http://www.springerprotocols.com/cdp/access/showWebFeedListing This feed provides the latest 25 protocols in the given category. http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_1 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_10 Although the role of invasion in the virulence of Candida albicans has been demonstrated, the mechanism that governs fungal invasion is not fully understood. Among the tools that exist to fill these gaps in knowledge, in vitro tissue models based on reconstituted human epithelia (RHE) have already been developed. Such models are designed to study more reproducably the fungus-host relationship, as they eliminate the complexity and variability found in vivo. Herein we describe the preparation of these RHE and their application in study of the invasion properties of C. albicans by further histologic processing and microscopic observation. For this purpose, different epithelial cell lines are grown on a collagen gel to build up models of intestinal (Caco-2 cell line), vaginal (A431 cell line), and oral (TR146 cell line) mucosa. The use of these in vitro models applied to test the invasiveness of C. albicans strains (clinical isolates or gene deleted mutants) and to identify changes in gene expression during the invasion of the RHE will help to advance our knowledge of pathogenesis and to study specific mechanisms used by C. albicans to adapt to changing environments present in different epithelia. Furthermore, because these models are useful to study the host response during the challenge with the pathogen, they will also offer important new insights into host cell biology and identify new targets for treatment. 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_11 The incidence of invasive fungal diseases has increased over the past decades, particularly in relation with the increase of immunocompromised patient cohorts (e.g., HIV-infected patients, transplant recipients, immunosuppressed patients with cancer). Opportunistic fungal pathogens such as Candida spp. are most often associated with serious systemic infections. Currently available antifungal drugs are rather unspecific, often with severe side effects. In some cases, their prophylactic use has favored emergence of resistant fungal strains. Major antifungal drugs target the biosynthesis of lipid components of the fungal plasma membrane or the assembly of the cell wall. For a more specific and efficient treatment and prevention of fungal infection, new therapeutic strategies are needed, including strengthening or stimulation of the residual host immune response. Achieving such a goal requires a better understanding of factors important for the defense and the survival of the host combating Candida spp. Where possible, primary cultures of mammalian immune cells of the innate immune system constitute a better suited model than transformed cell lines to study host-pathogen response and virulence. Hence, in vitro primary cell culture systems are a good strategy for a first screening of mutant strains of Candida spp. to identify virulence traits with regard to host cell response and pathogen invasion. 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_12 Invasive fungal infections comprise a group of serious and life-threatening diseases affecting immunocompromised patients. Molecular analysis of fungal virulence involves the deletion of genes that are suspected for contributing to fungal pathogenesis. Phenotypic analysis of the generated mutants includes in vivo infection experiments in order to assign a function during fungal disease to a gene of interest. 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_13 Aspergillus species are infamous for causing several plant and animal diseases that directly (e.g., invasive aspergillosis) or indirectly (e.g., consumption of toxic food supplies) can lead to high rates of morbidity in humans and animals worldwide. Despite progress in molecular information and manipulation of Aspergillus spp., including genome sequence availability and suitable transformation methodologies, efforts to control Aspergillus diseases are still far from satisfactory, due in part to lack of knowledge of fungal virulence attributes. In order to obtain meaningful insights on the disease mechanism(s), it is essential to detect virulence gene expression during host invasion. Here, we describe two PCR-based detection methods of Aspergillus gene expression in both plant and mammalian tissues. Moreover, these techniques can be employed for routine screening of large numbers of aspergilli to improve diagnosis, disease monitoring, and therapy of fungal disease. 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_14 Investigators have long used antibody-based screening strategies to identify Candida albicans immunogenic proteins and the genes that encode them during infections. With the recent availability of the C. albicans genome sequence and the development of genomic and proteomic technologies, it is now possible to efficiently conduct large-scale screening in standard research labs. C. albicans proteins and genes identified with a variety of screening methods have been implicated as important determinants of candidal virulence and exploited as vaccine and therapeutic targets. In this chapter, we describe methods used in our lab, in which sera recovered from patients with candidiasis are used to screen a C. albicans genomic DNA expression library. Immunoreactive colonies are detected by reaction with anti-human immunoglobulin, and the corresponding open reading frames are identified using the genome sequence database. The methods are also suitable for use with cDNA expression libraries, and they are complementary to proteomic screening strategies described elsewhere in this volume. 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_15 Over the past two decades, mass spectrometry (MS) has ceased to be a fairly exotic technique banished from the protein scientists’ mind to become a seminal tool for deciphering the information encoded in the genomes of many biological species. Clues to this shift in the modus operandi for characterizing their proteomes stem from the progressive availability of full genome sequences and well-annotated protein databases of many model (micro)organisms, the development both of soft ionization methods for large biomolecules (peptides and proteins) and of innovative instrumentation designs, and the introduction of sophisticated search algorithms able to correlate MS information with sequence databases, to name but a few. Here we integrate the typical MS-based strategy for identifying proteins of Candida albicans, an opportunistic fungal pathogen of humans, which have proved to be present during systemic infection and targeted by the immune system as a consequence of its interaction with the host (i.e., the C. albicans immunome). 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_16 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_17 Parasitic helminths are a major cause of disease worldwide, yet the molecular mechanisms of host-helminth interaction and parasite development are only rudimentarily studied. A main reasons for this lack of knowledge are the tremendous experimental difficulties in cultivating parasitic helminths under defined laboratory conditions and obtaining sufficient amounts of parasite material for molecular analyses. For one member of this neglected group of pathogens, the fox-tapeworm Echinococcus multilocularis, we have established and optimized in vitro cultivation systems by which the major part of the parasite’s life cycle, leading from early metacestode vesicles to the production of protoscoleces, can be mimicked under laboratory conditions. The methodology comprises co-cultivation systems for host cells and parasite larvae by which large amounts of parasite vesicles can be generated. Furthermore, we have established an axenic (host cell–free) cultivation system that allows studies on the influence of defined host factors on parasite growth and development. On the basis of this system, the isolation and maintenance of primary Echinococcus cells that are devoid of overgrowing host cells is now possible. The availability of the primary cell culture system constitutes a first step toward the establishment of genetic manipulation methods for the parasite that will be of great interest for further research on infection strategies and development of Echinococcus and other cestodes. 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_18 The two most prevalent human malaria parasites, Plasmodium falciparum and Plasmodium vivax, cause the majority of malaria-related morbidity and mortality. Compared with our knowledge about the erythrocytic stages, we understand little about the liver exoerythrocytic (EE) stages of the human malaria parasites. Our recent development of a hepatocyte line from normal human liver tissue is crucial for successful culturing of the liver stages of both P. falciparum and P. vivax. This technical advancement should be an important tool for directly studying developmental biology of the EE stages of the human malaria parasites and developing drugs against parasite liver stages. 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_19 Apoptosis plays crucial roles for the outcome of infection with various infectious agents. The host’s apoptotic program may be modulated after infection in order to combat the pathogen or to restrict the immune response. In addition, distinct microorganisms alter the apoptotic program of the host in order to meet the requirements for their further distribution. The activation of caspases (i.e., cysteine proteases with specificity for aspartic acid residues) preludes the disassembly of the cell in response to apoptosis-inducing stimuli. This depends on the proteolytic cleavage of inactive proforms into catalytically active subunits. Analyses of the proteolysis and the enzymatic activity of caspases therefore represent valuable tools to study apoptotic programs during infection. The apicomplexan parasite Toxoplasma gondii interferes with the caspase cascade of its host cell in order to facilitate intracellular survival. The modulation of caspase activation by T. gondii is determined by SDS-PAGE and immunoblotting with caspase-specific antibodies. Furthermore, the impact of the parasite on caspase activity is fluorimetrically determined by measuring the cleavage of caspase-specific substrate analogues. 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_2 As in many other areas of basic and applied biology, research in infectious diseases has been revolutionized by two recent developments in the field of genome biology: first, the sequencing of the human genome as well as that of many pathogen genomes; and second, the development of high-throughput technologies such as microarray technology, proteomics, and metabolomics. Microarray studies enable a deeper understanding of genetic evolution of pathogens and investigation of determinants of pathogenicity on a whole-genome scale. Host studies in turn permit an unprecedented holistic appreciation of the complexities of the host cell responses at the molecular level. In combination, host-pathogen studies allow global analysis of gene expression in the infecting bacterium as well as in the infected host cell during pathogenesis providing a comprehensive picture of the intricacies of pathogen-host interactions. This chapter briefly explains the principles underlying DNA microarrays including major points to consider when planning and analyzing microarray experiments and highlights in detail their practical application using the interaction of Neisseria meningitidis with endothelial cells as an example. 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_20 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_21 Neutrophils are professional phagocytes of the innate immune system that are essential to control bacterial and fungal infections. These cells engulf and kill invading microbes. Additionally, activated neutrophils are able to release neutrophil extracellular traps (NETs). These fibers consist of chromatin decorated with antimicrobial proteins to trap and kill microbes. Appropriate quantitative methods are required to understand the nature of interactions of neutrophils with pathogens. Here we present assays to measure killing mediated by phagocytosis, by NETs, by a combination of both, and by granular extract. As examples, we use Candida albicans for fungal and Shigella flexneri for bacterial pathogens. 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_22 The opportunistic fungal pathogen Candida albicans enters the bloodstream and causes hematogenously disseminated infection in hospitalized patients. During the initiation of a hematogenously disseminated infection, endothelial cells are one of the first host cells to come in contact with C. albicans. Endothelial cells can significantly influence the local host response to C. albicans by expressing leukocyte adhesion molecules and proinflammatory cytokines. Thus, it is of interest to investigate the response of endothelial cells to C. albicans in vitro. We describe the use of real-time PCR and enzyme immunoassays to measure the effects of C. albicans on the endothelial cell production of E-selectin and tumor necrosis factor α in vitro. 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_23 This protocol describes the setup, maintenance, and characteristics of models of oral and vaginal candidiasis based on well-established three-dimensional organotypic tissues of human oral and vaginal mucosa. Infection experiments are highly reproducible and can be used for the direct analysis of pathogen/epithelial cell interactions. Using the models, the several stages of infection by wild-type Candida albicans strains, the consequence of gene disruption of putative virulence factors in mutant cells, and the evaluation of the host immune response can be evaluated by histologic, biochemical, and molecular methods. As such, the models provide clear answers regarding protein and gene expression that are not complicated by nonepithelial factors. To study the impact of several host components, the mucosal infection models can be supplemented with immune cells, saliva, and probiotic bacteria, which might be relevant for host defense. It requires at least 3 days to be established and can be maintained thereafter for 2 to 4 days. 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_24 Phagocytosis is a highly conserved aspect of innate immunity. Drosophila melanogaster has an innate immune system with many similarities to that of mammals and has been used to successfully model many aspects of innate immunity. The recent availability of Ribo Nucleic Acid interference (RNAi) libraries for Drosophila has made it possible to efficiently screen for genes important in aspects of innate immunity. We have screened an RNAi library representing 7216 fly genes conserved among metazoans to identify proteins required for the phagocytosis of the human fungal pathogen Candida albicans. 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_25 Controlled studies on the immunopathogenesis of mucosal candidiasis in HIV infection have been hampered by the lack of a relevant animal model. We have previously reported that oral Candida infection in CD4C/HIV transgenic mice expressing gene products of HIV-1 in immune cells and developing an AIDS-like disease closely mimics oropharyngeal candidiasis in human HIV infection. The role of defective dendritic cells and CD4+ T cells in impaired induction of protective immunity and in the phenotype of chronic oral carriage of C. albicans can now be investigated under controlled conditions in these transgenic mice. 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_26 Candida albicans is a commensal inhabitant of the normal human microflora that can become pathogenic and invade almost all body sites and organs in response to both host-mediated and fungus-mediated mechanisms. Serologic responses to C. albicans that underlie its dichotomist relationship with the host (host-commensal and host-pathogen interactions) display a high degree of heterogeneity, resulting in distinct serum anti-Candida antibody signatures (molecular fingerprints of anti-Candida antibodies in serum) that can be used to discriminate commensal colonization from invasive disease. We describe the typical proteomic strategy to globally and integratively profile these host antibody responses and determine serum antibody signatures. This approach is based on the combination of classic immunoproteomics or serologic proteome analysis (two-dimensional electrophoresis followed by quantitative Western blotting and mass spectrometry) with data mining procedures. This global proteomic stratagem is a useful tool not only for obtaining an overview of different anti-Candida antibodies that are being elicited during the host-fungus interaction and, consequently, of the complex C. albicans immunome (the subset of the C. albicans proteome targeted by the immune system), but also for evaluating how this pathogen organism interacts with its host to trigger infection. In contrast with genomics and transcriptomics, this proteomic technology has the potential to detect antigenicity associated with posttranslational modification, subcellular localization, and other functional aspects that can be relevant in the host immune response. Furthermore, this strategy to define molecular fingerprints of serum anti-Candida antibodies may hopefully bring to light potential candidates for diagnosis, prognosis, risk stratification, clinical follow-up, therapeutic monitoring, and/or immunotherapy of candidiasis, especially of its life-threatening systemic forms. 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_3 Surface-exposed proteins of pathogenic bacteria are considered as potential virulence factors through their direct contribution to host-pathogen interactions. The specific interaction of bacterial proteins with host proteins often subverts the physiologic function of host-derived proteins, and therefore the bacterial proteins are considered as key players in the infectious process. The direct binding of host proteins is exploited by the pathogens for colonization, host tissue invasion, or immune evasion. Strikingly, surface proteins such as ABC transporters are also implicated in bacterial pathogenesis through their role in maintenance of bacterial fitness. Here, we are interested in surface-exposed proteins of Streptococcus pneumoniae, which interact with host proteins including proteins of the extracellular matrix, serum proteins, or ectodomains of cellular host receptors. These bacterial proteins are termed collectively adhesins or MSCRAMMs (microbial surface components recognizing adhesive matrix molecules). We have shown that choline-binding proteins and proteins that lack classic features of surface proteins such as a signal peptide that is required for protein secretion or a membrane anchor motif represent a major class of adhesins produced by S. pneumoniae. 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_4 The social soil amoeba Dictyostelium discoideum has been established as a host model for several human pathogens including Legionella pneumophila. The complete genome sequence, the genetic tractability, and the phagocytic characteristics of Dictyostelium generate many opportunities for the study of host-pathogen interactions. Important applications of this haploid model organism are (i) the use of Dictyostelium cells as a screening system for bacterial virulence, (ii) the use of Dictyostelium mutant cells to identify genetic host determinants of susceptibility and resistance to infection, and (iii) experiments that allow the dissection of the complex cross-talk with infectious agents. Accordingly, this chapter describes a plaque assay to identify attenuated pathogens, an infection assay for the analysis of host cell mutants and pathogens, and a screening method for the isolation of Legionella mutants that are defective in the reprogramming of the phagolysosomal maturation of the host. 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_5 A critical determinant of host range and specificity relies on the ability of pathogenic bacteria to recognize eukaryotic cell surface molecules via specialized adhesins. The specific adhesin-receptor interaction allows pathogens to tightly bind to their target cells, thereby facilitating the colonization of host tissues. Therefore, the identification and characterization of bacterial adhesins is a major topic in infection biology. This chapter focuses on a rapid and simple method for the analysis of adhesin-receptor interactions that permits the characterization of receptor binding properties at the level of single bacteria. Accordingly, this methodological approach is ideally suited for the analysis of adhesins expressed in a phase-variable manner and for the study of heterogeneous bacterial populations. Besides focusing on the receptor-binding assay, this chapter describes the production of fluorescence-tagged soluble host receptor domains required for conducting this assay. 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_6 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_7 The encapsulated fungal pathogens Cryptococcus neoformans and Cryptococcus gattii are significant agents of life-threatening infections, particularly in persons with suppressed cell-mediated immunity. This chapter provides detailed methodology for the purification of two of the major antigen fractions of C. neoformans: glucuronoxylomannan (GXM) and mannoprotein (MP). GXM is the primary component of the polysaccharide capsule, which is the major cryptococcal virulence factor. In contrast, MPs have been identified as key antigens that stimulate T-cell responses. Purification of GXM and MP should assist investigators studying the antigenic, biochemical, and virulence properties of Cryptococcus species. 2009-01-01T05:00:00Z http://www.springerprotocols.com/Abstract/doi/10.1007/978-1-59745-204-5_8 Recognition of pathogenic yeasts by host cells is based on components of the yeast cell wall, which are considered part of its virulence attributes. Cell wall glycans play an important role in the continuous interchange that regulates the balance between saprophytism and parasitism and between resistance and infection. Flow cytometry is a useful method for probing surface yeast glycans in order to compare their expression depending on strains and growth conditions. By using different monoclonal or polyclonal antibodies, levels of β- and α-linked mannosides as well as β-glucans can be successfully evaluated by flow cytometry methods. The cytometric method we describe here represents a useful tool to investigate to what extent yeasts are able to regulate their glycan surface expression and therefore modify their virulence properties. 2009-01-01T05:00:00Z