Cellular Microbiology, Second Edition

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.

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.

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 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.

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.

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.

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.

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.

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).

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 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.