微生物学技术

This chapter describes the techniques used to study nucleolar-localized proteins. The chapter starts with cloning of viral proteins for expression in mammalian cells as fusion proteins to well-characterized tags such as enhanced green fluorescence protein (EGFP). This follows on ...

Co-immunoprecipitation is a common method used to determine protein—protein interactions. This method typically exploits the highly specific interaction between a monoclonal antibody and a protein of interest. Providing that the antibody—protein interaction does not int ...

Adenoviruses (Ads) are small DNA tumor viruses that have played a pivotal role in understanding eukaryotic cell biology and viral oncogenesis. Among other cellular pathways, Ad usurps cell cycle progression following infection. Likewise, progression of the viral infection is infl ...

The encapsidation of adenovirus DNA into virus particles depends on cis-acting sequences located at the left end of the viral genome. Repeated DNA sequences in the packaging domain contribute to viral DNA encapsidation, and several viral proteins bind to these repeats when analyzed using ...

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 ...

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 cu ...

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 t ...

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 bact ...

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, ...

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 Borr ...

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 rel ...

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-acetylglu ...

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 ...

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 whi ...

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 ne ...

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 path ...

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 s ...

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 effic ...

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 describ ...

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. aure ...