INTRODUCTION

As an interface with the outside world, epithelial cells play an important role in host defense against the vast number of microbes within our midst. A central feature in such microbial–epithelial cell interactions is the use of host pattern-recognition receptors (PRR) to recognize microbes, thus enabling an appropriate response. This chapter reviews the PRRs that mediate epithelial responses, considers the mechanisms by which they function, and discusses the role of these receptors in host defense and homeostasis. The chapter is based primarily on the intestinal mucosa in light of the authors' knowledge in this area, but many of the concepts should be applicable to other mucosal surfaces. Due to the rapid pace of advancement in this area of research, PRRs are often described and partially characterized well ahead of full determinations of the tissues and cells that actually express these PRRs; thus, this chapter proceeds with a general review of PRRs and then discusses direct and indirect PRR activation of epithelial cells and the role of these activation events in host defense.

PATTERN-RECOGNITION RECEPTORS: MEDIATORS OF HOST–BACTERIAL INTERACTIONS

The immune system traditionally has been divided into innate and adaptive components, which are differentiated functionally on the basis of whether their antimicrobial action requires previous exposure to the microbe or its components. Innate immunity has long been thought to rely on some germline-encoded mechanisms to recognize foreign products.

INTRODUCTION

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide, and commonly colonizes the upper respiratory tract. In some colonized individuals the organism translocates to other tissues and causes life-threatening diseases including pneumonia, bacteremia, and meningitis. Rates of disease are especially high in the very young and old and in patients with predisposing conditions including HIV infection, cardiopulmonary or co-morbidities, renal diseases including nephrotic syndrome, and sickle cell disease and other causes of hyposplenia.

Approximately 5 million children under the age of 5 years die with a respiratory tract infection every year; the major causative pathogen in these cases is S. pneumoniae (Williams et al. 2002). Pneumococcal infection caused approximately 45,000 deaths in adults in the United States in 1998, about two years prior to the introduction of the 7-valent pneumococcal conjugate vaccine for infants (Robinson et al. 2001). Streptococcus pneumoniae also causes less serious but extremely common diseases such as otitis media, sinusitis and exacerbations of chronic obstructive pulmonary disease. It has been estimated that there are 7 million cases of otitis media in the United States every year (Stool & Field 1989).

OVERVIEW OF PATHOGENESIS

Nasopharyngeal carriage

The first step in the pathogenesis of pneumococcal disease is nasopharyngeal colonization, with individuals often carrying more than one serotype at a time. Asymptomatic nasopharyngeal carriage is established when surface components of the pneumococcus bind to nasal epithelium (Tuomanen & Masure 1997; Weiser et al., 1996).

INTRODUCTION

The quintessential event that initiates an infection is defined by the pathogen encountering a targeted tissue of a host. Mucosal surfaces often serve as an entry point for pathogens, and the pathogens frequently access epithelial cells at these sites with the aid of flagellar-mediated motility. Flagella are common to a diversity of pathogenic bacteria, and their structure is well conserved (Harshey and Toguchi, 1996). The number of flagella produced by a bacterium is tightly regulated, ranging from one up to several dozen per cell depending on the species (Aldridge and Hughes, 2002). Likewise, these organelles may be localized to a single pole or both poles of a cell or may be in a peritrichous arrangement. In some cases, flagellar number and cellular position undergo a regulated change in response to specific environmental conditions. Directional control of motility, either towards a favorable situation or away from a stressful circumstance, is achieved by a chemosensory phosphorelay system, which integrates environmental signals and adjusts the frequency of motor reversals that cause reorientation of bacterial movement (Sournik, 2004; Wadhams and Armitage, 2004). The cumulative affect invoked by the chemosensory system in response to chemical, physical, and physiological cues offers the bacterium a behavioral activity to fine tune its progression through an environment and consequently enhances the ability of many bacteria to infect their hosts.

INTRODUCTION

The intracellular pathogen Listeria monocytogenes exploits a number of normal host cell functions to survive and spread; its intracellular lifestyle explains many of the unique clinical characteristics of this deadly food-borne pathogen (Southwick & Purich 1996). Listeria is able to grow on refrigerated foods and also multiplies readily at room temperature. Humans with defects in cell-mediated immunity who ingest foods stored for prolonged periods in the refrigerator are at risk of contracting Listeria. When ingested in high numbers, this bacterium can quietly enter through the gastrointestinal tract, seed the bloodstream, and subsequently invade the meninges, causing serious and often fatal bacterial meningitis. Based on epidemiologic studies, defects in humoral immunity are not associated with an increased risk of contracting Listeria; however, defects in cell-mediated immunity (particularly in patients with CD4 counts below 200) confer an increased predisposition for listeriosis. Pregnant women, neonates, patients receiving corticosteroids and other immunosuppressants to prevent the rejection of organ transplants or to treat connective tissue disease, and patients with AIDS, are all at increased risk of developing Listeria infection (Lorber 1997). Because Listeria can grow within the cytoplasm of host cells and spread from cell to cell without ever coming in contact with the extracellular milieu, this pathogen is able to avoid antibodies as well as extracellular antibiotics, and can only be killed by cell-mediated immune mechanisms.

INTRODUCTION

Colonizing host epithelia represents a formidable challenge to bacterial pathogens. To a large extent, epithelial surfaces are designed to shield the multicellular organism from the environment and to protect the body interior from potentially harmful microbes. Where epithelial surfaces permit exchange of molecular components with the external world, a multitude of innate and acquired host defence mechanisms keep microorganisms in check. In addition, invading pathogens either have to compete successfully with resident commensal bacteria for space and nutrients or have to reach and establish themselves in otherwise sterile parts of the body. As we will discuss in this chapter, the specific interaction with host surface components and the tight adhesion to epithelial cells form one of the common ways in which bacterial pathogens have evolved to successfully accomplish the colonization of their respective host organism. It is important to emphasize that in many cases, this initial host–microbe encounter at the epithelial barrier is not only a critical determinant of pathogen–host specificity and range but also, to a large extent, a decisive point for the infection process as a whole.

Adhesion of pathogenic bacteria to host cells had been observed at the beginning of the twentieth century in the early days of investigations into the microbiological origin of infectious diseases (Guyot, 1908). However, the concept that bacterial adhesion to host surfaces often represents an essential step in the development of infection matured only several decades later (Beachey, 1981; Duguid, 1959; Eden et al., 1976; McNeish et al. 1975; Punsalang and Sawyer, 1973).

INTRODUCTION

Urinary tract infections (UTI) are among the most common human bacterial infections. The prevalent pathogens are the uropathogenic Escherichia coli (UPEC) strains. A great deal of information concerning the genetic, virulence, and innate immune host responses against those bacteria have been obtained. Furthermore, the knowledge of uroepithelium cell biology and physiology, in particular at the level of the urinary bladder, has made considerable progress since 1995, improving our understanding of the strategies used by UPEC to colonize and invade this tissue.

UTIs account for significant morbidity and high medical costs. In 1997, the National Ambulatory Medical Care Survey and the National Hospital Medical Care Survey Report estimated that UTIs in the USA result in nearly seven million doctor visits per year, excluding visits to hospital emergency departments. The overall costs associated with UTIs have been estimated to reach upwards of two billion US dollars a year (Foxman, 2002). Moreover, nosocomial acquired urinary tract infections (NAUTIs) account for up to 40% of all hospital-acquired infections in European countries and represent the most frequent nosocomial infection (Eriksen et al., 2004; Johansen, 2004; Zotti et al., 2004). NAUTIs, which are frequently associated with medical procedures (the most important risk factor being an indwelling catheter), may result in significant acute morbidity, medical complications, and legal issues (Johansen, 2004). Moreover, one of the main concerns with UTIs is that they are frequently sources of recurrent infections.

The severity of UTIs depends on the spread of the infection.

Applications of high-value proteins

Proteins used in industrial enzyme technology, e.g. detergent proteinases or enzymes applied in the food industry, are in most cases rather crude preparations and usually mixtures of different enzymes. In contrast, there are a number of commercial applications where highly purified (and therefore high-value) proteins are needed. Examples are:

• Analytical enzymes and antibodies: Used in medical diagnostics, food analysis, as well as biochemical and molecular biological analysis (see Section 21.2).

• Enzymes used as tools in genetic engineering technology: Gene technology has become possible through the availability of highly purified restriction endonucleases, DNA and RNA polymerases, nucleases and modifying enzymes (see Chapters 4 and 5). Similarly, glycohydrolases and glycosyl transferases are used increasingly in glycobiotechnology in order to modify the sugar residues of glycoproteins.

• Therapeutic proteins: Growth factors, antibodies and enzymes are used as the active drug ingredients for the treatment of diseases (see Section 21.3).

Furthermore, proteins with proven or supposed biological relevance in pathomechanisms are needed as targets for the search of new ligands (agonists or antagonists) or inhibitors and for X-ray or nuclear magnetic resonance (NMR) structural analysis in order to design novel interacting compounds by structure-based molecular modelling. This requires the production of these proteins on a relatively small scale (10 to 100 mg), but often with high purity depending on the intended use.

Analytical enzymes

Enzymes are highly specific both in the reaction catalysed as well as in their choice of substrates.

Introduction

The biological production of active compounds, ranging from small molecules, such as organic acids, vitamins or antibiotics, through to macromolecules, such as therapeutic proteins or plasmid gene therapy vectors, is of great commercial and social value. The cornerstone of any such bioprocess is the cell cultivation step where a highly selected, and usually engineered, cell-line is grown under carefully controlled conditions. The term cell-line is used here to represent both microbial and mammalian cells. The aim of the cultivation step is to yield the product in as an efficient and cost-effective manner as possible. However, the design and implementation of a cell cultivation process is often a complex, lengthy and costly task. The development of a cell cultivation process typically involves four stages, as shown in Fig. 12.1. Stage 1 involves the initial identification of a native, or wild-type, cell-line that produces the compound of interest, though usually slowly and at low levels. This is followed by Stage 2 in which the productivity of the chosen cell-line [g product (g cells)−1 h−1] is enhanced using a variety of microbiological and molecular biology techniques. Stage 3 involves optimisation of the growth media composition and culture conditions; while in Stage 4, scale-up of the process from laboratory, through pilot plant to manufacturing scale occurs.

High-throughput experimentation

Traditionally cell culture development has been carried out following a sequential series of experiments, using conventional apparatus with a high labour requirement. Conventional experimentation usually involves carrying out one or only a few experiments at a time.

Introduction

Quantitative description of cellular processes is an indispensable tool in the design of fermentation processes. Thus, the two most important quantitative design parameters, yield and productivity, are quantitative measures that specify how the cells convert the substrates to the product. The yield specifies the amount of product obtained from the substrate (or raw material) and the productivity specifies the rate of product formation. These two design parameters can easily be derived from experimental data, e.g. from measurement of the substrate consumption and the product formation. However, what is more difficult is to predict how they change with the operating conditions, e.g. if the medium composition changes or the temperature changes. In order to do this it is necessary to set up a mathematical model (see Box 6.1), which may be anything from a simple empirical correlation that specifies the product formation rate as a function of the medium composition, to a complex model that accounts for all the major cellular reactions involved in the conversion of the substrates to the product. Independent of the model structure, the process of defining a quantitative description of a fermentation process involves a number of steps (Fig. 6.1).

A key aspect in setting up a model is to specify the model's complexity. This depends much on the aim of the work, i.e. what the model is going to be used for, as discussed in Section 6.2.1.

Introduction

Biotransformation deals with the use of biological catalysts to convert a substrate into a product in a limited number of enzymatic steps. The establishment of an efficient biotransformation process requires the extensive examination of factors affecting the development of optimal biocatalysts, reaction media and bioreactors (Fig. 24.1).

The chemical industry currently takes advantage of enzyme technology in various sectors, namely in the food, pharmaceutical and detergents sectors. An on-going trend towards the implementation of commercial processes based on the use of biocatalysts in other areas (e.g. polymers, fine and agricultural chemicals, and miscellaneous chemicals) is noticeable. It is foreseeable that in the near future the use of biocatalysts in these fields will be enhanced which, coupled to the already established bioprocesses, will further broaden the overall impact of biocatalysis in the chemical industry. When compared to equivalent chemical processes, bioprocesses are simpler, less demanding in raw materials and in energy, lead to higher quality products (i.e. with fewer impurities) and produce higher yields, lower the involvement of toxic wastes and decrease the emission of wastewater. Such features decrease production costs and, since bioprocesses easily comply with the stringent environmental legislation in highly regulated countries, these processes are given a competitive edge when matched up to conventional (chemical) methods. In many cases, near-term benefits will increase as the market penetration of the product and process continues to grow, leading to further cost reduction and performance improvements relative to competing products and processes.

Introduction

Biotechnology is the exploitation of biological processes for industrial or other purposes. In this chapter we will discuss the biotechnology industry's development from its initial manifestation – the biotech start-up company through its maturation and development to yield integrated companies contributing products on a global scale – and what factors contribute to the success and failure of the entrepreneurial application of the science described elsewhere in this volume. This industry appeared in the 1970s, initially in the USA (an opportunity created by intellectual resource and inherent entrepreneurial spirit allied to a powerful capitalist environment) and soon afterwards in Europe and Asia as well. Our aim in this chapter is to give you a feel for what you need to have, and what you need to do, if you are to start up and successfully run a biotechnology company.

What is biotechnology used for?

As an introduction, we will review what companies are using biotechnology for, to see why some areas have been successful and others have failed.

The applications: medicine

The significant proportion of biotechnology investment has been directed to health-care, and specifically in the discovery of new drugs. Patents provide legal protection and mean that an effective new drug can be sold at high price for as long as the patent on it prevents someone else from making and selling it at a lower price. Once a drug comes off patent it can be manufactured as a generic, and sales and profit margins for the originator rapidly plummet.

Glossary

Adjuvants Substances that when mixed with an antigen will make them more immunogenic, i.e. they enhance the immune response. Adjuvants cause inflammation and irritation and help to activate cells of the immune system.

Affinity The measured binding constant of an antibody for its antigen at equilibrium.

Alloimmunisation Immunisation of an animal with cells or tissues derived from another animal of the same species where there are allelic differences in their genes.

Antibody Adaptive proteins in the plasma of an immune individual with binding specificity for antigens (cf. immunoglobulin).

Antigen A molecule, or complex of molecules, that is recognised by an antibody (immunoglobulin) by binding to the antibody's variable or V-regions.

Antigen-presenting cells (APCs) An antigen-presenting cell is a specialised cell (dendritic cells and macrophages) that can ingest, degrade and then present on its cell surface, fragments of pathogens and other antigens, to other cells of the immune system (e.g. B-cells and T-cells).

Autoimmune Immunity to molecules (antigens) within an animal's own body that can lead to a disease, e.g. rheumatoid arthritis, or some forms of diabetes.

Avidity Antibodies frequently interact with antigen using multiple antigen-binding sites and thus they have a functional affinity termed avidity which is a complex function of the individual binding affinities.

B-cells A sub-set of white cells (lymphocytes) in the blood which produce antibodies.

Introduction

Bacteria and yeasts have been the organisms of choice for the industrial production of heterologous recombinant proteins for many years. The ability to cultivate bacterial strains to high cell density at large scale has become an increasingly important technique throughout the field of biotechnology, from basic research programmes (structural or kinetic studies) to large-scale pharmaceutical production processes. Escherichia coli remains one of the most attractive organisms for the production of recombinant proteins (see Chapters 4, 5, and 21 for examples) where no complex post-translational modifications (e.g. glycosylation or disulphide bond formation) are required for biological activity and because its genetics and physiology are well understood. However, there are important drawbacks associated with the use of prokaryotic organisms. The low percentage of GC nucleotides in their genomes, when compared to mammalian genes, and the existence of rare codons that often result in low expression levels or inactive truncated forms and, in many cases, proteins are expressed as insoluble inclusion bodies in the bacterial periplasmic space. Bacteria are also incapable of carrying out any post-translational modifications, which strongly influences protein stability, folding, solubility and, hence, its biological activity. Yeasts (e.g. Saccaromyces cerevisiae or Pichia pastoris), though, can perform some post-translational modifications similar to those of the more complex eukaryotic cells (see Chapter 5). However N-glycosylation of mammalian proteins in yeast seems to be very inefficient. Additionally, both bacteria and yeast cells are surrounded by a mechanically strong cell wall that may hinder recovery of any non-secreted proteins.

Introduction

The First Law of Biology (if there was one) could be: the purpose of a microorganism is to make another microorganism.

In some cases biotechnologists, who seek to exploit the microorganism, may wish this to happen as frequently and as quickly as possible; in other words they wish to have as many microorganisms available at the end of the process as possible. In other cases, where the product is not the organism itself, the biotechnologist must manipulate it in such a way that the primary goal of the microbe is diverted. As the microorganism then strives to overcome these restraints on its reproductive capacity, it produces the product which the biotechnologist desires. The growth of the organism and its various products are therefore intimately linked by virtue of its metabolism.

In writing this chapter, I have not attempted to explain the structure of the main microbial cells: the bacteria, the yeasts, the fungi and the microalgae. These are available in most biology textbooks and these should be consulted if there are uncertainties about cell structures. However, biology textbooks rarely explain the chemistry that goes on in the living cell (i.e. their biochemistry) in simple terms but, as the biochemistry of the cell is fundamental to the exploitation of the organism, it is important to be acquainted with the basic systems that microbial cells use to achieve their multiplication.

The biochemistry of the cell is therefore described as an account of the chemical changes that occur within a cell as it grows and multiplies to become two cells.