Current protocols in microbiology

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ISSN 1934-8533

Publications in this journal

  • Current protocols in microbiology 05/2015; 37. DOI:10.1002/9780471729259.mc15c06s37
  • Current protocols in microbiology 05/2015; 6F. 2.1-6F. 2.14. DOI:10.1002/9780471729259.mc06f02s37
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    ABSTRACT: Vaccination has a proven record as one of the most effective medical approaches to prevent the spread of infectious diseases. Traditional vaccine approaches involve the administration of whole killed or weakened microorganisms to stimulate protective immune responses. Such approaches deliver many microbial components, some of which contribute to protective immunity, and assist in guiding the type of immune response that is elicited. Despite their impeccable record, these approaches have failed to yield vaccines for many important infectious organisms. This has prompted a move towards more defined vaccines ('subunit vaccines'), where individual protective components are administered. This unit provides an overview of the components that are used for the development of modern vaccines including: an introduction to different vaccine types (whole organism, protein/peptide, polysaccharide, conjugate, and DNA vaccines); techniques for identifying subunit antigens; vaccine delivery systems; and immunostimulatory agents ('adjuvants'), which are fundamental for the development of effective subunit vaccines. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.
    Current Protocols in Microbiology, Edited by Richard Coico, 02/2015: chapter UNIT 18.1 Progress in vaccine development.: pages 18.1.1-18.1.26; John Wiley & Sons., ISBN: 9780471729259
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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 11/2014; 35:6A.4.1-6A.4.12. DOI:10.1002/9780471729259.mc06a04s35
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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. DOI:10.1002/9780471729259.mc14e06s35
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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. DOI:10.1002/9780471729259.mc14e05s35
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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 08/2014; 34:1F.3.1-1F.3.16. DOI:10.1002/9780471729259.mc01f03s34
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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 08/2014; 34:4A.3.1-4A.3.17. DOI:10.1002/9780471729259.mc04a03s34
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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. DOI:10.1002/9780471729259.mc09b05s33
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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. DOI:10.1002/9780471729259.mc1804s33
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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. DOI:10.1002/9780471729259.mc14b03s34
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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. DOI:10.1002/9780471729259.mc14b02s33
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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. DOI:10.1002/9780471729259.mc09c03s32