Colonization of Mucosal Surfaces

The mucosal surfaces of the body are the areas where important absorptive and excretive functions occur. The innate defense system consists of three components: mechanical, chemical, and cellular. The first defense that an invading pathogen would encounter is the preepithelial barrier. Mucins form two lines of preepithelial defense, such that the secreted gel overlies the mucins forming part of the glycocalyx on the apical surface of the epithelial cells. The most important chemokine released from the epithelial cells is interleukin-8 (IL-8), which, as well as being an effective chemoattractant for granulocytes, stimulates mucin secretion by goblet cells. Some microorganisms can utilize the mucus layer for protection. Several proteinase inhibitors are produced by epithelial cells and, where present, submucosal glands, e.g., in the airways. These inhibitors form an important part of the preepithelial defenses and the innate immune system. Secretory leukocyte proteinase inhibitor (SLPI) is one such epithelial secretion, along with elafin (SKALP), which is an elastase inhibitor with 42% sequence homology to SLPI. A key role for the preepithelial barrier is to prevent microbial adherence by interfering with microbial adhesins and toxins. Several enzymes are secreted by the epithelial cells into the external secretions. A key role for the preepithelial barrier is to prevent microbial adherence by interfering with microbial adhesins and toxins. As well as cell membrane-associated mucin, the apical surface of the epithelial cells has other molecules presented to approaching microbes.

This chapter provides an in-depth account of defensins, and ties together the roles of other antimicrobial peptides and proteins that together contribute to mucosal innate host defense. While the focus is on defensins and antimicrobial peptides from humans, peptides from other species are also included where appropriate. The structural and functional relationships between these three classes of alpha-defensins, beta-defensins, theta-defensins are discussed in this chapter. The antibacterial and antifungal activities of the resulting retrocyclin were modest and were similar to those of synthetic and native rhesus theta-defensins. The noticeable salt sensitivity of ELR- CXC chemokines was similar to that of defensins and many other antimicrobial peptides. High concentrations of alpha-defensins are frequently observed in chronically inflamed tissues. Certain factors within airway fluid, including electrolytes and anionic macromolecules, decrease the activity of many antimicrobial peptides, including defensins. Even though the processing of human neutrophil alpha-defensins occurs during neutrophil maturation prior to cellular release from the bone marrow, matrilysin may participate in the activation of beta-defensins or other antimicrobial peptides in the human airways. Defensins and other antimicrobial peptides and proteins coat mucosal surfaces and are among the primary early mediators of host defense against colonization and tissue invasion by pathogenic microbes. In vitro and in vivo studies indicate that most antimicrobial peptides probably act as endogenous antibiotics.

This chapter discusses the chief mechanisms of adaptive immunity in mucosal tissues with particular emphasis on the gut, the best understood of the mucosal immune organs. It describes the organization and main components of the mucosal immune system and the adaptive host defense mechanisms that prevent microbial infection. It also gives schematic representation of the organizational structure of the gut-associated lymphoid tissue (GALT) and the key elements involved in mucosal immunity. It focuses on induction of adaptive immunity at mucosal surfaces, and antibody- and B-cell-mediated immunity in mucosal tissues. Antibodies bound to surface antigens form large immune complexes that prevent colonization and invasion by microbes, facilitating their entrapment in the mucus and subsequent peristaltic or ciliary clearance. The chapter then talks about cell-mediated immunity in mucosal tissues. One of the biggest challenges that lies ahead is the unraveling of the extraordinarily complex mechanisms that underlie the generation of effective immune responses to potentially pathogenic organisms while controlling inflammatory responses to commensal organisms and food antigens. This knowledge will greatly enhance one's ability to prevent inflammatory diseases in organs lined by large mucosal surfaces. Moreover, this information will also be invaluable in designing new generations of vaccines that can be administered via mucosal surfaces, such as attenuated live vectors, which have the potential of inducing strong mucosal and systemic immune responses.

The transfer of molecular biology methods and concepts to environmental microbiology and microbial ecology has provided new insights into microbial complexity and activity in many different types of natural settings, and the rapidly expanding genomic databases have further accelerated the development. Comparative genomics, meta-genomes, techniques for in situ metabolic activity monitoring, and DNA chips for rapid identification of hundreds of species as well as for transcriptomic investigations are tools which have recently been added to those of fluorescent in situ hybridization (FISH) and reporter gene techniques. This chapter reviews the application of some of these in situ methods and tools in the context of mucosal colonization, after which one specific example—monitoring of quorum sensing-based cell-cell communication in colonized lungs of cystic fibrosis (CF) animal models. The first fluorescent protein to be useful as a tool for gene expression was the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. The last 10 years of development of new molecular tools for microscopic investigations, along with the rapid development and dispersal of advanced fluorescence microscopy methods, have resulted in greatly improved techniques for studies of microbial performance in very complex settings, including those found in connection with both commensal and pathogenic bacteria in animals and even human patients.

The majority of the homologous sequences were “housekeeping genes” that are widely distributed among both respiratory and nonrespiratory tract bacterial species. It appears that the expression of choline phosphate (ChoP) phosphorylcholine among respiratory tract organisms is an example of convergent evolution since ChoP is present on distinct bacterial structures in different species. An additional consideration is that in each of these species, ChoP is exposed on the bacterial cell surface, suggesting that it plays a role in host-bacterium interaction. The chapter focuses on phosphorylcholine expression. Exchanging the licD genes between the two strains with ChoP on different chain extensions was sufficient to switch its position. Therefore, in addition to the mechanism involving phase variation, structural rearrangements within the oligosaccharide have the potential to aid the evasion of an immune response targeting ChoP. The chapter also discusses the role of ChoP in the host-bacterium interaction and the advantages and disadvantages that expression of this host-like structure may confer on bacterial survival. A series of experiments examined whether bacteria are predominantly ChoP phase-on or phase-off during colonization and systemic infection. The efficacy of the human anti-ChoP IgG was assessed by using in vitro assays that correlate with protection against H. influenzae and S. pneumoniae infection. The surface expression of ChoP appears to be particularly common among species that colonize predominantly the upper respiratory tract. This allows for bacterial mimicry of the host cell surface since choline is a prominent feature of the host cell membrane.

Sialic acids (NeuAc) and its derivatives are found on cell membranes and in body fluids in all mammals and many higher-order animals, as well as pathogenic microorganisms. Based on an understanding of the biosynthesis of sialic acid and the evolving elucidation of the genomes of multiple microbes, researchers have described at least four mechanisms of microbial surface sialylation. These include de novo synthesis, donor scavenging, trans-sialylation and precursor scavenging. To understand the role of sialic acids in pathogenesis, it is important to examine their role in eukaryotic systems. Sialic acid-dependent receptors play an important role in adhesion to mammalian cells. Two examples of the receptors, the selectin and sialoadhesin families, are discussed in this chapter. There is a correlation between sialic acid levels and the development of cancer. A tumor cell has an increased amount of sialylation and sialyltransferase activity. The biological effects of sialylation, which mediate antiphagocytosis, anticomplement activity, and protection against bactericidal killing, have the potential to act with sialic acid binding immunoglobulinlike lectins (siglecs) on the surface of hematopoietic and immune system cells. Many bacteria produce neuraminidases, which can modify the sialylation of microbial and human tissues. Bacterial biofilms and their role in pathogenicity have generated considerable interest because of their role in antimicrobial resistance and pathogenesis. Recent studies demonstrating the potential for cooperative behavior between bacteria suggest that in complex communities, the disadvantages of surface sialylation may be obviated by neuraminidase production by a neighboring microbial partner.

This chapter reviews aspects of interaction among the members of the respiratory microflora by considering relevant laboratory models and examples of both cooperation and competition. In general, the author focuses on the anatomic area between the nasal mucosa and the epiglottis, where several bacterial species coexist and may cocolonize for extended periods. The lower airway, below the epiglottis, is a sterile environment under normal conditions. In contrast, another neighbor of the nasopharynx, the oral cavity, is home to literally hundreds of distinct species of bacteria. Specific data regarding the anatomic localization of colonizing bacteria in the nasopharynx and the relative burdens of each species are limited; however, evolving concepts of interaction among the members of the commensal flora are furthering one's understanding of carriage. Evidence from colonization rates of children transferred from other nurseries suggested that the latter was the more likely possibility, so the investigators took the striking approach of directly testing the phenomenon of bacterial interference. In addition, many of the members of the upper respiratory flora are auxotrophs. Competition for nutrients among members of mixed bacterial populations has been proposed. Artificial alteration of colonization through vaccination or antibiotic therapy may disrupt the ecological balance in this microenvironment and have unintended consequences. Further research in this area, especially if relevant in vivo models are employed, will contribute to one's evolving understanding of the ecology of the upper respiratory tract.

This chapter presents a comparative overview of several major adhesins and invasins of four human-tropic respiratory bacteria and includes examples of frequent to rare colonizers, namely, Haemophilus influenzae, Moraxella catarrhalis, Neisseria meningitidis, and Bordetella pertussis, and their extensively studied host tissue-targeting mechanisms. It considers one's current understanding of the bacterial adhesion factors that determine their tropism in the human respiratory tract, although it has to be noted that other factors important for bacterial survival also constitute determinants of host and tissue tropism. Several ligands of the organisms described in this chapter target extracellular matrix (ECM) proteins and directly or indirectly target the RGD-binding integrins. In addition, heparan sulfate proteoglycans (HSPGs) and members of Ig superfamily, especially the carcinoembryonic antigenrelated cell adhesion molecules (CEACAMs), are targeted by multiple mucosal bacteria and are specifically discussed. The adhesins that form layers above the bacterial outer membrane due to their extended morphology are categorized in this section and include polymeric as well as monomeric or small multimeric filamentous or protruding structures visible by electron microscopy (EM). Pili are generally regarded as the most important adhesins in capsulate phenotypes of N. meningitidis and are thought to determine host and tissue tropism mediating primary interactions with human epithelial and endothelial cells. Pili thus allow the bacteria to make an initial contact with a host cell surface, leading to more intimate interactions via nonfilamentous adhesins. It is therefore also credible that the primary adhesins such as pili in general determine host and tissue tropism.

The three principal causes of bacterial meningitis, Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae, produce an IgA1 protease. In addition, IgA1 proteases are produced by two urogenital pathogens, five species of commensal gram-positive cocci found in the pharynx and oral cavity, H. parahaemolyticus, and all human-associated species of the genera Capnocytophaga and Prevotella. In accordance with the substrate specificity of IgA1 proteases, humans and hominoid primates are the exclusive hosts of the bacteria that produce these enzymes. This chapter presents the hypothesis that invasive infection in occasional individuals is a result of nonsynchronized induction of the two types of antibodies by successive encounters with two different microorganisms: (i) colonization in the gut or upper respiratory tract with bacteria expressing surface epitopes similar or identical to those of the respective pathogen (e.g., E. coli K100 in the case of H. influenzae type b, and E. coli K1 or Moraxella nonliquefaciens in the case of N. meningitidis group B), and (ii) subsequent colonization with the actual pathogen. As a result of the prior colonization with a cross-reactive microorganism, the pathogen encounters preexisting IgA1 antibodies to its surface epitopes but no antibodies that will neutralize its IgA1 protease. IgA1s of humans and hominoid primates were the only known substrates of IgA1 proteases until recently. Now other permissive substrates have been revealed. Most of these are proteins of immunological relevance, but for some of the substrates, their accessibility to IgA1 protease in vivo is a matter of speculation.

This chapter focuses on three important pathogens and the role that genetic exchange has played in their continuing evolution. There are numerous examples where genetic exchange has been responsible for the evolution of antibiotic resistance and virulence determinants and wholesale acquisition of pathogenicity islands, and there is increasing evidence that recombination is important in populations of both naturally transformable and nontransformable organisms. The prevalence of genetic exchange varies among different bacteria, as does the major mechanism of exchange. If these mechanisms enable the transmission of extrachromosomal elements, plasmids transposons, or lysogenic bacteriophage, bacteria can acquire completely novel resistance or virulence determinants. For chromosomal DNA, genetic exchange is mediated primarily by homologous recombination. Genetic exchange involving the acquisition of plasmids or conjugative transposons carrying novel resistance determinants is undoubtedly the most widespread route by which bacteria have acquired resistance to antibiotics. In many countries, lack of susceptibility to tetracyclines is the most frequently observed resistance phenotype in pneumococci. It would appear that different bacterial species are genetically variable for most of these factors. For example, restriction systems which differ widely between organisms function primarily against incoming double-stranded DNA but may also play a role in recombination. Although the future for the pathogenic commensal organisms of the upper respiratory tract is uncertain, it is clear that genetic exchange will continue to play an important role in helping to shape that future.

This chapter discusses what is currently known regarding how and why a few of the most important human respiratory pathogens control gene expression in response to environmental conditions. Even less well characterized is the ability of Haemophilus influenzae to control gene expression in response to environmental conditions. Like Neisseria meningitidis and H. influenzae, Streptococcus pneumoniae expresses a polysaccharide capsule that plays a major role in pathogenesis. Understanding how these regulators coordinate to mediate precise control of capsule and other virulence factors in response to specific environmental conditions awaits further study. Comparative analyses between S. pneumoniae and Streptococcus pyogenes should facilitate understanding the structure and function of the Mga/MgrA regulons of these two streptococcal species. Roles for the other two, or for the C-terminal domain of the protein, remain to be determined, but there is presently no evidence that Mga protein activity is controlled in response to environmental conditions. Bordetella pertussis does not cause severe invasive or systemic disease, although serious complications, including pneumonia, seizures, encephalopathy, and even death, sometimes occur. Natural-host animal models were recently developed to allow the study of the entire infectious cycle in the laboratory. Comparison of mutants altered in signal transduction and/or in Bvg-mediated control of specific factors in these models is expected to allow one to gain a better understanding of the importance of precise control of gene expression in the establishment, maintenance, and transmission of Bordetella infection.

The terminal ileum and the large bowel (cecum and colon) are hospitable places for bacterial proliferation, and a complex and numerous bacterial community resides in this site. This community is often referred to as the normal gut microbiota (microflora). Many of the numerically important members of the gut microbiota have not yet been cultivated under laboratory conditions and are known and detected on the basis of their 16S rRNA gene sequences. Comparisons of the characteristics of germfree and conventional animals have clearly demonstrated that the gut microbiota has considerable influences on host biochemistry, physiology, immunology, and low-level resistance to gut infections. Cytoskeletal rearrangements within the enterocyte form a socket by which the filament becomes permanently attached to the mucosal surface. The influence of the gut microbiota of neonates on the immune system is of especial interest because of the observed increase in the incidence of allergies in children in affluent countries over recent decades. The adherence of Lactobacillus cells to, and proliferation on, epithelial surfaces in rodents, pigs, and poultry has tempted some researchers to consider that the same phenomenon occurs in the human gut. This overlooks the differences in the anatomy and histology of the human gut relative to that of other monogastric animals. In other words, the immune systems of different humans or other animals recognize different epitopes. This is apparent from experimental-animal studies because the composition of the gut microbiota of HLA-B27 rats and interleukin-10-deficient mice is different, yet colitis results in both types of animals.

This chapter surveys recent information on the roles of the commensal intestinal flora and provides an overview of how the natural symbiosis can be enhanced. The dominant microbial genera of the human gastrointestinal tract include Bacteroides, Bifidobacterium, Eubacterium, Lactobacillus, Clostridium, Fusobacterium, Peptococcus, Peptostreptococcus, Escherichia, and Veillonella. The ability of the commensal flora to persist in the intestinal lumen stands in stark contrast to the abundance and vigor of the intestinal immune system. The intestinal mucosa must maintain a highly selective barrier function, capable of permitting the absorption of highly variable nutrients and the sampling of antigens while excluding pathogenic microorganisms. Just as the presence of the commensal flora is needed to drive the maturation of the immune system, several studies have suggested that the flora is required to establish normal epithelial barrier function. The studies by Gordon and Hooper have illuminated dramatically the contributions of the commensal flora to ontogeny of the intestinal mucosa. The study of health-promoting effects conferred by administration of a live commensal flora, so called probiotic species, has a long but often confusing history. The contribution of the enteric commensal flora to human health is only beginning to be appreciated, and many more studies are required. The availability of molecular approaches will greatly accelerate laboratory investigations, but careful clinical observations are required to ascertain the full scope of these effects.

This chapter reviews the published literature concerning quorum sensing (QS) among bacteria of the gastrointestinal (GI) tract, with particular emphasis on pathogenic species that cause infection in the GI tract. Three major QS circuits have been described: one used primarily by gram-negative bacteria, one used primarily by gram-positive bacteria, and one that is universal. The gram-negative bacterial QS system involves the use of acyl homoserine lactones (AHLs) as autoinducers, which then bind to response regulators that affect gene expression. The first evidence that QS could be involved in the regulation of virulence factors of GI pathogens was found with enteropathogenic E. coli (EPEC), which causes nonbloody diarrhea primarily in infants in developing countries, and enterohemorrhagic E. coli (EHEC), which causes bloody diarrhea and hemolytic-uremic syndrome. The major virulence factors for V. cholerae are cholera toxin (CT) and the toxin-coregulated pilus (TCP), both of which are regulated as part of the ToxR regulon. The discovery of QS in human pathogens has led to considerable interest in developing new therapeutic interventions to interfere with the signaling molecules. There are at least three major strategies for the development of drugs that interrupt bacterial QS: (i) inhibition of QS signal synthesis, (ii) destruction or degradation of the signal, and (iii) inhibition of signal reception. A fundamental property of QS is that the greater the density of bacteria, the greater the density of signaling molecules and the greater the opportunity for cell-to-cell communication.

This chapter focuses on the mucus layer of the intestinal tract and its role in colonization of the intestine by enteric bacteria. The gel-like mucus layer of the intestine is dynamic in that it is continuously being renewed by secretion of stored or newly synthesized components, sloughed or eroded by mechanical forces, and degraded by the indigenous flora. In addition to the major gel-forming mucins present, the mucus layer contains a variety of proteins, carbohydrates, lipids, nucleic acids, and other components that originate from a number of sources. The presence of the intestinal mucus layer as a viscous physical barrier and the need for bacteria to reach and maintain themselves in an appropriate environmental niche has led many investigators to propose chemotaxis and motility as important aspects of colonization for the microorganisms that exhibit these properties. In the case of the normal flora of the large intestine, association with the mucosal surface appears to be a critical aspect of colonization and, depending on the organism, may or may not involve direct adhesion to epithelial cells. The lumen of the upper intestine may contain a number of microorganisms, but the rate of bacterial growth in the lumen in these regions does not does not appear to be high enough to compensate for the rapid flow of luminal contents. The types of experiments being used to elucidate the carbon nutrition for colonization of Escherichia coli can and should be extended to include other members of the intestinal microflora.

In recent years, different studies of bacterial flagella have unmasked novel features regarding their complex and sophisticated structure as well as their biological relevance beyond motility. This chapter focuses on these new structural and functional features of flagella, with emphasis on their ability to favor adherence, colonization, penetration, and translocation by bacterial pathogens and the resulting activation of innate immunity. For most bacterial pathogens, flagella and flagellum-driven motility are recognized as essential elements in their virulence scheme. Klose and Mekalanos constructed an rpoN (encoding s54)-null mutant of Vibrio cholerae and found that this strain was defective in motility, flagellation, and colonization in the infant-mouse colonization assay. In this study, they also identified three flagellar regulatory genes (flrABC), among which flrA and flrC encode σ54-activators; mutations in these two genes yielded mutants defective in colonization. Flagella purified from enterohemorrhagic Escherichia coli (EHEC) and E. coli K-12 showed similar levels of interleukin-8 (IL-8) induction as those for H6 flagella, suggesting that this is a property of flagella of some pathogenic bacteria as well as some members of the normal flora. It is possible that the conserved regions play an important role in generating an optimal conformation of the hypervariable domain within the flagellin molecule and, in turn, on the flagellum filament in order to display proinflammatory epitopes effectively. Flagellar genes are highly conserved among gram-negative bacteria, and much similarity in structure and function exists.

This chapter reviews the current understanding of the tissue tropism of bacterial pathogens, with a focus on virulent types of Escherichia coli. As surface-expressed organelles involved in bacterial attachment to the host mucosa, bacterial pili are likely to contribute to the tissue tropism of many bacterial pathogens. The enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) translocon directs the delivery of selected proteins into the internal milieu of the host cell. Loss of invasin-mediated integrin-binding activity leads to the colonization of other epithelial surfaces rich in mucus, suggesting that invasin is absolutely required for bacterial translocation into Peyer’s patches. Hence, similar to intimin, invasin modulates the site of initial mucosal colonization by an enteric pathogen. Tir from EPEC and the pili of uropathogenic E. coli (UPEC) allowed the gain of significant insight into the complexity of bacterial tissue tropism and its importance for pathogenesis. Importantly, the E. coli pathogens have shown that the specificity and affinity of bacterial surface attachment underlie the tissue tropism of mucosal pathogens and depend not only on timely expression of a specific bacterial adhesin but also on its binding affinity for a cognate host cell receptor and the tissue distribution and availability of the host receptor.

This chapter considers two additional facets of adherence: (i) that in addition to adhering to a surface, bacteria often adhere to each other; and (ii) that colonization is sometimes promoted by relinquishing adherence. It focuses on bacterial aggregation. Bacteria adhering to mucosal surfaces commonly exhibit interbacterial aggregation or agglutination, perhaps with relatively few points of anchorage to the substratum. On the mucosa, interbacterial aggregation presumably plays the same biophysical role as it does on abiotic substrata, although data to this effect are few. Recent studies demonstrated that Escherichia coli aggregation mediated by antigen 43 (Ag43) protects bacteria from H2O2-dependent killing. The study of aggregation and biofilm formation by staphylococcal species continues to represent the paradigm for bacterial infections. Bacterial aggregation and microcolony formation are considered to be an initial stage of biofilm formation. Biofilms of Pseudomonas aeruginosa represent bacteria embedded in an alginate polysaccharide matrix. Instead of a shortening and thickening of the fimbriae as seen with enteropathogenic E. coli (EPEC), meningococci (MC) shed their fimbriae completely and initiate a complex signal transduction cascade within the cell. Dispersin mutants are deficient in colonization of the streptomycin-treated mouse model, suggesting that enteroaggregative E. coli (EAEC) mutants exhibiting collapsed fimbriae and hyperaggretation are not able to establish colonization of the intestine. Although each bacterium interacting with a mucosal surface must modify its colonization approach to fit its particular life-style and survival strategy, comparative biology continues to indentify new conserved general functions such as aggregation and its counterpoint, dispersal.

This chapter considers the remarkable signal transduction networks that have evolved between intestinal microbes and their host in trying to maintain the balance of health and disease. Resident bacteria serve a central line of resistance to colonization by exogenous microbes and thus assist in preventing the potential invasion of the intestinal mucosa by an incoming pathogen. A number of enteric pathogens and some opportunistic commensal bacteria possess the means to provoke NF-κB activation and, subsequently, intestinal inflammation. Innate epithelial defense mechanisms provide a rapid response whereby microbial pathogens in the host are quickly detected and signals are generated that activate mucosal antimicrobial defense mechanisms. The intimate interaction between enteropathogenic Escherichia coli (EPEC) and the intestinal epithelium causes the induction of phosphate fluxes within the host cells, as well as the activation of protein kinase C (PKC), phospholipase C, and NF-κB. Chloride secretion in the intestinal mucosa involves the collaborative effort of several transporters. The current paradigm postulates that intestinal epithelial cells respond to Salmonella enterica serovar Typhimurium by the polarized release of distinct proinflammatory chemoattractants, which sequentially orchestrate neutrophil movement across the intestinal epithelium. Speculatively, microorganisms intimately associated with the intestinal mucosa may have evolved such mechanisms to dampen the host proinflammatory and immune responses without provoking apoptotic death. A more complete understanding of the signal transduction cascades that exist between the intestinal bacteria and the human host in the intestinal mucosa may uncover new insights into human diseases and reveal novel approaches to treating them.

This chapter discusses what is known about bacterial gene expression during infection of the intestinal tract in animal models of disease. Despite the publication of numerous studies investigating gene expression by pathogens that colonize the intestinal tract, many of these studies were performed in vitro or in cell culture models of infection. While the importance of these investigations cannot be disputed, these systems do not fully represent the complex milieu of the intestinal tract, on which the chapter is focused. In addition, since many intestinal pathogens have the ability to cause serious systemic disease, bacterial gene expression has been examined at sites of systemic infection. DNA microarrays have been recently used to examine Vibrio cholerae gene expression in rabbit ligated ileal loops. Genes encoding transcriptional regulatory factors, including phoP, were absent from the set of Salmonella enterica serovar Typhimurium ivi genes induced specifically in the small intestine. Of the three Yersinia species that infect humans, Y. enterocolitica and Y. pseudotuberculosis are enteric pathogens. Yersinia enterocolitica harbors a virulence plasmid, known as pYV, that carries many genes required for virulence. Using a strain lacking this plasmid, Y. enterocolitica chromosomal intestinal ivi genes were identified in the PP of mice 24 h after oral infection. The examination of gene expression in diverse bacterial pathogens during intestinal infection has revealed much about the conditions experienced during this process.

The serotype associated most frequently with Salmonella, a diarrheal disease, is Salmonella enterica serotype Typhimurium. Serotype Typhimurium infection in calves is an excellent model for the intestinal pathology, host response, and disease syndrome observed in humans. The interaction of S. enterica serotype Typhimurium with the intestinal mucosa in calves and humans results in the recruitment of neutrophils whose presence is the histopathologic hallmark for the acute phase of Salmonella-induced enterocolitis. This neutrophilic infiltrate is associated with necrosis of the upper mucosa in large areas of the terminal ileum and colon. Serotype Typhimurium initiates interaction with epithelial cells by causing the formation of membrane ruffles, a process that results in bacterial internalization. This invasion process is mediated by a type III secretion system (T3SS-1) encoded by genes located on Salmonella pathogenicity island 1. The main function of the T3SS-1 is to translocate effector proteins into the cytosol of a host cell. Persistence of serotype Typhimurium in the mesenteric lymph nodes has also been described for apparently healthy cattle. The lipopolysaccharide (LPS) of serotype Enteritidis does not contain the O4 antigen but instead carries a tyvelose branch (O9 antigen) as the immunodominant epitope on the trisaccharide backbone of its O-antigen repeat unit.

Entamoeba parasites have a two-stage life cycle consisting of a disease-causing trophozoite stage and an infectious cyst stage. Ingestion of Entamoeba histolytica cysts from fecally contaminated food or water initiates infection in humans. The ingested cyst is quadrinucleate and is resistant to chlorination, gastric acidity, and desiccation. The first line of defense against E. histolytica invasion is provided by the intestinal mucins, which are high-molecular-weight glycoproteins lining the mucosal epithelium. DNA fragmentation characteristic of apoptosis is observed in host cells upon intestinal invasion by E. histolytica. Apoptosis occurs when a signal activates effectors such as caspases (cysteine proteinases with specificity for aspartate residues) to degrade cell components. The role of caspases in vivo on amoebic liver abscesses has been studied by using a mouse model of infection. Infection with E. histolytica usually results in asymptomatic colonization, but there is an approximately 10% risk of developing invasive disease. Interestingly, there was actually an increased rate of new E. histolytica infections in children with serum IgG antibodies to the lectin: children with serum anti-lectin IgG antibodies had 53% more new infections. The discovery that humans naturally acquire partial immunity gives hope that an effective vaccine can be made, and the Gal/GalNac lectin, which mediates adherence, is an intriguing vaccine candidate. Importantly, since humans are the only significant reservoir for E. histolytica infections, a vaccine that blocks colonization could lead to the elimination of amoebiasis.

Phase-variable expression of different versions of the same gene, as in the case of opa genes, or of genes that contribute to the structure of the same macromolecule, as occurs with lipooligosaccharide (LOS) biosynthesis genes, results in reversible changes in the antigenic makeup of the bacterial surface. Pilin antigenic variation, the result of new genetic information recombining into the pilin gene, is perhaps the most fascinating example of true antigenic variation in Neisseria gonorrhoeae. Despite the experimental challenges inherent in studying this human-specific pathogen, evidence that variable expression of surface molecules plays a critical role in gonococcal pathogenesis is strong. The depth of variability created by the size of the pilin repertoire and the seemingly random manner by which cassettes are inserted make Neisseria pilus antigenic variation one of the most fascinating stories of genetic diversity in bacterial pathogenesis. The purpose of pilus phase variation in bacterial pathogenesis is less intuitive than that of antigenic variation. Experimental infection of mice may be a useful tool for investigating the kinetics of gonococcal opacity (Opa) expression in vivo. Recovery of Opa-positive variants occurs following vaginal inoculation of mice with a predominantly Opa-negative inoculum. Acquisition of iron for growth and as a cofactor of several key enzymes in the low-iron environment of the host is important for successful colonization by most microbes.

One of the clearest examples of pathoadaptive mutation can be found in the allelic variation of the FimH lectin adhesin of type 1 fimbriae. This chapter reviews evidence for the role of type 1 fimbriae as urovirulence factors. While the focus is on the FimH lectin and the occurrence of mutations that cause some fimH alleles to be pathoadaptive, the discussion on allelic variation of FimH is presented within the broader context of type 1 fimbrial biology in the chapter. Type 1 fimbriae bearing the FimH lectin are expressed on the surfaces of virtually all Escherichia coli strains and most other members of the family Enterobacteriaceae. Importantly, zonal analysis of fimC alleles from the same strains did not reveal any similar signs of adaptive selection. No striking differences could be found between the highest binding and lowest binding of the strains in terms of fimbrial number, fimbrial length, and relative amounts of FimH protein incorporated into fimbriae. These results suggested that conformational differences in the FimH subunit alone were responsible for the differences in E. coli adhesion. It was logical to hypothesize, on the basis of the in vitro studies, that the ability to bind effectively to Man1 receptors was a key factor in the pathogenesis of cystitis.

This chapter describes the signaling pathways in the urinary tract and their relevance to asymptomatic carriage, acute symptomatic disease, and chronic infection with tissue damage. There is evidence that P fimbriae enhance bacterial virulence by promoting both intestinal colonization and spread to the urinary tract, by promoting the establishment of bacteriuria, by facilitating the establishment of bacteremia, by activating the innate host response, and by resisting neutrophil killing. Escherichia coli P fimbriae use glycosphingolipid receptors (GSLs) as primary receptors to adhere to the host cells and use TLR4 as coreceptors in transmembrane signaling and cell activation. The nonfimbriated E. coli strain did not induce a host response in either Tlr4+/+ or Tlr4-/- mice, demonstrating that P fimbriae and Tlr4 both were needed to trigger the innate host response. The fimbriae have been identified as virulence factors in the murine experimental model of urinary tract infection (UTI) and as colonization factors of the large intestine, but a role in virulence is potentially difficult to reconcile with the occurrence of type 1 fimbriae in both virulent and commensal strains. The GSL recognition receptors are essential for P fimbriae to adhere and to recruit TLR4 for signaling. The expression of receptors for P fimbriae reflects the P blood group, since the receptor structures also act as the P blood group of the host.

In urinary tracts with functional or anatomical abnormalities in which the normal flow of urine is disrupted or in which residual urine cannot be expressed from the bladder, a distinct group of bacterial species may cause infection. These infections are most frequently polymicrobial. The placement of a long-term indwelling catheter ensures that a urinary tract infection (UTI) will develop. Proteus mirabilis, a dimorphic gram-negative bacterium, commonly causes UTI in individuals with structural abnormalities or long term catheterization, i.e., complicated UTI. Flagella and the mannose-resistant, Proteus-like (MR/P) fimbriae and P. mirabilis fimbriae (PMF) have been identified as virulence factors of P. mirabilis that contribute to its colonization of the urinary tract in a murine model. Urease inhibitors have been used to treat patients with urolithiasis. The level of bladder colonization by the urease-negative mutant was >200-fold higher in the catheterized mice than in the uncatheterized mice. Despite the successful colonization of the bladders of the catheterized mice by the urease-negative mutant, urolithiasis or death was never observed. The low-level urease activity detected in the uninduced culture of the wild type is consistent with a previous finding that the expression of UreD is not as tightly regulated in P. mirabilis as it is in Escherichia coli expressing cloned urease genes.

Indwelling catheters are used to control the release of urine from the bladder. Various types of stents maintain the flow of urine through the ureters and the urethra. They are not inert, however, and their surfaces are capable of physical, chemical, and biological interactions. In addition, they have none of the defense mechanisms which protect mucosal tissue surfaces from bacterial colonization. The surface irregularities common on these devices also induce the passive entrapment of bacterial cells. These foreign bodies are thus extremely vulnerable to colonization by any contaminating microbes that might be in their vicinity. If infection occurs in the first week of catheterization, it is usually caused by a single species such as Staphylococcus epidermidis, Enterococcus faecalis, or Escherichia coli. The longer the catheter remains in place, the greater the variety of organisms that accumulate in the bladder. Polymicrobial bacteriuria is thus characteristic of patients enduring longterm bladder management by indwelling catheter. Results of a study found that biofilms were visible on 46% of 72 stents, most of which had been in place for 12 weeks. Enterococci and coagulase-negative staphylococci were again reported as the organisms commonly colonizing the stents. The crystalline biofilms that form on catheters usually contain Proteus mirabilis and several other species. Vaccines have been developed against uropathogenic E. coli and shown to induce effective prophylaxis against infections in patients with “normal” urinary tracts, but whether they are capable of preventing the colonization of the catheter and the urine in the catheterized urinary tract is unknown.

This chapter examines the current knowledge of urogenital bacterial microbiota in women and explores methods to manipulate the organisms to restore and maintain health. One study that effectively monitored colonization of two newborns from birth showed that the primary colonizers in the first week of life in vaginally born, breast-fed babies are Escherichia coli, Clostridium, Enterobacter, Streptococcus, and Enterococcus, with bifidobacteria not appearing until the second week. In terms of urogenital colonization, most studies have focused on the transfer of pathogens, such as group B streptococci, from mother to newborn. Proteomic studies have shown that the protein composition of the urine fluctuates, with stone formers having higher concentrations of albumin. There is considerable knowledge about how sexually transmitted disease pathogens such as gonococci, herpes simplex virus, and human immunodeficiency virus (HIV) interact with the host, but the focus of this chapter is on bacterial and yeast populations on the vaginal and urethral mucosa. Given that Candida colonizes up to 60% of women deemed to have a healthy vaginal mucosa, more studies are needed to understand which hosts react in an inflammatory and symptomatic manner and to determine the triggers for this response. Vaccine development such as the recent use of L. jensenii recombined to express the CD4 receptor for HIV will continue for some pathogens, as will the search for new remedies for viral infections.