Current protocols in microbiology

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  • ISSN
    1934-8533

Publications in this journal

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    ABSTRACT: As with all Herpesviruses, Herpes simplex virus (HSV) has both a lytic replication phase and a latency reactivation cycle. During lytic replication, there is an ordered cascade of viral gene expression that leads to the synthesis of infectious viral progeny. In contrast, latency is characterized by the lack of significant lytic gene expression and the absence of infectious virus. Reactivation from latency is characterized by the re-entry of the virus into the lytic replication cycle and the production of recurrent disease. This unit describes the establishment of the mouse sensory neuron model of HSV-1 latency-reactivation as a useful in vivo system for the analysis of mechanisms involved in latency and reactivation. Assays including the determination of viral yields, immunohistochemical/immunofluorescent detection of viral antigens, and mRNA quantitation are used in experiments designed to investigate the network of cellular and viral proteins regulating HSV-1 lytic infection, latency, and reactivation.
    Current protocols in microbiology 11/2014; 35(14E.6):14E.6.1 - 14E.6.21.
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    ABSTRACT: Herpes Simplex Virus (HSV) is a human pathogen that establishes latency and undergoes periodic reactivation, resulting in chronic recurrent lytic infection. HSV lytic infection is characterized by an organized cascade of three gene classes; however, successful transcription and expression of the first, the immediate early class, is critical to the overall success of viral infection. This initial event of lytic infection is also highly dependent on host cell factors. This unit uses RNA interference and small molecule inhibitors to examine the role of host and viral proteins in HSV lytic infection. Methods detailing isolation of viral and host RNA and genomic DNA followed by quantitative real-time PCR allow characterization of impacts on viral transcription and replication, respectively. Western blots can be used to confirm quantitative PCR results. This combination of protocols represents a starting point for researchers interested in virus-host interactions during HSV lytic infection.
    Current protocols in microbiology 11/2014; 35(14E.5):14E.5.1 - 14E.5.27.
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    ABSTRACT: Listeria monocytogenes is frequently encountered in foods but often at low concentrations and typically in the presence of other microbiota, including nonpathogenic Listeria spp. The potential of L. monocytogenes to cause severe human disease mandates sensitive, accurate, and rapid detection in foods. Isolation of L. monocytogenes from foods is critical, not only for routine surveillance, but also for epidemiologic investigations. Isolation of the pathogen from water (especially surface water used for irrigation) is similarly important, as produce has been implicated in listeriosis outbreaks and contaminated water can be involved in contamination of produce. This unit provides basic protocols for the isolation of L. monocytogenes from foods and water. Curr. Protoc. Microbiol. 33:9B.5.1-9B.5.19. © 2014 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2014; 33:9B.5.1-9B.5.19.
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    ABSTRACT: Acinetobacter baumannii is a Gram-negative nosocomial pathogen of clinical importance. A lack of genetic tools has hindered the research of this organism in the past; however, recently, various methods have been designed, modified, and optimized to facilitate the genetic manipulation of A. baumannii. This unit describes some of the recent genetic advances and new recombinant tools developed for this pathogen, including standard transformation and conjugation techniques specifically developed for the bacteria. As the need to understand the basic biology of A. baumannii increases with the prospect of developing new therapeutics, the use of the basic genetic methods herein can provide the critical first step to identify genes required for infection. © 2014 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2014; 35:6G.2.1-6G.2.11.
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    ABSTRACT: Stenotrophomonas maltophilia is a ubiquitous soil bacterium that is increasingly recognized as an emerging nosocomial pathogen. This unit includes protocols for the in vitro growth and maintenance of S. maltophilia. Curr. Protoc. Microbiol. 32:6F.1.1-6F.1.6. © 2014 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2014; 32:6F.1.1-6.
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    ABSTRACT: Immunological methods for quantitative measurement, antigenic characterization, and monitoring the stability of active immunogenic component(s) are a critical need in the vaccine development process. This unit describes an enhanced chemiluminescence-based western blot for quantitative detection of Plasmodium falciparum circumsporozoite protein (PfCSP), a major malaria candidate vaccine antigen. The most salient features of this assay are its high sensitivity and reproducibility; it can reliably detect ∼5 to 10 pg PfCSP expressed on native parasites or recombinantly expressed in Escherichia coli. Although described for a specific vaccine antigen, this assay should be applicable for any antigen-antibody combination for which relevant detection reagents are available. Detailed stepwise experimental procedures and methods for data acquisition and analysis are described. Curr. Protoc. Microbiol. 33:18.4.1-18.4.11. © 2014 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2014; 33:18.4.1-18.4.11.
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    ABSTRACT: Papillomaviruses have a strict tropism for epithelial cells, and they are fully reliant on cellular differentiation for completion of their life cycles, resulting in the production of progeny virions. Thus, a permissive environment for full viral replication in vitro-wherein virion morphogenesis occurs under cooperative viral and cellular cues-requires the cultivation of epithelium. Presented in the first section of this unit is a protocol to grow differentiating epithelial tissues that mimic many important morphological and biochemical aspects of normal skin. The technique involves growing epidermal cells atop a dermal equivalent consisting of live fibroblasts and a collagen lattice. Epithelial stratification and differentiation ensues when the keratinocyte-dermal equivalent is placed at the air-liquid interface. The apparent floating nature of the cell-matrix in this method led to the nickname "raft" cultures. The general technique can be applied to normal low passage keratinocytes, to cells stably transfected with papillomavirus genes or genomes, or keratinocytes established from neoplastic lesions. However, infectious papillomavirus particles have only been isolated from organotypic epithelial cultures initiated with cells that maintain oncogenic human papillomavirus genomes in an extrachomosomal replicative form. The second section of this unit is dedicated to a virion isolation method that minimizes aerosol and skin exposure to these human carcinogens. Although the focus of the protocols is on the growth of tissues that yields infectious papillomavirus progeny, this culture system facilitates the investigation of these fastidious viruses during their complex replicative cycles, and raft tissues can be manipulated and harvested at any point during the process. Importantly, a single-step virus growth cycle is achieved in this process, as it is unlikely that progeny virions are released to initiate subsequent rounds of infection. Curr. Protoc. Microbiol. 34:14B.3.1-14B.3.18. © 2014 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2014; 34:14B.3.1-14B.3.18.
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    ABSTRACT: Staphylococcus aureus is a facultative anaerobic Gram-positive coccus and a member of the normal skin flora, as well as that of the nasal passages of humans. However, S. aureus can also gain entry into the host and cause life-threatening infections or persist as disease foci that develop into suppurative abscesses. While genetically tractable, the manipulation of S. aureus remains challenging. This unit describes methods developed in our laboratory for gene disruption by allelic replacement and transposition. We also provide a protocol for bacteriophage-mediated transduction of mutants marked with selectable alleles and describe plasmid utilization for complementation studies. Curr. Protoc. Microbiol. 32:9C.3.1-9C.3.19. © 2014 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2014; 32:9C.3.1-9C.3.19.
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    ABSTRACT: Papillomavirus genomes replicate as extrachromosomal plasmids within infected keratinocytes, requiring the regulated expression of early viral gene products to initially amplify the viral genomes and subvert cell growth checkpoints as part of a complex path to immortalization. Building on contemporary keratinocyte transfection and culture systems, the methods described in this unit form a detailed approach to analyzing critical events in the human papillomavirus (HPV) life cycle, utilizing physiologic levels of viral gene products expressed from their native promoter(s) in the natural host cells for HPV infection. A quantitative colony-forming assay permits comparison of the capacities of various transfected HPV types and mutant HPV genomes to initially form colonies and immortalize human keratinocytes. In conjunction with additional methods, these protocols enable examination of genomic stability, viral and cellular gene expression, viral integration, and differentiation patterns influenced by HPV persistence in clonal human keratinocytes that effectively mimic early events in HPV infection. Curr. Protoc. Microbiol. 33:14B.2.1-14B.2.13. © 2014 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2014; 33:14B.2.1-14B.2.13.
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    ABSTRACT: Neisseria gonorrhoeae (GC) is a strict human pathogen and the agent of the sexually transmitted disease gonorrhea. Gonococcal infections have been successfully treated with antibiotics; however, GC has repeatedly developed resistance to each new antibiotic used. Currently, third-generation cephalosporins are recommended, and resistance to these antimicrobials is emerging worldwide. Additionally, no vaccine is available to prevent GC infections. With the dire possibility of untreatable gonorrhea, there is a critical need to identify new therapeutic targets. Cell envelope and membrane vesicle proteins are key factors in pathogenesis, antibiotic resistance, biofilm formation, and general bacterial fitness. Here we describe methods for isolation and purification of GC cell envelopes and spontaneously released membrane vesicles. The isolated proteome fractions can be used in multiple downstream applications, including gel-based and gel-free quantitative proteomics, studies focused on subcellular localization of proteins, transmission electron microscopy, or strain characterization. Presented methods may be easily adapted to other bacterial species. Curr. Protoc. Microbiol. 34:4A.3.1-4A.3.17. © 2014 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2014; 34:4A.3.1-4A.3.17.
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    ABSTRACT: Acinetobacter baumannii has recently drawn great interest in the microbiology research community due to the increase in clinical antibiotic resistance of this organism, and persistence of this bacterial species in the hospital environment. This unit outlines protocols for the growth and maintenance of A. baumannii in the laboratory. © 2014 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2014; 35:6G.1.1-6.
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    ABSTRACT: These protocols apply to all currently known genotypes of avian bornavirus (ABV). First, they include four basic protocols for molecular techniques that should enable an investigator to detect ABV infection in a live or dead bird. These include both reverse transcriptase and real-time PCR assays. Second, they include three protocols enabling ABV infections to be diagnosed by serologic techniques including indirect immunofluorescence assays, western blotting, and enzyme-linked immunoassays. Third, they also include methods by which ABV can be isolated from infected bird tissues by culture in primary duck embryo fibroblasts, as well as in other avian cell lines. Finally, as part of a diagnostic workup, any virus detected should be genotyped by sequencing, and a protocol for this is also provided. Curr. Protoc. Microbiol. 34:15I.1.1-15I.1.33. © 2014 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2014; 34:15I.1.1-15I.1.33.
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    ABSTRACT: Many bacteria can become naturally competent to take up extracellular DNA across their outer and inner membranes by a dedicated competence apparatus. Whereas some studies show that the DNA delivered to the cytoplasm may be used for genome repair or for nutrition, it can also be recombined onto the chromosome by homologous recombination: a process called natural transformation. Along with conjugation and transduction, natural transformation represents a mechanism for horizontal transfer of genetic material, e.g., antibiotic resistance genes, which can confer new beneficial characteristics onto the recipient bacteria. Described here are protocols for quantifying the frequency of transformation for the human pathogen Vibrio cholerae, one of several Vibrio species recently shown to be capable of natural transformation. © 2014 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2014; 35:6A.4.1-6A.4.12.
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    ABSTRACT: Borrelia burgdorferi sensu lato is a group of spirochetes belonging to the genus Borrelia in the family of Spirochaetaceae. The spirochete is transmitted between reservoirs and hosts by ticks of the family Ixodidae. Infection with B. burgdorferi in humans causes Lyme disease or Lyme borreliosis. Currently, 20 Lyme disease-associated Borrelia species and more than 20 relapsing fever-associated Borrelia species have been described. Identification and differentiation of different Borrelia species and strains is largely dependent on analyses of their genetic characteristics. A variety of molecular techniques have been described for Borrelia isolate speciation, molecular epidemiology, and pathogenicity studies. In this unit, we focus on three basic protocols, PCR-RFLP-based typing of the rrs-rrlA and rrfA-rrlB ribosomal spacer, ospC typing, and MLST. These protocols can be employed alone or in combination for characterization of B. burgdorferi isolates or directly on uncultivated organisms in ticks, mammalian host reservoirs, and human clinical specimens. Curr. Protoc. Microbiol. 34:12C.1-12C.31. © 2014 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2014; 34:12C.5.1-12C.5.31.
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    ABSTRACT: Proteins localized to the cell envelope and naturally released membrane vesicles (MVs) play diverse functions in physiology and pathogenesis of Gram-negative bacteria. Study of these proteome fractions is essential for better understanding the basic physiological processes, development of vaccines, and identification of potential drug targets. This unit presents gel-free quantitative proteomic methods for comprehensive proteomic profiling of the cell envelopes and MVs. The procedure starts with the precipitation of the isolated proteome fractions to remove any potential compounds that may interfere with downstream experimental steps. Subsequently, the proteins are reduced, alkylated, and subjected to trypsin digestion. The trypsinized peptides are labeled using isobaric tagging for relative and absolute quantification (iTRAQ), and analyzed samples are pooled and subjected to rigorous prefractionations by strong cation exchange (SCX) and reversed-phase (RP) liquid chromatography (LC). Finally, the tandem mass spectrometry (MS/MS) fragmentation enables peptides identification and quantification. Curr. Protoc. Microbiol. 34:1F.3.1-1F.3.16. © 2014 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2014; 34:1F.3.1-1F.3.16.
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    ABSTRACT: Murine norovirus (MNV) is a positive-sense, plus-stranded RNA virus in the Caliciviridae family. It is the most common pathogen in biomedical research colonies. MNV is also related to the human noroviruses, which cause the majority of nonbacterial gastroenteritis worldwide. Like the human noroviruses, MNV is an enteric virus that replicates in the intestine and is transmitted by the fecal-oral route. MNV replicates in murine macrophages and dendritic cells in cells in culture and in the murine host. This virus is often used to study mechanisms in norovirus biology, because human noroviruses are refractory to growth in cell culture. MNV combines the availability of a cell culture and reverse genetics system with the ability to study infection in the native host. Herein, we describe a panel of techniques that are commonly used to study MNV biology. Curr. Protoc. Microbiol 33:15K.2.1-15K.2.61. © 2014 by John Wiley & Sons, Inc.
    Current protocols in microbiology 01/2014; 33:15K.2.1-15K.2.61.
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    ABSTRACT: Poliovirus (PV) is the prototypical picornavirus. It is a non-enveloped RNA virus with a small (∼7.5-kb) genome of positive polarity. It has long served as a model to study RNA virus biology, pathogenesis, and evolution. cDNA clones of several strains are available, and infectious virus can be produced by the transfection of in vitro transcribed viral genomes into an appropriate host cell. PV infects many human and non-human primate cell lines including HeLa and HeLa S3 cells, and can grow to high titer in culture. Protocols for the production, propagation, quantification, and purification of PV are presented. Curr. Protoc. Microbiol. 29:15H.1.1-15H.1.27. © 2013 by John Wiley & Sons, Inc.
    Current protocols in microbiology 05/2013; Chapter 15H:Unit15H.1.
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    ABSTRACT: Poliovirus (PV) is the prototypical picornavirus. It is a non-enveloped RNA virus with a small (∼7.5-kb) genome of positive polarity. cDNA clones of several strains are available, and infectious virus can be produced by the transfection of in vitro-transcribed viral genomes into an appropriate host cell. The ease of genetic studies in poliovirus is a primary reason that it has long served as a model to study RNA virus biology, pathogenesis, and evolution. Protocols for the generation and characterization of PV mutants are presented. Curr. Protoc. Microbiol. 29:15H.2.1-15H.2.32. © 2013 by John Wiley & Sons, Inc.
    Current protocols in microbiology 05/2013; Chapter 15:Unit15H.2.
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    ABSTRACT: The incorporation of a fluorescent reporter gene into a replication-competent influenza A virus (IAV) has made it possible to trace IAV infection in vivo. This protocol describes the process of inserting a green fluorescent protein (GFP) reporter into the IAV genome using the established reverse genetics system. The strategy begins with the reorganization of segment eight of the IAV genome, during which the open reading frames of nonstructural protein 1 (NS1) and the nuclear export protein (NEP) are separated to allow for GFP fusion to the NS1 protein. The NS1, GFP, and NEP open reading frames (ORF) are then cloned into the IAV rescue system backbone. Upon construction of the GFP-encoding segment eight rescue plasmid, recombinant NS1-GFP influenza virus can be rescued via co-transfection with the remaining seven rescue plasmids. The generated NS1-GFP IAV can subsequently be used to visualize infected cells, both in vitro and in vivo. Curr. Protoc. Microbiol. 29:15G.4.1-15G.4.16. © 2013 by John Wiley & Sons, Inc.
    Current protocols in microbiology 05/2013; Chapter 15:Unit15G.4.