INTRODUCTION

The epithelial surfaces of the skin and the intestinal, respiratory, and reproductive tracts constitute the outer frontiers of the body and are exposed continuously to the myriad microorganisms present in the external environment. Like any good frontier guard, the cells of these epithelia must carry out two important functions – establish barriers against microbial intruders and raise the alarm if the barriers are breached. The nuclear factor kappa B (NF-κB) family of transcription factors plays a vital role in these functions by controlling the expression of a number of genes involved in antimicrobial defense and in the inflammatory response. Central to this role is the ability of NF-κB to be regulated by cellular signaling pathways that are activated by a wide variety of microorganisms. This sensitivity to microbial signals allows NF-κB function to be modulated appropriately in response to any changes in the flora that is in contact with the epithelium.

This chapter reviews what is currently known about the major NF-κB-dependent inflammatory responses elicited in mammalian epithelial cells as a result of interactions with bacteria. We start with an overview of the NF-κB family and its basic regulation. In subsequent sections, we discuss the mechanisms of modulation of NF-κB function by bacteria-derived signals, the consequent alterations in cell function, and the clinical abnormalities that can result from genetic defects in NF-κB activation.

THE YERSINIA INFECTION

There are three human pathogenic Yersinia species: Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis (Smego et al. 1999; Sulakvelidze 2000). Y. pestis is the causative agent of bubonic plague and has been responsible for the deaths of millions of people over the years. This pathogen is transmitted to humans by the bite of an infected rodent flea. Once inside, the bacteria initially invade and proliferate in lymphatic tissue. Y. enterocolitica and Y. pseudotuberculosis cause enteric infections (yersinosis) in humans. These are transmitted to humans by infected beverages and food or by direct contact with infected mammals; pigs are the major reservoir (Bottone 1999; Smego et al. 1999). Despite having a different route of infection, the orally transmitted non-plague Yersinia species also exhibit tropism for lymphoid tissue. The infection route occurs through the ileal mucosa in the gastrointestinal tract, where they are taken up into the lymphoid follicles through M-cells. These specialized cells cover the lymphoid follicles of Peyer's patches and engulf bacteria in a way that resembles active phagocytosis (Grassl et al., 2003). The bacteria multiply within the Peyer's patches, which are intestinal lymphoid nodules that contain B and T lymphocytes and phagocytes, and then drain to mesenteric lymph nodes. At this location, Yersinia encounters cells of the innate immune system, and can exert a block on the customary antimicrobial functions of these cells, including phagocytosis (Hanski et al. 1989; Simonet et al. 1990).

INTRODUCTION

Bacterial pathogens utilize invasive pathways and/or toxins to subvert the innate and acquired immune systems in order to damage the host epithelium. The infectious process requires that the pathogen can adhere and proliferate in the host. The capacity to colonize and cause disease varies among bacterial pathogens. For example, Clostridium tetani has only a limited ability to bind and proliferate within the host but is pathogenic due to the production of a potent neurotoxin, but the streptococcus and staphylococcus have strong adhesion factors that allow efficient colonization, with virulence due to the production of a multitude of virulence factors, including superantigens that simultaneously bind the major histocompatibility complex (MHC) and T-cell receptor of immune cells to stimulate production of antigen-independent cytokines.

The basic distinction between a member of our normal flora and a pathogen lies in the capacity to damage the host. However, this distinction is grayed by the immune status of the host, where host compromise converts commensal bacteria or even saprophytic bacteria into potent opportunistic pathogens. Pseudomonas aeruginosa is an opportunistic pathogen in many clinical situations but does not elicit disease in healthy individuals despite its ability to produce both a classical exotoxin and type III cytotoxins. Clostridium difficile can cause pseudomembrane colitis in patients undergoing antibiotic therapy. C. difficile pathogenesis is related to the ability to produce the exotoxins, toxin A and toxin B. Escherichia coli, a component of our normal gut flora, becomes a pathogen upon the acquisition of accessory genes that can encode several classes of toxins.

INTRODUCTION

Bacteria colonize the gastrointestinal tract as early as a few hours after birth. This relationship that develops at an early stage between humans and bacteria is shared with other mammals. Gastrointestinal epithelial cells play a crucial role in maintaining a quiescent environment while being bathed with normal flora, and yet at the same time they must possess functions that allow them to participate in immune surveillance. In addition to screening for and responding to the presence of pathogens in the intestinal lumen, gastrointestinal epithelial cells provide barrier function and transport of ions and solutes.

Enteric pathogens, as opposed to normal flora, cause disease by exploiting the host cytoskeleton or signaling pathways, which ultimately alters the physiologic functions of the intestinal epithelium. For example, pathogens can induce or suppress inflammatory responses, alter the transport of fluid, solutes, and ions, perturb the tight-junction barrier, and activate programmed cell death (apoptosis). This chapter summarizes the cross-talk between bacterial pathogens and host cells that leads to gastrointestinal symptoms.

ENTERIC PATHOGENS AND INTESTINAL EPITHELIAL CELL RECEPTORS

The interaction that occurs between pathogenic or non-pathogenic bacteria and intestinal epithelial cells begins with the adherence of bacteria to the cellular surface. This is a common mechanism by which bacteria cause disease, not only in the gastrointestinal tract but also in other systems where epithelial cells face the external environment, such as the genitourinary and respiratory systems. Adherence of bacteria to the cellular surface is essential to reduce the washout effect caused by intestinal secretion and peristalsis.

INTRODUCTION

Microbes populate virtually every square inch of the earth's surface. This success is due in large part to the numerous adaptation strategies they have developed to facilitate survival/persistence. The practical requirement for microbial resilience is particularly true of pathogenic microorganisms, as they must cope with host environments that are often actively adversarial. This is the case with Helicobacter pylori, which colonizes in the unlikely niche of the human stomach. Once in the stomach, H. pylori establishes a chronic infection in a manner that shows many similarities to what we typically think of as behavior of the host-adapted flora – “with an eye towards persistence rather than towards causing disease” (Merrell and Falkow, 2004). This is evidenced by the fact that severe disease typically takes decades to develop. This delayed development of overt pathology suggests that there is a balance shift that causes colonization to go awry and leads to disease. This shift is likely due to a combination of physiological and genetic factors for both participants of the host–pathogen interaction. On the bacterial front, H. pylori interacts with gastric mucosal cells and expresses a repertoire of factors that result in alterations in host cell signaling. These changes in host cell signaling are likely ultimately responsible for H. pylori-induced disease. Thus, H. pylori represents a model organism in terms of its ability to chronically exploit the gastric niche as well as to manipulate gastric epithelial cells (Figure 12.1).

To survive in any given niche, bacteria must be capable of sensing, interacting with, and responding to their environment. The method and extent to which bacteria interact with their environment are governed to a large degree by the proteinaceous molecules located on the bacterial cell surface or released into the extracellular milieu. Due to differences in cell-envelope architecture, this process of protein secretion is markedly different between Gram-positive and Gram-negative organisms.

GRAM-POSITIVE VERSUS GRAM-NEGATIVE BACTERIA

Gram-positive bacteria possess a single biological membrane termed the cytoplasmic membrane, which is surrounded by a thick cell wall. The majority of proteins targeted for secretion possess an N-terminal amino-acid signal peptide and utilize the Sec-dependent pathway (Holland, 2004). The Sec machinery is composed of several membrane-associated proteins, including an ATPase (SecA), the Sec translocon (SecYEG), which appears to be the basic unit of cellular life forms, several integral membrane proteins (e.g. SecD, SecF), and a signal peptidase that removes the signal peptide during translocation of the proteins across the cytoplasmic membrane (Dalbey and Chen, 2004). In addition to the Sec pathway, several alternative protein-secretion systems have been recognized in Gram-positive organisms, including the Tat (twin arginine translocation) and ESAT-6/WXG-100 pathways (Pallen, 2002; Robinson and Bolhuis, 2004). However, the role of these systems in protein secretion in Gram-positive bacteria is minor in comparison with the Sec-dependent pathway. Once translocated across the cytoplasmic membrane, the mature protein either can be released into the extracellular milieu or may remain in contact with the cell wall.

Through their capacity to recognize, phagocytose and inactivate invading microorganisms, phagocytic cells have a key role in the innate immune response and host defense. During this process there is an intimate interplay between different recognition mechanisms displayed by both the host cells and the microorganisms. Understanding the complex process of phagocytosis requires insight into the mechanisms of receptor function, signal transduction, actin-based movements, membrane and vesicle trafficking, and oxidative activation, as well as how pathogens interfere with and subvert these processes. The complexity is thus in part due to the diversity of receptors capable of stimulating phagocytosis, and in part due to the capacity of different microbes to influence their own fate, as they are recognized and internalized. It is now evident that pathogens are not passive bystanders evading phagocytosis and intracellular killing, but have evolved specific means of subverting the process of phagocytosis through different mechanisms, involving inhibition of opsonization and receptor recognition, inactivation of specific GTPases, dephosphorylation, inhibition of PI-3 kinases, and actin polymerization. Studies of the pathogenicity strategies of bacteria such as Salmonella, Helicobacter pylori, Streptococcus pneumoniae, Shigella, Mycobacterium tuberculosis, Yersinia pseudotuberculosis, and Listeria monocytogenes have not only shed light on microbial pathogenicity but have also been useful tools for elucidating the phagocytic process per se. Understanding how Listeria escapes from the phagosome by forming an actin-rich tail has revealed how actin polymerization is initiated and controlled.

Helicobacter pylori is a spiral-shaped, flagellated, microaerophilic Gram-negative bacterium that colonizes the gastric epithelium of c.50% of humans (Blaser & Berg 2001). These organisms elicit a strong immune response that does not resolve the infection; once acquired, bacteria persist for a lifetime in the absence of antibiotic treatment. All persons infected with H. pylori have gastritis and 20%–30% will develop severe disease that includes gastric and duodenal ulcers, gastric adenocarcinoma or MALT lymphoma (Covacci et al. 1999). A characteristic feature of H. pylori infection is the massive recruitment of phagocytes (particularly neutrophils) to the gastric mucosa (Allen 2000). Of interest here is the fact that H. pylori survives for years in a phagocyte-rich environment, and a growing body of data demonstrates that these organisms modulate the host inflammatory response and phagocyte function. By this mechanism H. pylori evades phagocytic killing and promotes host tissue damage.

COLONIZATION OF THE GASTRIC EPITHELIUM

Helicobacter pylori is the only microbe that survives in the hostile environment of the human stomach (Blaser & Berg 2001, Montecucco & Rappuoli 2001). Urease is a nickel-containing enzyme that is essential for colonization; ammonia generated by this enzyme buffers H. pylori as it passes through the highly acidic gastric lumen. Bacteria establish residence in the mucus layer over the epithelium; motility in this milieu is enhanced by the spiral shape of the organism and multiple polar flagella.

INTRODUCTION

The evolution of bacterial pathogens is essentially the story of how all life evolves. It is a story of mutation and selection, of adaptation and survival, of incremental genetic changes and quantum leaps in genome content. All organisms evolve, but the evolution of microbes is the best studied. The short generation times of microbes and the ability to grow individual populations to large numbers allow researchers to study rare events over many generations – something that is next to impossible with larger, more complex organisms such as fruit flies, mice, and humans.

This chapter begins with a brief overview of the principles of mutation and selection in bacteria. This background will prepare the reader for the subsequent sections that will discuss the various forms of horizontal gene transfer (HGT) and how they each contribute to bacterial evolution. Specific examples are presented to give the reader insight into the enormous power of genetic selection as well as the great diversity of pathways that bacteria take in adapting to their environment. We also highlight some recurrent themes in bacterial pathogenesis. The chapter concludes with a discussion of a new paradigm of bacterial pathogen evolution that involves the loss of gene function as an adaptation to colonization of the host. The concept of pathoadaptation is introduced and expanded to include selection for gene loss as the pathogen improves its fitness within the host niche.

INTRODUCTION

Mycobacterium tuberculosis, the cause of tuberculosis, has infected an estimated one-third of the world's human population and causes more deaths per year than any other single bacterial pathogen (Corbett et al. 2003). Although tuberculosis is most frequently an infection of the lungs, it can affect virtually any organ of the body (Raviglione & O'Brien 2004). In most individuals the infection remains latent without symptoms or transmission, but in approximately 10% the infection progresses to active disease and kills at least half of these. Untreated, active disease provides the opportunity for transmission of M. tuberculosis to other individuals through coughing up of the bacteria by an infected person, which provides droplet nuclei that are inhaled into the lung alveoli and establish a new infection. Tuberculosis is most common in developing countries; because T-lymphocyte-mediated cellular immunity is essential for control of the infection, the ongoing epidemic of HIV infection in regions with a high prevalence of tuberculosis is worsening an already severe problem. Moreover, the development of multiple drug resistance in M. tuberculosis has amplified the problems of treatment of tuberculosis in many parts of the world.

LIFE CYCLE OF M. TUBERCULOSIS

Although bacteria are not classically considered to have morphologically distinct stages representing phases of their life cycle as eucaryotic parasites do, it is clear that pathogenic bacteria such as M. tuberculosis adapt to distinct environmental niches by major alterations in their patterns of gene expression (Schnappinger et al. 2003).

INTRODUCTION

Lipid membranes and the individual lipids that comprise them were initially considered to solely provide eukaryotic cells with organized hydrophobic barriers used to separate cytoplasmic and extracellular environments. Additional studies demonstrated that these lipid bilayer structures also acted as boundaries for discrete intracellular structures, e.g. mitochondria, endosomes, and endoplasmic reticulum. Although this capacity to separate aqueous compartments clearly is an essential feature of normal cell structure and function, more recent studies have demonstrated that lipid components in these bilayer membranes also provide cells with substrates to produce a spectrum of intra- and extracellular messengers. Metabolism of membrane lipid components has been shown to produce bioactive lipids that participate in numerous signaling mechanisms. Many of these bioactive lipids, such as prostaglandins, leukotrienes, hydroperoxy acids, hepoxilins, lipoxins, and thromboxanes, are derived from the metabolic processing of arachidonic acid.

Arachadonic acid, a 20-carbon fatty acid that contains four carbon–carbon double bonds, is the precursor substrate used for the production of a large family of bioactive lipids known as eicosanoids (Fitzpatrick and Soberman, 2001; Lieb, 2001) (Figure 10.1). By itself, arachidonic acid can act as a second messenger by its ability to interact with GTP-binding proteins (Abramson et al.., 1991), inhibit GTPase-activating protein regulated by RAS (Ras-GAP) function (Han et al., 1991), cause the release of Ca2+ ions stored in the sarcoplasmic reticulum (Dettbarn and Palade, 1993), and modulate protein kinase C (PKC) activity (Khan et al., 1995).