http://www.springerprotocols.com/cdp/access/showWebFeedListing This feed provides the latest 25 protocols in the given category. http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_1 Lipopolysaccharide (LPS) is the signature glycolipid isolated from almost all Gram-negative bacteria. LPSs are well known for their ability to elicit the release of cytokines from eukaryotic cells including macrophages, neutrophils, and epithelial cells. LPS can be isolated free of contaminating nucleic acids and proteins by various techniques. In this review, we outline approaches for the isolation and preparation of LPSs for structural studies as well as preparation of very highly purified material for biological studies. Methods are also provided for the analysis of the purity and the structural composition of the LPSs. Finally, three methods for the isolation of lipid A are described. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_10 We developed PCR assays to detect and quantitate Yersinia pestis, the bacterial agent of plague, in flea vector and mammalian host tissues. Bacterial numbers in fleas, fleabite sites, and infected lymph nodes were determined using real-time PCR with primers and probes for a gene target on a multi-copy plasmid specific to Y. pestis. Tissue-matched standard curves used to determine absolute bacterial numbers in unknown samples were linear over at least five orders of magnitude. The methods were applied to studies of transmission of Y. pestis by the rat flea Xenopsylla cheopis, but should be generally useful to investigate the transmission dynamics of any arthropod-borne disease. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_11 Intracellular bacterial pathogens have evolved sophisticated strategies to survive and proliferate within cells of their hosts. Studying their intracellular life cycle is key to understanding virulence and requires methodologies that can identify the compartments in which they localize and characterize the replicative niche they generate. Here, we describe immunofluorescence-based microscopy techniques applied to the intracellular pathogens Brucella abortus and Francisella tularensis during their respective intracellular cycles inside murine bone marrow-derived macrophages. Standard immunofluorescence techniques are used to define the intracellular localization of the pathogens based on their co-localization with specifically labeled macrophage organelles. In addition, we describe an assay to assess the integrity of Francisella-containing phagosomes and bacterial release into the macrophage cytoplasm, which is a hallmark of Francisella intracellular pathogenesis. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_12 Helicobacter pylori is a Gram-negative bacterium that colonizes the gastric epithelium and plays a causative role in the development of peptic ulcers and gastric cancer. Phagocytosis is an element of innate defense used by macrophages and neutrophils to engulf microorganisms. We and others have shown that strains of H. pylori that contain the cag pathogenicity island actively retard their entry into phagocytes. Consequently, there is a lag of several minutes between bacterial binding and the onset of engulfment, and relative to other particles and microbes, the rate of internalization is slow. Herein, we describe in detail the use of synchronized phagocytosis and indirect immunofluorescence microscopy to quantify the rate and extent of H. pylori phagocytosis. This method is appropriate for primary phagocytes as well as transformed cell lines. More importantly, the effects of opsonins, virulence factors, and other agents on infection can be measured independent of bacterial viability or intracellular locale. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_13 Anaplasma phagocytophilum is the etiologic agent of granulocytic anaplasmosis, a tick-borne, zoonotic, emerging infectious disease. A. phagocytophilum is an obligate intracellular pathogen that primarily resides within membrane-bound, cytoplasmic vacuoles of host neutrophils. Closely related to Ehrlichial and Rickettsial organisms, A. phagocytophilum is a small, fragile, Gram-negative bacterium that presents unique challenges for culture, isolation, enumeration, and labeling. This chapter delineates pathogen-specific considerations for culture and labeling of this organism for subsequent use in assays to examine mechanisms of host cell–pathogen interactions. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_14 Electron microscopy of bacterial pathogens and interactions between bacteria and host cells and tissues provides valuable insights into structural and molecular properties and processes involved in pathogenesis. Applications for electron microscopy in bacterial pathogenesis range from discovering etiologic agents and following chronological events during infections by conventional examination of clinical samples to assessing molecular host–cell responses to infection and in situ interactions between receptors and ligands using specific immune-labeling techniques. This chapter focuses on techniques for preparing samples of bacteria and host cells for conventional transmission (TEM) and scanning electron microscopy (SEM) and use of luminescent nanocrystals or “quantum dots” as specific probes for correlative light and electron microscopy. Conventional TEM and SEM are well established tools for high resolution examination of structural effects and chronological events associated with bacterial infections. The recent development of quantum dots as physiological and immunological probes in biology has provided a powerful technique for bridging fluorescent analyses of fixed and live material with preparation and examination by TEM and SEM. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_15 Coxiella burnetii, the agent of Q fever, is an obligate intracellular bacterium that has a tropism for cells of the mononuclear phagocyte system. Following internalization, C. burnetii remains in a phagosome that ultimately matures into a vacuole with lysosomal characteristics that supports pathogen replication. Most in vitro investigations of Coxiella –macrophage interactions have employed continuous cell lines. Although these studies have been informative, genetic alterations of immortalized cells may result in attenuated biological responses to infection relative to primary cells. Consequently, primary macrophages are preferred as in vitro model systems. Here, we describe procedures for propagation and isolation of C. burnetii from cell culture and the use of these preparations to infect primary macrophages derived from human peripheral blood monocytes. Both virulent phase I and avirulent phase II C. burnetii productively infect human monocyte-derived macrophages (MDMs) and replicate with approximately the same kinetics, thereby providing a more physiologically relevant in vitro model system to study the infectious process of this pathogen. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_16 Salmonella enterica serovars cause a variety of diseases ranging from self-limiting gastroenteritis to severe systemic infections. Virulence of these facultative intracellular pathogens is dependent on their ability to invade and replicate within non-phagocytic cells, and cultured epithelial cell systems have been used extensively to dissect the molecular mechanisms involved. For efficient invasion in vitro, the bacterial cell growth conditions as critical since the invasion associated type III secretion system (T3SS1) must be expressed and functional. The ability of Salmonella to invade, and replicate within, epithelial cells can be easily assessed using a gentamicin protection assay or immunofluorescence microscopy. Here, the protocols used in our laboratory are described in detail. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_17 The response of Borrelia burgdorferi to the challenge of reactive oxygen species (ROS) is a direct result of its limited biosynthetic capabilities and lack of biologically significant levels of intracellular Fe. In other bacteria, the major target for oxidative damage is DNA as a consequence of the reaction of “free” intracellular with ROS through the Fenton reaction. Therefore, cellular defenses in these bacteria are focused on protecting this essential cellular component. This does not seem to be the case for B. burgdorferi. In this chapter, we describe methods that were used to analyze the potential targets for ROS in B. burgdorferi. Surprisingly, membrane lipids (e.g., linoleic and linolenic acids) derived from host are the major target of ROS in the Lyme disease spirochete. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_18 Whole body biophotonic imaging (BPI) is a technique that has contributed significantly to the way researchers study bacterial pathogens and develop pre-clinical treatments to combat their ensuing infections in vivo. Not only does this approach allow disease profiles and drug efficacy studies to be conducted non-destructively in live animals over the entire course of the disease, but in many cases, it enables investigators to observe disease profiles that could otherwise easily be missed using conventional methodologies. The principles of this technique are that bacterial pathogens engineered to express bioluminescence (visible light) can be readily monitored from outside of the living animal using specialized low-light imaging equipment, enabling their movement, expansion and treatment to be seen completely non-invasively. Moreover, because the same group of animals can be imaged at each time-point throughout the study, the overall number of animals used is dramatically reduced, saving lives, time, and money. Also, as each animal acts as its own control over time, the issues associated with animal-to-animal variation are circumvented, thus improving the quality of the biostatistical data generated. The ability to monitor infections in vivo in a longitudinal fashion is especially appealing to assess chronic infections such as those involving implanted devices. Typically, bacteria grow as biofilms on these foreign bodies and are reputably difficult to monitor with conventional methods. Because of the non-destructive and non-invasive nature of BPI, the procedure can be performed repeatedly in the same animal, allowing the biofilm to be studied in situ without detachment or disturbance. This ability not only allows unique patterns of disease relapse to be seen following termination of antibiotic therapy but also in vivo resistance development during prolonged treatment, both of which are common occurrences with device-related infections. This chapter describes the bioluminescent engineering of both Gram-positive and Gram-negative bacteria and overviews their use in device-associated infections in several anatomical sites in a variety of animal models. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_19 Staphylococcus aureus nasal colonization is a well-known risk factor for development of S. aureus infections in humans, but despite this established association, we are only beginning to understand the factors, both host and pathogen, that play a role in the colonization of the nares by S. aureus. The cotton rat is a model for many human respiratory pathogens and has proved its utility as a robust model for S. aureus nasal colonization. In this animal model, S. aureus is instilled in the nostrils of adult cotton rats, the bacteria rapidly colonize, and 7 days later S. aureus nasal colonization is enumerated by surgical removal of the nose and recovery of the colonizing S. aureus. This model is an excellent animal model to allow for the evaluation of the efficacy of various therapies, including semi-solid formulations, for determination of their ability to eradicate S. aureus nasal colonization. Further, the cotton rat model allows for assessment of the ability of defined genetic mutants of S. aureus to colonize mucosal surfaces. Finally, this model has demonstrated its utility for the assessment of various antigens as vaccine candidates to protect against S. aureus nasal colonization. This chapter will discuss in detail the method to establish nasal colonization, treatment and eradication of colonization, and recovery of the colonizing bacteria from the nose. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_2 Streptococcus pyogenes secretes various proteins to the extracellular environment. During infection, these proteins interact with human macromolecules and contribute to pathogenesis. We describe a proteomic approach routinely used in our laboratory to characterize culture supernatant proteins using small-format two-dimensional gel electrophoresis. Proteins are collected after overnight growth of the bacteria in broth media. Compounds that inhibit isoelectric focusing, such as salts, are removed by enzymatic treatment and precipitation with trichloroacetic acid and acetone. Following resuspension in denaturing solution, the proteins are separated by isoelectric focusing using a 7-cm immobilized strip with a pH gradient of 4–7. Subsequently, proteins are further separated with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and stained with SYPRO Ruby. The small-gel format requires less time, reagents, and smaller culture volumes compared with large-format approaches, while still resolving and detecting a large proportion of the exoprotein fraction. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_20 Humana Press, Totowa, NJ This chapter describes methods for using non-human primates as a model of group A streptococcal (GAS) pharyngitis. This model has been used successfully to study host–pathogen interactions occurring during pharyngeal GAS infections. The protocol as described will compare two different GAS strains for their ability to cause clinical symptoms of pharyngitis. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_21 The continued increase in antibiotic resistance among bacterial pathogens, coupled with a decrease in infectious disease research among pharmaceutical companies, has escalated the need for novel and effective antibacterial chemotherapies. While current agents have emerged almost exclusively from whole-cell screening of natural products and small molecules that cause microbial death, recent advances in target identification and assay development have resulted in a flood of target-driven drug discovery methods. Whether genome-based methodologies will yield new classes of agents that conventional methods have been unable to is yet to be seen. At the end of the day, perhaps a synergy between old and new approaches will harvest the next generation of antibacterial treatments. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_22 The analyses of numerous prokaryotic and eukaryotic genomes have revealed the presence of variable number tandem repeats (VNTRs). VNTR analysis is currently widely used to sub-speciate many bacterial, fungal, and viral pathogens and has facilitated a number of molecular epidemiology studies. In this chapter, we focus on spa typing which is based on sequence analysis of VNTRs in the polymorphic X region of the Staphylococcus aureus protein A gene Staphylococcus aureus. As the specific methods for spa typing, detailed in this chapter, are well-established and routine procedures (e.g., DNA isolation, PCR and DNA sequencing) for most molecular biology laboratories, we highlight the analytic methods used to interpret the genotyping data generated by sequence analysis and their potential applications in local and global epidemiologic investigations. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_3 The analysis of the expression of virulence genes and the elucidation of metabolic and regulatory pathways of Staphylococcus aureus provide us with important information about the interaction between the pathogen and its host, mechanisms by which this organism causes diseases, and the resistance to antibiotics. In order to investigate regulatory networks of S. aureus, we analyze the cytoplasmic and extracellular proteome by using two-dimensional (2D) gel analyses combined with matrix-assisted laser ionization–time-of-flight mass spectrometry (MALDI–TOF MS). Gel-based proteomics is an extremely valuable tool in microbial physiology that can, in combination with various visualization and quantitation software packages, very rapidly provide comparative and quantitative data for multi-sample comparison. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_4 Comparative genomic hybridization analyses have contributed greatly to our understanding of bacterial evolution, population genetics, and pathogenesis. Here, we describe a robust protocol for microarray-based comparison of genome content, which could be applied to any bacterial species of interest. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_5 Bacteria communicate with other members of their community through the secretion and perception of small chemical cues or signals. The recognition of a signal normally leads to the expression of a large suite of genes, which in some bacteria are involved in the regulation of virulence factors, and as a result, these signaling compounds are key regulatory factors in many disease processes. Thus, it is of interest when studying pathogens to understand the mechanisms used to control the expression of virulence genes so that strategies might be devised for the control of those pathogens. Clearly, the ability to interfere with this process of signaling represents a novel approach for the treatment of bacterial infections. There is a broad range of compounds that bacteria can use for signaling purposes, including fatty acids, peptides, N-acylated homoserine lactones, and the signals collectively called autoinducer 2 (AI-2). This chapter will focus on the latter two signaling systems as they are present in a range of medically relevant bacteria, and here we describe assays for determining whether an organism produces a particular signal and assays that can be used to identify inhibitors of the signaling cascade. Lastly, the signal detection and inhibition assays will be directly linked to the expression of virulence factors of specific pathogens. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_6 The lack of a system for genetic manipulation has hindered studies on the molecular pathogenesis of relapsing fever Borrelia. The focus of this chapter is to describe selectable markers, manipulation strategies, and methods to electro-transform and clone wild-type infectious Borrelia hermsii. Preliminary studies suggest that the variable tick protein (Vtp) of B. hermsii is involved in tick-to-mammal transmission. To address this hypothesis, we have developed a system for genetic manipulation and have constructed clones of a Vtp mutant and an isogenic reconstituted strain. The methods described here are applicable for the inactivation of other loci in B. hermsii and should be adaptable for other species of relapsing fever spirochetes. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_7 Borrelia burgdorferi, the causative agent of Lyme disease, is an obligate parasite that cycles between vertebrate hosts and tick vectors. Attempts to understand the genetic factors that allow B. burgdorferi to sense, adapt to, and survive in different environments have been limited by a relatively low transformation rate. Here, we describe a mariner-based transposon system that achieves saturating levels of random mutagenesis in B. burgdorferi. In comparison with allelic exchange, which targets a single locus, transposon mutagenesis can create libraries of mutants encompassing disruptions of all genes. Suitably designed screens or selections of such a library permit the recovery of mutants exhibiting a desired phenotype. The system described here allows rapid identification of the genetic locus responsible for the mutant phenotype. With appropriate modifications, this mariner-based transposon can be adapted to other spirochetes and bacteria with inefficient genetic transformation methods. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_8 Exopolysaccharides play a crucial role in the formation of biofilms and biofilm resistance to antimicrobials and innate host defense. Here we describe methods to analyze and quantify polysaccharide intercellular adhesin (PIA), a biofilm exopolysaccharide made of N-acetylglucosamine that is found in staphylococci and many other bacterial biofilm-forming pathogens. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-60327-032-8_9 Staphylococcus aureus is a leading cause of human infections worldwide and causes a variety of diseases ranging in severity from mild to life-threatening. The ability of S. aureus to cause disease is based in part on its ability to subvert the innate immune system. Advances in genome-wide analysis of host–pathogen interactions have provided the necessary tools to investigate molecular factors that directly contribute to S. aureus pathogenesis. This chapter describes methods to analyze gene expression in S. aureus during interaction with human neutrophils. 2008-02-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-457-5_1 The insect cell culture/baculovirus system has three primary applications: (1) recombinant protein synthesis, (2) biopesticide synthesis, and (3) as a model system (e.g., for studying apoptosis). The fundamental techniques involved in these applications are described throughout this book. In this chapter, the most widely techniques are summarized and the reader is directed to detailed information found elsewhere in this book. Furthermore, many useful tips and the author’s personal preferences that are rarely published are discussed in this chapter along with quantitative methods to characterize cell growth, baculovirus infection, and metabolism. 2007-07-06T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-457-5_10 Improved methods of baculovirus cloning and insect cell culture and their commercialization have made the use of the baculovirus expression vector system (BEVS) a routine tool for the production of preparative quantities of recombinant protein. This chapter outlines basic techniques for small-scale protein production using the BEVS, including protocols for expression from adherent and suspension insect cell cultures, titer estimation, and expression optimization. 2007-07-06T04:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-457-5_11 The production of recombinant proteins using the baculovirus expression vector system in large-scale agitated bioreactors is discussed in this chapter. Detailed methods of the key stages of a batch process, including host cell growth, virus stock amplification and quantification, bioreactor preparation and operation, the infection process, final harvesting, and primary separation steps for recovery of the product are presented. Furthermore, methods involved with online monitoring and bioreactor control, which have a significant impact on the overall success of the process, are provided, including advanced online monitoring of physiological parameters such as biovolume and respiration activity for batch and fed-batch insect cell cultures along with their role in operating high cell density cultures. 2007-07-06T04:00:00Z