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The term “cellular microbiology” was coined in 1996 to identify an emerging discipline integrating the fields of cell biology and microbiology. The advent of genomics, proteomics, and postgenomics has resulted in more widespread understanding of the field of cellular microbiology among scientists. Cellular Microbiology, 2nd Edition, considers genomic information and advances in technology in an updated examination of this burgeoning area of important research.
The second edition of this successful textbook offers five new chapters that comprehensively cover bacterial human pathogen genomes; structure and dynamics of the host cell membrane; study of prokaryotic biology using genome-wide approaches; cell biology of virus infection; and use of simple nonvertebrate hosts to model mammalian pathogenesis. All chapters are revised and organized like the first edition to provide fundamental details of each discipline along with the cellular microbiology aspects.
This chapter provides an introduction to the main human microbial pathogens, with a description of the clinical features of the disease and emphasis on the cell biology of the infectious process. There is increasing evidence for association of Chlamydia pneumoniae with atherosclerosis. Chlamydia psittaci is primarily an animal pathogen, and only in rare cases is it responsible for human respiratory tract infections, oropharyngitis, and atypical pneumonia. Chlamydia has a biphasic developmental cycle with two morphologically different forms: the elementary body (EB), which is the infectious form and is metabolically inactive, and the reticulate body (RB), which results from the differentiation of the EB in the parasitophorous vacuole. The genus Entamoeba includes two related species, the commensal Entamoeba dispar and the pathogenic Entamoeba histolytica, the latter being the agent of amebic dysentery and visceral amebiasis, the second leading cause of death due to parasitic disease. Continuous cell growth leads to ulceration of the intestinal mucosa, causing diarrhea and severe intestinal cramps. The diarrhea is then replaced by a condition referred to as dysentery, characterized by intestinal bloody and mucoid exudates. If the condition is not treated, trophozoites of E. histolytica can migrate to the liver, lungs, bones, and brain, where large abscesses may appear. Infections caused by the encapsulated basidiomycetous yeast Cryptococcus neoformans are initiated by inhalation of the yeast into the lungs and show a remarkable propensity to spread to the brain and meninges.
This chapter provides an overview of what has been learned from analysis of bacterial human pathogen genomes. Bacterial human pathogens such as alphaproteobacteria, spirochetes, actinobacteria, firmicutes and gammaproteobacteria are studied. Haemophilus influenzae is a common commensal of the human respiratory tract. Three genes of the tricarboxylic acid (TCA) cycle are missing (citrate synthase, isocitrate dehydrogenase, and aconitase). This explains the requirement for high levels of glutamate in culture media. The relatively large size of the Mycoplasma penetrans genome is attributed to the presence of a number of paralogous gene families. M. genitalium exists in parasitic association with ciliated epithelial cells of human genital and respiratory tracts. It can be seen from the summaries presented that bacterial pathogen genomes have provided researchers with an unprecedented wealth of fundamental information on the functional (and nonfunctional) content of pathogen genomes. In terms of the benefits to basic science, the payoff has been huge. Genome sequencing has fundamentally altered the rate and range of projects that can be undertaken at the bench. It has allowed novel insights into fundamental questions of bacterial metabolism and evolution. The chapter ends with concluding remarks looking at the major findings from bacterial pathogen genomics to date, the impact on treatment and disease control, and finally a discussion of possible directions for future research.
Mechanisms controlling basic cellular functions, such as cell division, motility, adherence, differentiation, cell death, and the detection of potential cell dangers, are extremely highly conserved throughout the animal kingdom. This chapter aims to introduce some of these basic cell biology mechanisms. Cell membranes are fluid structures at physiological temperatures due to the cis double bond present in glycerophospholipids, which prevent the close packing of the lipidic acyl chains. Membrane receptors can be divided into three main classes according to their response to ligand binding: the ligand-gated ion channels open a selective pore; the seven transmembrane receptors are linked to heterotrimeric guanine triphosphate (GTP)-binding proteins, and various receptors are linked to enzyme cascades. Endocytosis is mediated by different mechanisms. The endoplasmic reticulum (ER) produces glycerolipids and cholesterol, and the Golgi apparatus produces sphingolipids. Lipids travel between organelles by the secretory and endocytic pathways via vesicle-mediated transport or via lipid-carrier proteins present in the cytosol. Apoptosis is divided into three phases. Detection of the apoptotic signal by sensors is followed by conversion of the signal so that it can trigger the execution phase of apoptosis. When a cell prepares to divide, it must coordinate the duplication and division steps. To ensure good quality control, the transitions between phases must be governed by a decision-making process that assesses such things as the quality of the duplicated DNA and the successful reorganization of the cellular machinery.
Bacteria express surface-associated adhesion molecules, generally termed adhesins, recognize the eukaryotic cell surface, the extracellular matrix (ECM) protein, or carbohydrate structures. The major structural components of the eukaryotic ECM are collagens that form different types of interstitial or basement membrane networks, including fibril-forming collagens (types I, II, and III) and the two-dimensional collagen type IV network. Due to the tight association of other adhesion proteins, such as fibronectin, vitronectin, von Willebrand factor, laminin, nidogen, and proteoglycans with collagens to form supramolecular aggregates of variable structure and composition, the interaction with host cells or bacteria is not determined solely by the collagen component. Although most characterized interactions with gram-negative bacteria involve recognition of lectins and carbohydrate structures in the host tissue, several types of fimbriae of enterobacteria exhibit specific interactions with fibronectin, laminin, or other adhesion proteins. Importantly, thrombospondin binds Plasmodium falciparum-parasitized erythrocytes and, together with its cell surface receptor, CD36, mediates their adherence to endothelial and other cells. Chlamydia trachomatis expresses a heparan sulfate-like glycan that links the bacterium to host cell heparin-binding proteins, thereby using a trimolecular complex for adherence and invasion. Finally, the epithelial-cell mucosal barriers in the body provide different high-molecular weight mucin glycoproteins (containing 50 to 80% carbohydrate) which exhibit considerable genetic polymorphism among individuals.
An essential step in the successful colonization and production of disease by microbial pathogens is their ability to adhere to host cell surfaces and the underlying extracellular matrix. The choice of host cell substrate that a pathogen can adhere to is large. The mammalian cell surface contains many proteins, glycoproteins, glycolipids, and other carbohydrates that could potentially serve as a receptor for an adhesin. Additionally, the extracellular matrix provides a rich source of glycoproteins for adhesins to bind to and even initiate signaling, and implanted devices remain a major target for bacterial adherence. Bordetella pertussis, the causative agent of whooping cough, is a respiratory mucosal pathogen that possesses several potential adherence factors that exemplify the complexity of bacterial adherence to host cell surfaces. N. gonorrhoeae and N. meningitidis are two mucosal pathogens that have developed sophisticated and overlapping mechanisms to adhere to host cell surfaces. Two high-molecular-weight adhesins (HMW1 and HMW2) belong to the autotransporter family and mediate bacterial adherence. Adhesins play an important role in disease and represent the interface between the pathogen and the host cell. In many cases the precise role that individual adhesins play in the pathogenesis of specific diseases has been established. For example, inactivation of the gene which encodes Cna, a collagen-binding adhesin of S. aureus, results in a mutant with a considerably diminished capacity to cause septic arthritis in an animal model.
This chapter addresses the complex molecular interrelationships between cell adhesion and the transduction of transmembrane signals that affect cell adhesion and fate. It is shown here that adhesion sites such as focal contacts and cell-cell adherens junctions contain multimolecular protein complexes that participate both in the physical assembly of adhesion sites and the associated cytoskeleton and in the transduction of long-range growth, differentiation, and survival signals. The network of molecular interactions of the different adhesions, their involvement in the interaction with the cytoskeleton, and their particular role in adhesion mediated signaling are discussed in this chapter. Cell-cell adhesion is also mediated by a multitude of transmembrane receptor molecules including immunoglobulin superfamily cell adhesion molecules (CAMs), selectins, and cadherins. The transmembrane domain of matrix adhesions consists of adhesion receptors, mainly different members of the integrin superfamily. As may be expected from the fact that these receptors can interact with different matrix molecules, this domain is also quite heterogeneous with respect to the integrin composition. The importance of tension for triggering adhesion-dependent signal transduction is supported by recent findings where external forces were directly applied to cell-extracellular matrix (ECM) adhesion sites by a microneedle, by stretching an elastic substrate, or by laser trapping of cell surface-attached beads covered with adhesion ligands. Definitive molecular mechanisms responsible for microtubule directing to focal adhesions are not clear, but the Rho effector, Diaphanous (Dia1), might be involved in this process based on its effects on microtubule dynamics.
This chapter focuses on a few examples of the involvement of cell adhesion molecules during infection, in interactions involved in processes such as adhesion or invasion, or in controlling the process of host inflammation. Adhesion molecules are cell surface receptors that establish cell-to-cell interactions, or interactions between cells and the extracellular matrix. The immunoglobulin superfamily of adhesion molecules is mostly involved in cell-to-cell interactions and consists of receptors with extracellular domains sharing homology with immunoglobulins. It is possible that interactions of proteoglycans with bacterial ligands participate in the initial stages of adhesion and that other types of interactions are involved either in stabilization of the bacterial adhesion processes or in invasion. Also, other Neisseria Opa proteins bind to CD66 carcinoembryonic antigens, which are members of the Ig superfamily, and this interaction mediates bacterial internalization into host cells. For both Yersinia invasin and Listeria internalin, the entry process appears to be driven by incremental interactions between bacterial surface ligands and cell adhesion molecules. Lipid rafts have changed our view of the classical membrane fluid mosaic model into a more complex system. Raft microdomains are described as dispersed liquid-ordered phase microdomains that diffuse laterally within the two-dimensional liquid-disordered phase membrane. The fact that cell surface receptors are dispensable for this type of bacterial signaling is not totally true because interactions between components of the Shigella translocon and the α5β1-integrin or the CD44 hyaluronic acid receptor were reported.
This chapter focuses on the nature and function of these two-dimensional membrane domains and highlights several examples of microbial exploitation of host cell membrane organization. Cholesterol is an important component of most membranes of mammalian cells, and disruption of its homeostasis or distribution in intracellular membranes leads to debilitating disease in humans. For dynamic analyses of cholesterol distribution in living cells, it is necessary to have a fluorescent analogue that faithfully mimics cholesterol, as opposed to a fluorescent probe, like filipin, that binds to cholesterol and potentially perturbs its distribution. Dehydroergosterol (DHE) is a naturally occurring fluorescent sterol found in sponge and fungal cells. In a fluorescence correlation spectroscopy (FCS) experiment, a small region of the plasma membrane is monitored for fluctuations in fluorescence intensity. Fluorescence resonance energy transfer (FRET) requires two fluorophores with distinct but overlapping excitation and emission spectra; the emission spectrum of the donor fluorophore must overlap with the excitation spectrum of the acceptor fluorophore so that energy transfer can occur. Many bacterial toxins exert their action in the cytosols of host cells, but first they enter host cells by endocytosis and then they are translocated to the cytosol. Cholera toxin (CT) is a pentavalent toxin that binds to surface-expressed ganglioside GM1 and causes its aggregation. When CT is bound to GM1, it can be visualized in caveolae in several cell types, and this has led to speculation that caveolae mediate its internalization.
Eukaryotic cells need to be in constant communication with their environment in order to perform most of their functions, such as the transmission of neuronal, metabolic, and proliferative signals and the uptake of nutrients or to protect the organism from microbial invasion, to name only a few. Macropinocytosis involves remodeling of the actin cytoskeleton, a process regulated by the small GTPases Ras, Rac, and Cdc42, and leads to massive membrane internalization. Phagocytosis requires important actin rearrangements and pseudopod extension under the control of Rho GTPases. Several pathways have been proposed to mediate membrane traffic between endosomes and the biosynthetic pathway. In particular, several lines of evidence support the view that direct transport routes mediate anterograde and retrograde transport between early endosomes and the trans-Golgi network (TGN). PI3K activity and, hence, PI(3)P are required for autophagy in yeast, where Vps34 has been shown to coimmunoprecipitate with proteins required for autophagy. Early endosomes are important sorting stations along the endocytic pathway. After receptor-ligand uncoupling at the mildly acidic pH (pH 6.2), housekeeping receptors are transported along the recycling route, whereas ligands follow the degradation pathway together with downregulated receptors and fluid phase markers. Phosphorylation of the inositol ring of phosphatidylinositol in the 3, 4, and 5 positions generates seven different phosphoinositides at the cytosolic face of cellular membranes. During the past few years, phosphoinositides have emerged as regulators of membrane traffic by regulating the localization and/or activity of effector proteins.
This chapter describes a series of interconnected problems for a range of bacterial, protozoal, and fungal pathogens and explores, in the order in which they are encountered by the pathogen, the consequences of each decision point in the establishment of an intracellular infection. There are three basic mechanisms of invasion: (i) phagocytosis, i.e., entry into professional phagocytes such as macrophages, monocytes, and neutrophils via a process dependent on the host cell contractile system; (ii) induced endocytosis and phagocytosis, i.e., entry into nonprofessional phagocytes by the active induction of internalization through the activity of the host cell contractile system; and (iii) active invasion, i.e., active entry into a passive host cell without triggering any contractile event in the host cell cytoskeleton. The niches exploited by intracellular pathogens fall readily into three different groupings. The first is intralysosomal, in which pathogens persist in acidic, hydrolytic compartments that interact with the endosomal network of the host. The second is intravacuolar, in which pathogens persist in nonacidic vacuoles that exhibit modified or little interaction with the endosomal system of the host. The third is cytoplasmic, in which pathogens exit the phagosome and reside within the host cell cytosol. This chapter has attempted to present the major points in the biology of these pathogens within a thematic framework from the time of initial infection, through the choice of intracellular niche, avoidance or exploitation of the immune response, and culminating in the metastasis or spread of the infection.
Eukaryotic cells possess three kinds of cytoskeletal elements: 5- to 9-nmdiameter actin filaments, 24-nm-diameter microtubules, and 10-nmdiameter intermediate filaments. All these polymer networks are assembled reversibly from monomers. This chapter concentrates on actin. Reorganization of the actin cytoskeleton is required for leukocytes to migrate to sites of infection, for fibroblasts and endothelial cells to migrate to areas of wound healing, and for platelets to plug leaking vessels. Actin polymerization not only drives the motility of leukocytes, fibroblasts, keratocytes, and amoebae, but also provides the propulsive force for the movement of intracellular bacteria including Listeria, Shigella, and Rickettsia as well as the poxvirus, vaccinia. The severing of actin filaments reduces the viscosity of the peripheral cytoplasm and allows the actin cytoskeleton to be rapidly remodeled. When cells are stimulated to move and change shape, the number of free barbed filament ends markedly increases. This rapid rise in the number of free barbed ends can be accomplished in two ways. First, the barbed ends of preformed actin filaments can be uncapped and second, nucleating proteins can serve as a template to initiate the elongation of new actin filaments. The precise mechanisms regulating the formation of new actin filaments in the motile cell remain to be determined. In addition, filaments can be severed by gelsolin. The combination of uncapping and severing can greatly increase the number of free barbed-filament ends available for rapid actin assembly.
This chapter illustrates several modes of bacterial manipulation of the host cell cytoskeleton using a few well-studied examples and explores interactions of various pathogenic bacteria with the actin cytoskeleton of both phagocytic and nonphagocytic host cells. These interactions are generally mediated by specific bacterial gene products, virulence factors, whose sole function is to mimic or interfere with normal host cell signals, in this case, those that regulate actin filament dynamics. A section focuses on the ability of Shigella to invade epithelial cells by a process that triggers global changes in the cellular actin cytoskeleton that result in membrane ruffles and macropinocytosis. Importantly, both zipper and trigger uptake mechanisms proceed using energy derived from the host cell. The cytoskeletal and membrane rearrangements that are responsible for bacterial invasion require no energetic input from the bacterium once the type III secretion apparatus is made (for the trigger mechanism of uptake); they invade by persuasion rather than by force. The chapter further focuses on four well-characterized cytoskeletal manipulators: YopE, YopH, YopT, and YpkA/YopO. Intracellular motility allows pathogens to spread directly from the cytoplasm of one cell into the cytoplasm of an adjacent cell. The many processes used by bacterial pathogens of cellular invasion, inhibition of internalization, cellular adhesion, and intercellular spread may appear diverse, but they are linked by common molecules: the components of the host cell cytoskeleton. In becoming better pathogens, these bacteria have come to a highly evolved appreciation of the subtlety required to regulate it.
This chapter discusses the molecules that have been classically known as bacterial toxins; the last section mentions some recently identified molecules that cause cell intoxication and have many but not all of the properties of classical toxins. A section shows the subunit composition and the spatial organization of toxins whose structures have been solved either by X-ray crystallography or by quick-freeze deep-etch electron microscopy. For simplicity, the toxins have been divided into three main categories: (i) those that exert their powerful toxicity by acting on the surface of eukaryotic cells simply by touching important receptors, by cleaving surface-exposed molecules, or by punching holes in the cell membrane, thus breaking the cell permeability barrier; (ii) those that have an intracellular target and hence need to cross the cell membrane (these toxins need at least two active domains, one to cross the eukaryotic cell membrane and the other to modify the toxin target); and (iii) those that have an intracellular target and are directly delivered by the bacteria into eukaryotic cells. Depending on their target, these toxins can be divided into different groups that act on protein synthesis, signal transduction, actin polymerization, and vesicle trafficking within eukaryotic cells. The toxins that inhibit protein synthesis, causing rapid cell death, at extremely low concentrations are diphtheria toxin (DT), Pseudomonas exotoxin A (ExoA), and Shiga toxin.
At least four properties of bacterial protein toxins make them suitable as cell biological and pharmacological tools. First, the toxins enter cells without damaging the cell integrity. Second, the toxins possess high specificity. A high cell specificity is most often based on a toxin-specific membrane-binding domain and on specific receptors present on the surface of eukaryotic target cells. Actin, another important eukaryotic substrate for ADP-ribosylation by bacterial toxins, is not a GTP-binding protein but an ATP-binding protein. Because all these nucleotide-binding proteins are functionally important cellular proteins, the toxins, which allow their selective covalent modification, are widely used as tools. The actin cytoskeleton is the target of various bacterial toxins that affect the microfilament protein either directly by ADP-ribosylation or indirectly by modifying the regulatory mechanisms involved in the organization of the actin cytoskeleton. Actin, which is one of the most abundant proteins in eukaryotic cells, is the major component of the microfilament system. The toxin effect should occur with some delay of at least 15 to 30 min. This time is necessary for the translocation of the toxin. Moreover, it should be tested whether actin is in fact ADP-ribosylated by the toxin. Hydrolysis of bound GTP terminates the active state of the GTPases. It has been shown that especially Rho subfamily GTPases are targets for bacterial protein toxins. Recently, the genes for toxins were introduced into some crop plants in an effort to protect them from insect attack.
This chapter discusses the type III secretion system (TTSS) possessed by several gram-negative bacteria that live, for at least part of their life cycle, in close association with eukaryotic cells. The intestinal pathogen Salmonella provides an illustrative example of the adaptation of type III gene clusters. This pathogen possesses two entirely separate TTSSs, encoded by different gene clusters, which, based on genetic evidence, apparently were acquired at different times during evolution. Secretion and assembly of the external needle only occurs once the bacterial envelope-spanning components, including the outer membrane-associated parts of the needle complex, are assembled. Another feature common to both systems is that protein secretion appears to occur by a continuous process without any detectable periplasmic protein intermediates. The proteins secreted by the TTSS interact with eukaryotic cells in several ways. In most systems studied so far, the effector proteins are translocated across the eukaryotic plasma membrane into the cytoplasm by extracellular bacteria. For Yersinia and enteropathogenic E. coli (EPEC), it is thought that one function of the effector proteins is to either prevent phagocytosis or form a tight adherence between the bacterium and the target cell that induces the formation of so-called pedestal structures. Triggering apoptosis also depends on the Yersinia TTSS, suggesting that, like apoptosis-inducing proteins of plant-interacting bacteria, YopJ/YopP is recognized, or its activity occurs, inside the eukaryotic cell and that this recognition or activity triggers apoptosis.
Pathogenic bacteria of humans and plants have coopted conjugation systems to export virulence factors to eukaryotic host cells. Although this is a functionally diverse family, there are some unifying themes: (i) exporters are assembled at least in part from subunits of DNA conjugation systems, and (ii) the known substrates recognized by these transporters are large macromolecules such as nucleoprotein particles, scaffolding proteins, guanine nucleotide exchange factors, or multisubunit toxins. The type IV secretion family is composed of toxin exporters used by several bacterial human pathogens. Bordetella pertussis, the causative agent of whooping cough, uses the Ptl transporter to export the AB type pertussis toxin across the bacterial envelope. The growing list of pathogens that utilize type IV secretion system (TFSS) for delivery of effector molecules into the host cell environment, comprising species like Brucella, Actinobacillus, Ehrlichia, Wolbachia, and Xilella, is under continuous revision. The existence of a subset of VirB homologues in the Helicobacter pylori cag and Legionella pneumophila icm/dot systems underscores the functional importance of these types of proteins in macromolecular export. The Agrobacterium tumefaciens T-DNA-processing reaction resembles the conjugative DNA-processing reaction, resulting in formation of the T-strand/ VirD2/VirE2 nucleoprotein particle or T complex. Perhaps the most compelling evidence that conjugation machines recognize proteins as translocationcompetent substrates is that VirE2 SSB can be exported to plant cells independently of the T-strand/VirD2 complex. The evolution of a family of secretion systems from ancestral DNA conjugation machines raises many interesting questions and exciting new research directions.
There are two criteria to distinguish apoptosis from necrosis: morphology and DNA fragmentation. Typical morphological changes that occur during apoptosis include cell shrinkage and loss of normal cell-to-cell contacts, blebbing at the cell surface, and intense cytoplasmic vacuolization. Interestingly, the morphological changes, the fragmentation of DNA, and the expression of markers for recognition by phagocytes are very similar across different cell types and species. Caspases are a family of cysteine proteases that play a central role in the apoptotic pathway. Among cysteine proteases, caspases are unique in requiring an aspartate at the cleavage site. Bcl-2 is homologous to the Caenorhabditis elegans apoptosis inhibitor gene ced-9. bcl-2 complements the ced-9 mutation in worms and inhibits apoptosis in many different instances when overexpressed in mammalian cells. Many bacterial pathogens induce apoptosis in host cells. This chapter groups microbial pathogens by the mechanisms they use to induce apoptosis. Bordetella pertussis kills macrophages by apoptosis in vitro by secreting adenylate cyclase-hemolysin (AcHly) toxin. This toxin has two domains: (i) a potent adenylate cyclase activity, which is activated by calmodulin, and (ii) a hemolysin activity, which is a pore-forming protein that is thought to allow the translocation of the cyclase into the host cell cytoplasm.
The host response to pathogenic microorganisms is extraordinarily diverse. The extent and degree of the host response depend on the nature of the pathogen itself and the route and extent of the infection. Some general features of host-pathogen interactions are discussed in this chapter. Probably the best experimental approach to the analysis of the innate response is to examine mutant mice that can use only the innate system, by virtue of the absence of lymphocytes. The activated macrophage is found as a result of activation of either the innate immune system or the T-cell system. The innate system is activated when macrophages interact with microbes and release early cytokines that induce natural killer (NK) cells to produce interferon (IFN)-γ. Neutrophils are essential in infections with extracellular bacteria, which are rapidly eliminated by the oxidative and nonoxidative neutrophil microbicidal mechanisms. The most extensively studied infection that has led to insights into the activation of the innate immune system is Listeria monocytogenes infection in the SCID mouse. The peptide-major histocompatibility complex (MHC) molecular complex represents the molecular substrate that engages the T-cell receptor (TCR) for antigen. The composition of the peptide-MHC complex reflects the intracellular milieu of the antigen-presenting cells (APC).
Pathogens have evolved a wide variety of strategies to circumvent the host microbicidal activities and to use the cellular machinery to their own advantage. This chapter is devoted to electron microscopy, which is the approach of choice for optimal resolution and precision. A wide variety of cytochemical and immunoelectron microscopy methods can be used to characterize pathogens, analyze the intracellular compartment in which they reside, and localize bacterial virulence factors or cell components involved in their survival. Some of the major morphological methods, recent and less recent, of special interest to host-pathogen interplay are reviewed in this chapter. Phagosomes that retain intermingling characteristics of early endosomes are considered to be immature; those that become mature lose their ability to fuse with early endosomes and fuse with lysosomes to become phagolysosomes. Endocytosis and phagocytosis both involve an extensive transfer of membrane in both directions between the cell surface and intracellular membrane compartments. Given the large amounts of membrane required during uptake of particles and also during replication of endoparasites within their phagocytic vacuole, one of the questions that have been raised recently was whether other cell compartments, and more especially the endoplasmic reticulum (ER), could be an additional source of membrane for forming and/or dividing phagosomes.
Early forays into identifying virulence genes used the basic tools of bacterial genetics: mutation and complementation. The fundamental biology of some host-pathogen interactions can limit the ability to apply signature-tagged mutagenesis (STM) and related approaches. One of the most versatile tools to probe host-pathogen interactions is the green fluorescent protein (GFP). Differential fluorescence induction (DFI)-based screens have yielded a high proportion of virulence genes relative to housekeeping genes. Part of this success results not from any particular advantage of the DFI technique per se but from a principle that is important when designing any screen based on analysis of differential expression, be it DNA microarrays or in vivo expression technology (IVET). Caenorhabditis elegans is one of the prevalent model systems used to study the development of multicellular organisms. The availability of whole genomic sequences presents another method of virulence gene discovery: bioinformatics. This chapter outlines a variety of approaches used to identify candidate virulence genes. The genome sequence of every major pathogen (and most minor ones) is or will soon be available. These genomic sequences, augmented by the gene discovery methods described in the chapter, should permit the discovery of novel virulence genes at an unprecedented pace.
This chapter summarizes the development and use of two broad experimental approaches directed toward defining the biological function of unknown proteins. The first approach is based on the well-known biological phenomenon of coordinate gene expression, which can be analyzed through transcriptional profiling. The second approach requires the identification and analysis of protein complexes in which unknown proteins participate. These strategies typically rely on mass spectrometry methods and interactive genomic databases. A DNA microarray consists of multiple unique DNA fragments attached to a solid support in a specific pattern. DNA microarray technology is most often used for two applications: the determination of gene expression levels and the identification of specific sequences in genomes, including the detection of mutations. The interactome of Saccharomyces cerevisiae was analyzed in two separate efforts. An array-based screen using 6,200 cloned full-length yeast open reading frames (ORFs) identified 841 interactions, the majority of which were novel. The use of DNA microarrays in postgenomic research was wildly successful and thus became common in many branches of molecular biology. The major obstacle to using whole-genome protein arrays is the requirement for extensive customization. Nucleic acid probes and targets are generally stable and can be generated in large amounts using standardized highthroughput procedures.
This chapter focuses on events in viral replication cycles that have similarity or relevance to the interactions of bacterial pathogens with cells, and the examples discussed will draw mainly on membrane-containing enveloped viruses. The transferrin receptor is a well-studied cellular protein that undergoes constitutive endocytosis through clathrin-coated vesicles (CCVs). Phagocytosis plays a key role in the clearance of cells infected by viruses, either following antibody or cell-mediated killing of these cells or after apoptosis of the cell in response to the virus infection. Macropinocytosis is similar to phagocytosis in that it is dependent on remodeling of cortical actin, but it is not dependent on the ligation of specific receptors. Viral fusion proteins can be grouped into several distinct classes based on the organization of the protein. Class 1 fusion proteins, which include influenza HA and HIV envelope glycoprotein (Env), are synthesized as single-chain transmembrane proteins that assemble into trimers in the ER of the infected cell. Within these factories, viral DNA and protein synthesis occur and progeny viral particles undergo assembly. Virus replication is tightly integrated into the properties of the host cell. Due to their ability to efficiently exploit specific cellular functions, viruses have been, and continue to be, very effective tools to study basic cellular functions. Indeed, virus systems of one sort or another underlie much of the current knowledge of molecular genetics and cell biology.
This chapter describes an alternative method for identifying pathogen virulence factors that is based on the finding that many mammalian pathogens are also capable of causing disease in simple nonvertebrate hosts. The ability of Pseudomonas aeruginosa to infect plants as well as animals was subsequently extended to show that the same clinical isolate of P. aeruginosa that can infect Arabidopsis, strain PA14, was also a pathogen of the nematode worm Caenorhabditis elegans as well as the insects Drosophila melanogaster and Galleria mellonella. Toxin-mediated killing is characterized by rapid worm killing, usually within one day, and by the ability of the conditioned media to kill in the absence of live bacteria. Infection-associated killing usually occurs over the course of several days and thus far has been characterized by the accumulation and, in some cases, proliferation of bacteria within the worm digestive tract. Drosophila is an ideal insect host for genetic analysis of the host innate immune response. However, as Drosophila is relatively small, it is difficult to inject bacteria directly into the body cavity. An alternative approach for investigating the function of microbial products that manipulate host cells is to express these bacterial toxins within a genetically tractable host system. Interactive genetic analysis was also used to define the role of P-glycoproteins (PGP) in protecting C. elegans from the toxic effects of phenazines.
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