repellents have been described and spatial gradient assays that permit the visualization of these responses are detailed in this unit. These assays are simple and can be readily implemented with minimum set ups. © 2018 by John Wiley & Sons, Inc.
Mahdi OS, Fisher NA
Curr Protoc Microbiol
· 2018 Feb · PMID 29512117
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Paenibacillus larvae is a Gram-positive, spore-forming bacterium and the causative agent of American foulbrood disease (AFB), a highly contagious, fatal disease affecting managed honeybee (Apis mellifera) colonies. As th...
Paenibacillus larvae is a Gram-positive, spore-forming bacterium and the causative agent of American foulbrood disease (AFB), a highly contagious, fatal disease affecting managed honeybee (Apis mellifera) colonies. As the etiological agent of American foulbrood disease, P. larvae is the most economically significant bacterial pathogen infecting honeybees. This unit includes protocols for the in vitro growth and laboratory maintenance of P. larvae. © 2018 by John Wiley & Sons, Inc.
Mahdi OS, Fisher NA
Curr Protoc Microbiol
· 2018 Feb · PMID 29512116
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Endospores are metabolically dormant cells formed by a variety of Gram-positive bacteria within the phylum Firmicutes in response to nutrient limiting or otherwise unfavorable growth conditions. American foulbrood diseas...
Endospores are metabolically dormant cells formed by a variety of Gram-positive bacteria within the phylum Firmicutes in response to nutrient limiting or otherwise unfavorable growth conditions. American foulbrood disease (AFB) is a serious disease of honeybees that is caused by the introduction of Paenibacillus larvae endospores into a honeybee colony. Progression to fulminant disease and eventual collapse of the colony requires multiple rounds of endospore germination, vegetative replication, endospore formation, and subsequent spread within the colony. This unit includes protocols for the in vitro sporulation and germination of P. larvae to assist investigators in the study of these processes. © 2018 by John Wiley & Sons, Inc.
Lin S, Yang L, Zhang YJ
Curr Protoc Microbiol
· 2018 Feb · PMID 29512115
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Hepatitis E virus (HEV) predominantly causes acute liver disease in humans and is transmitted via the fecal-oral route. HEV infection in pregnant women can result in grave consequences, with up to 30% fatality. The HEV s...
Hepatitis E virus (HEV) predominantly causes acute liver disease in humans and is transmitted via the fecal-oral route. HEV infection in pregnant women can result in grave consequences, with up to 30% fatality. The HEV strains infecting humans mainly belong to four genotypes. Genotypes 1 and 2 are restricted to human infection, while genotypes 3 and 4 are zoonotic. HEV genotype 3 (HEV-3) can cause both acute and chronic liver diseases. Several cell lines (mainly hepatocytes) have been developed for HEV propagation and biological study. However, HEV production in these cell lines is suboptimal and inefficient. Here, we present methods for the isolation, propagation, and quantification of HEV. © 2018 by John Wiley & Sons, Inc.
Viktorova EG, Khattar S, Samal S
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, Belov GA
Curr Protoc Microbiol
· 2018 Feb · PMID 29512114
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Poliovirus is a prototype member of the Enterovirus genus of the Picornaviridae family of small positive strand RNA viruses, which include important human and animal pathogens. Quantitative assessment of viral replicatio...
Poliovirus is a prototype member of the Enterovirus genus of the Picornaviridae family of small positive strand RNA viruses, which include important human and animal pathogens. Quantitative assessment of viral replication is very important for investigation of the virus biology and the development of anti-viral strategies. The poliovirus genome structure allows replacement of structural genes with a reporter protein, such as a luciferase or a fluorescent protein, whose signals can be detected and quantified in vivo, thus permitting observation of replication kinetics in live cells. This paper presents protocols for poliovirus replicon RNA production, purification, packaging and transfection, as well as techniques for monitoring Renilla luciferase replication signal in living cells. © 2018 by John Wiley & Sons, Inc.
Ma Z, Yang L, Zhang YJ
Curr Protoc Microbiol
· 2018 Feb · PMID 29512113
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Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the family Arteriviridae, order Nidovirale. PRRSV is an enveloped, single-stranded, positive-sense RNA virus with a genome around 15 kb in length...
Porcine reproductive and respiratory syndrome virus (PRRSV) is a member of the family Arteriviridae, order Nidovirale. PRRSV is an enveloped, single-stranded, positive-sense RNA virus with a genome around 15 kb in length. For propagation of PRRSV in vitro, the MARC-145 cell line is the most often used in a laboratory setting. Infectious cDNA clones of many PRRSV strains have been established, from which these viruses can be recovered. PRRSV titration is generally done in MARC-145 cells. PRRSV RNA copy numbers can be assessed by reverse transcription and real-time PCR. Here, protocols for PRRSV propagation, virus recovery from infectious cDNA clones, and quantification are presented. © 2018 by John Wiley & Sons, Inc.
Howe DK, Yeargan M, Simpson L
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, Dangoudoubiyam S
Curr Protoc Microbiol
· 2018 Feb · PMID 29512112
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Sarcocystis neurona is a member of the important phylum Apicomplexa and the primary cause of equine protozoal myeloencephalitis (EPM). Moreover, S. neurona is the best-studied species in the genus Sarcocystis, one of the...
Sarcocystis neurona is a member of the important phylum Apicomplexa and the primary cause of equine protozoal myeloencephalitis (EPM). Moreover, S. neurona is the best-studied species in the genus Sarcocystis, one of the most successful parasite taxa, as virtually all vertebrate animals may be infected by at least one species. Consequently, scientific investigation of S. neurona 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 & Sons, Inc.