ABC transporter. One of a family of transporter systems with common molecular architecture. The hydrolysis of ATP drives the import/export of substrates.

Accessory cells. Immune cells that form part of immune defences but are not antigen specific. Examples include mast cells, natural killer cells and phagocytes.

Acidophile. An organism that prefers an acidic environment for growth.

Aciduric. An organism that can survive exposure to low pH values.

Acquired immunity. As opposed to innate immunity. The immunity generated only after an individual has been exposed to a pathogen (or specific immunogen).

Actin filaments. Key components of the cytoskeleton of eukaryotic cells.

Acute disease (infection). A disease (infection) that lasts only a short period of time.

Acute phase proteins. A group of proteins including C-reactive protein (CRP) and serum amyloid protein (SAP) that have opsonic and other actions against bacteria and protozoans.

Acute phase response. Part of the innate defence mechanism against pathogens. Refers largely to the induction of a group of proteins that can increase in concentration, in the blood, by up to three log orders.

Acylation. The attachment of a lipid molecule to a protein.

Adapter proteins. In cell signalling – proteins that act to connect one cell signalling protein with another, thus allowing passage of the signal.

Adenocarcinoma. Malignant growth of endocrine (glandular) tissue.

Adenylyl cyclase. Enzyme responsible for synthesis of cyclic AMP (c-AMP).

Adhesin. The particular molecule (or part of the molecule) on a bacterium (usually on one of its organelles) that is responsible for mediating adhesion of the organism to a host cell, to another bacterium or to an inanimate surface.

Aims

The principal aims of this chapter are:

• to describe the various molecular approaches that have been developed (in both bacteria and eukaryotic cells) to identify bacterial virulence factors

• to highlight the advantages and limitations of each method

• to provide examples of the application of molecular techniques to answer questions about pathogen behaviour during infection

Introduction

As described in the Preface to this book, there has been an alarming rise in the incidence of antibiotic resistance amongst bacterial pathogens during the last few years. The boast of the Surgeon General, in his address to the USA Congress in 1969, that ‘we can close the book on infectious diseases’ sounds very hollow now as we witness the resurgence of diseases caused by pathogens such as Mycobacterium tuberculosis and Streptococcus pyogenes. We also face a myriad new infectious agents that are emerging as the world shrinks, through international travel and migration, and as population demographics change. Understanding how bacteria cause disease is, consequently, high on the agenda once again.

Some fundamental aspects of bacterial virulence have been investigated in great detail. These include bacterial adhesion, invasion and the production of exotoxins, and these topics are covered in later chapters in this book. However, the results arising from the use of new molecular techniques, described in this chapter, are forcing us to broaden our idea of what we mean by the term ‘virulence factor’.

Aims

The principal aims of this chapter are to describe:

• the concept of bacterial evasion of host defences

• the means by which bacteria overcome mucosal defence mechanisms

• how bacteria counteract cytokine-mediated host defences

• the mechanisms used by bacteria to evade the innate and acquired immune systems

• how bacteria use apoptosis and control the host cell cycle in order to evade host defences

Introduction

Chapters 5 and 6 have introduced the reader to the overlapping systems of cells and molecules that mammals have evolved to defend against bacterial infection. Our protective systems of immunity and inflammation involve a range of cells (macrophages, dendritic cells, T lymphocytes, endothelial cells, etc.), cellular receptors (CD14, Toll-like receptors (TLRs), T cell receptors (TCRs), etc.), humoral effector molecules (antibacterial peptides, complement components, acute phase proteins, antibodies, etc.) and a growing array of integrating signals (cytokines and lipid mediators such as prostaglandins and lipoxins). As we learn more about the bacteria that share their lives with us, we appreciate just how many counter-measures they have evolved to help defeat our myriad systems of immunity (Figure 10.1). However, it is important to realise that the evolution of counter-measures is a two-way process, in other words it is a co-evolutionary process, which results ultimately in the survival of both the pathogen and the host.

This chapter will begin by considering the mechanisms of microbial evasion of host immunity at mucosal surfaces, and will then turn to how viruses and bacteria interfere with the integrating signals of immunity – the cytokines.

Aims

To introduce the reader to:

• the concept of antibacterial defence

• the mucosal surfaces of the body

• the role of epithelial cells in antibacterial defence

• antibacterial biomolecules including antibacterial peptides

• the concept of the normal microflora as a defence mechanism

• the mucosal epithelium as an antibacterial watchdog for the whole body

Introduction

The planet we live on is alive with unimaginable numbers, and diverse types, of bacteria. It is only possible to give the roughest estimate of how many such species may exist and the figures that are derived are in the range of millions. As described in Chapter 1, our own bodies are not, as we imagine, single organisms, but are colonies containing between 1000 and 3000 (or more) different species of bacteria. Indeed, in the average human body there are approximately 10 bacteria for each human cell. In addition to bacteria, we are also host to a range of viruses, fungi, protozoa and multicellular creatures such as the mites that live on our skin and hair.

Our general view of bacteria as disease-causing organisms is incorrect. This is obvious, given that we normally share our bodies with huge numbers of diverse bacteria. What prevents these astronomical numbers of bacteria from causing disease is not understood. However, it is likely to involve the very complex systems of immunity that mammals have co-evolved with bacteria to enable both types of organism to live in harmony.

Aims

The principle aims of this chapter are:

• to provide a basic understanding of those aspects of bacterial structure and function that are particularly relevant to disease pathogenesis

• to describe the various mechanisms used by bacteria to export virulence factors and other proteins

• to provide an introduction to bacterial genetics, with emphasis on virulence mechanisms and the evolution of pathogens

• to introduce the concept of bacterial biofilms

Introduction

In the succeeding chapters we will, time and time again, refer to the means by which bacteria damage their host, interfere with host cell functions and avoid the defence systems mounted by the host. All of these features of the lifestyle of a pathogenic bacterium are attributable either to the molecules of which the organism is composed (mainly those located on its surface) or to those molecules that it secretes into its environment. A knowledge of bacterial ultrastructure and the means by which bacteria export molecules is, therefore, central to understanding bacterial virulence mechanisms. However, this is only part of the story. New developments in molecular and genetic techniques are enabling us to make rapid strides in determining how bacteria have acquired those genes that encode virulence factors as well as how these genes are regulated. Such information not only contributes to our knowledge and understanding of these fascinating organisms but also could form the basis of new approaches to controlling bacterial diseases.

Aims

The principal aims of this chapter are:

• to explain what is meant by the term ‘invasion’

• to describe the various mechanisms underlying bacterial invasion of host cells

• to emphasise that invasion involves bacterial manipulation of the signalling pathways and cytoskeleton of the host cell

• to provide examples of invasive organisms and the diseases associated with them

• to indicate the consequences of invasion for both the bacterium and the host cell

• to describe the different ways in which bacteria can survive within the host cell

Introduction

We have seen in Chapters 1 and 7 that the ability of a bacterium to adhere to a body surface (the mucosa or skin) or structure (e.g. a tooth) is an attribute of many organisms and is not limited to those species that induce disease. Hence, colonisation of epithelial surfaces by members of the normal microflora is, ultimately, of benefit to the host as it can protect against the adhesion of pathogenic species. The adhesion of such benign organisms rarely results in a pathological process, unless the host defences are defective in some way, for example in an immunocompromised individual. However, for some organisms, adhesion is only the first step in a pathological process that involves invasion of the host. Invasion of the epithelium may result in penetration of the organism to only the superficial layers of tissue, as in the case of cholera and gonorrhoea or it may, ultimately, involve deeper penetration, resulting in dissemination of the organism throughout the body.

Aims

The principal aims of this chapter are:

• to describe the various categories of bacterial exotoxins and their cellular effects

• to provide examples of toxins from each category and their role in pathogenesis

• to emphasise that exotoxins may have subtle effects at low concentrations that do not cause target cell death

• to describe the role that exotoxins have played in the dissection of eukaryotic signalling pathways

• to highlight some therapeutic uses of exotoxins

Introduction

Bacterial exotoxins are a diverse collection of proteins responsible for many of the symptoms caused by pathogens during infection. Exotoxins are actively exported by bacteria or else are released on cell lysis. Some toxins can kill cells outright by a number of different means. Others interfere with fundamental cellular activities to the ultimate benefit of the bacterium.

A number of bacteria produce a barrage of toxic activities, some of which may appear at first glance to be ‘blunt instruments’. These include toxins that degrade host cell membrane components, and the channel-forming toxins that punch holes in target cell membranes. Closer inspection of some of these toxins, however, reveals an underlying degree of sophistication in function that may result, for example, in interference with host immune defence mechanisms. Other toxins are perhaps more analogous to the stiletto dagger, being exquisitely specific in their target selection. This is perhaps best exemplified by the clostridial neurotoxins.

Aims

The main aims of this chapter are to introduce the reader to:

• the concept of signalling in biology

• the role of intracellular signalling in normal eukaiyotic cell control

• eukaryotic intracellular signalling mechanisms

• cell–cell signalling in eukaiyotes

• the role of cytokines as cell–cell signalling controllers

• prokaryotic intracellular signalling

• prokaryotic cell–cell signalling

• prokaryote–eukaryote cell signalling

• the concept that bacteria manipulate eukaryotic signalling mechanisms to induce pathology

Introduction

Communication – the transfer of information – is part of our everyday lives and we are all participants in a rapidly expanding planet-wide communication experiment. It is also obvious that animals communicate – by using smell, sound and visual cues. The dawn chorus is a good example of animals signalling to each other. However, it may be less apparent that the maintenance of a multicellular organism is absolutely dependent on the correct transmission and reception of signals. To maintain the mammalian body in a healthy state requires an enormous number of signals. There are three major signalling systems in multicellular organisms and these are classified as neural, endocrine and cytokine. These individual systems also interact with each other in ways that are only now becoming apparent.

One way of looking at disease is as a disturbance in the signalling that generates the dynamically stable state known as homeostasis. This term defines the optimal conditions of the multitude of body systems required for normal functioning. Disturbances of endocrine hormone secretion, for example, are well-known causes of disease.

It is unlikely that even Steven Spielberg could do justice to the history of Bacteriology, with its fascinating story of ignorance, blinding enlightenment, industrial success and an ultimate denouement that the reader is living through. This story is populated with some of the most fascinating characters in science such as Antonie van Leeuwenhoek, Louis Pasteur, Robert Koch, Élie Metchnikoff, Paul Ehrlich, Alexander Fleming and Selman Waksman, to name but a few.

With some 5000 years of recorded human history, it is stunning to realise that it was only in the 17th century that we saw bacteria for the first time, through the ‘magic’ of Antonie van Leeuwenhoek's single lens microscopes. It was only 150 years ago that Pasteur firmly established the concept of the microorganism. In the 30 years spanning the last two decades of the 19th century and the first decade of the 20th century, in what is the proverbial ‘twinkling of an eye’, the major human bacterial pathogens were identified. No sooner had they been identified than vaccination for bacterial diseases (e.g. anthrax) was developed. Ehrlich developed the first antibacterial, Salvarsan, in 1910 and penicillin was discovered by Fleming in 1929, although introduced into clinical practice only in the 1940s. In the 1940s and 50s, Selman Waksman and others developed new antibiotics such as streptomycin from soil organisms. However, antibiotics proved to be the death-knell of bacteriological research and by 1969 the American Surgeon General felt able to say ‘we can close the book on infectious diseases’.

Aims

To introduce the reader to:

• the concept of immunity against bacterial infections

• the cell populations involved

• the soluble mediators involved

• the organisation of the immune system

• innate immunity and recognition of pathogens

• mechanisms of phagocytosis and bacterial killing: linking innate and acquired immunity

• acquired immunity, generation of diversity and antigen presentation

• how these systems work together to kill bacteria

Introduction

Many of our defence mechanisms against infection function at mucosal surfaces, are continually active and are generally invisible to us. These various defence systems have been described in Chapter 5 and the linkage between epithelial ‘immunity’ and what would classically be called immunity has been made. This chapter will consider the antibacterial defence mechanisms that can be thought of as being ‘post-mucosal’ and will describe the systems of cells and mediators that are normally thought of when the term ‘immunity’ is used. The reader has to understand that, for the purposes of teaching, the various systems that defend us against bacterial infection are parcelled up into distinct categories (‘innate’ versus ‘acquired’, or ‘humoral’ versus ‘cellular’). However, in reality, they work as one seamless mechanism. Unfortunately, we do not yet understand all the links that exist between these systems and the challenge is to identify the connections.

Before entering the fascinating realm of antibacterial immunity, the reader must be warned that modern immunology, which has developed largely since the 1960s, was created at a period when the world thought itself safe from bacterial infections.

Aims

The principal aims of this chapter are to describe:

• the molecular basis of bacterial adhesion

• the range of adhesins produced by bacteria

• the types of bacterial structures involved in adhesion

• the nature of the host receptors for bacterial adhesins

• tissue tropism

• the consequences of adhesion for the bacterium and for the host cell

• the possibility of anti-adhesion approaches as prophylactic or therapeutic measures for bacterial infections

Introduction

The ability of a bacterium to adhere to its host is an often-overlooked aspect of virulence and in this chapter we will consider this crucial first stage in the induction of an infectious disease. It is difficult to understand why adhesion should be given such short shrift by many microbiologists as it is an essential preliminary to any interaction (including one of the most intimate interactions possible, disease) between a bacterium and its host. We would also like to stress that adhesion should not be regarded simply as a somewhat random, serendipitous ‘sticking’ of a bacterium to its host. Rather, the adhesion process is accomplished by highly specific molecular interactions and is accompanied by changes in the phenotype of the bacterium and, when the substratum is cellular, changes in the behaviour of the host cell.

Before we go any further, it is important to consider how we should go about investigating the interactions between bacteria and host cells. Practical bacteriology is traditionally based on the use of a ‘pure culture’ (axenic) of an organism.

The observation that a poor IFN response in clinical trials correlated with a high degree of variability of quasispecies (Kanazawa et al., 1994; Chayama et al., 1995; Koizumi et al., 1995) (Ch. 9) implies that mutations in one or more HCV gene products may alter the sensitivity of the virus to IFN. Further work revealed the presence of an IFN sensitivity-determining region (ISDR) within the carboxy-terminal region of the NS5A polypeptide that spans amino acid residues 2209–2248 (Enomoto et al., 1995, 1996), which may be selected for during the course of IFN therapy (Enomoto et al., 1994). Patients that failed to respond to IFN therapy had one sequence within this region, while patients that were IFN responders had a ISDR sequence that differed in 4–11 amino acid residues. Among HCV sequences reported in the literature, the NS5A sequence associated with IFN resistance was identical to that of subtype 1b (Kato et al., 1990), implying that subtype 1b was associated with IFN resistance. The existence of an ISDR was confirmed by independent studies in Japan (Chayama et al., 1997; Kurosaki et al., 1997), but the correlation broke down when similarly designed studies were reported from other countries (Hofgartner et al., 1997; Squadrito et al., 1997).

The replication of HCV in various lymphoid and hepatocellular cell lines suggests that these cell types are permissive for HCV replication. Lymphotrophism has been further documented by the finding of HCV in PMBCs (Meller et al., 1993; Gunji et al., 1994; Cribier et al., 1995; Lerat et al., 1996) and bone marrow (Gabrielli et al., 1994). HCV has also been found at higher titer in lymph nodes than in sera from individual infected patients (Sugiyama et al., 1997), although it is not known whether this reflects replication and/or immune-based accumulation of HCV in vivo. The transmission of NANBH to a chimpanzee using PMBCs from a patient with NANBH implies that either HCV is transmitted passively upon transfer of the lymphoid cells or that these cells actually support replication (Hellings et al., 1985). The high frequency of HCV markers in selected lympho-proliferative disorders (Strassburg et al., 1996; Zignego & Brechot, 1999), in EMC (Gabrielli et al., 1994), and in nonHodgkin's lymphoma (Luppi et al., 1996) is also consistent with an etiologic role for HCV in these lymphoid diseases, although these correlative studies do not establish cause and effect. HCV replication has also been reported in primary biliary epithelial cells isolated from infected patients and in a gall bladder epithelial cell line (Ahmed et al., 1995; Loriot et al., 1999), although at the present time there is no known association between the infection of this cell type and biliary disease.

The lack of tissue culture systems that consistently support HCV replication (Ch. 11) has prompted the development of other assays with individual virus gene products that may be converted into high throughput screens for drug screening and discovery. A powerful tool in the design of such assays has been the development of cell lines and cell-free systems that express one or more HCV proteins (Table 11.1). For example, the recent development of HCV subgenomic replicons in Huh7 cells provides opportunities to test antiviral compounds against the virus polymerase, which is responsible for the replication of the subgenomic plasmids in this system (Lohmann et al., 1999; Blight et al., 2000). In addition, the development of cell lines or chimeric viruses with replication dependent upon HCV NS3 expression (Hirowatari, Hijikata & Shimotohno, 1995; Hahm et al., 1996; Song et al., 1996; Filocamo, Pacini & Migliaccio, 1997) will allow screening against the NS3 protease and helicase. Another important contribution has been the publication of the crystal structure for several HCV proteins. This information will be central to the rational design of inhibitors based upon the three-dimensional active sites of the corresponding virus proteins. For example, the recent crystallographic structure of the NS3 helicase domain (Yao & Weber, 1998; Kwong, Kim & Lin, 2000) (Sections 2.4 and 2.5), which recognizes and unwinds double-stranded RNA in an NTP-dependent reaction during virus replication, has revealed amino acid residues that could be sites for inhibitor design and interaction.