The innate immune system in drosophila and mammals senses the invasion of microorganisms using the family of Toll receptors, stimulation of which initiates a range of host defense mechanisms. In drosophila antimicrobial responses rely on two signaling pathways: the Toll pathway and the IMD pathway. In mammals there are at least 10 members of the Toll-like receptor (TLR) family that recognize specific components conserved among microorganisms. Activation of the TLRs leads not only to the induction of inflammatory responses but also to the development of antigen-specific adaptive immunity. The TLR-induced inflammatory response is dependent on a common signaling pathway that is mediated by the adaptor molecule MyD88. However, there is evidence for additional pathways that mediate TLR ligand-specific biological responses.

Toll-like receptors.

Escherichia coli is the predominant nonpathogenic facultative flora of the human intestine. Some E. coli strains, however, have developed the ability to cause disease of the gastrointestinal, urinary, or central nervous system in even the most robust human hosts. Diarrheagenic strains of E. coli can be divided into at least six different categories with corresponding distinct pathogenic schemes. Taken together, these organisms probably represent the most common cause of pediatric diarrhea worldwide. Several distinct clinical syndromes accompany infection with diarrheagenic E. coli categories, including traveler’s diarrhea (enterotoxigenic E. coli), hemorrhagic colitis and hemolytic-uremic syndrome (enterohemorrhagic E. coli), persistent diarrhea (enteroaggregative E. coli), and watery diarrhea of infants (enteropathogenic E. coli). This review discusses the current level of understanding of the pathogenesis of the diarrheagenic E. coli strains and describes how their pathogenic schemes underlie the clinical manifestations, diagnostic approach, and epidemiologic investigation of these important pathogens.

Diarrheagenic Escherichia coli

Few microorganisms are as versatile as Escherichia coli. An important member of the normal intestinal microflora of humans and other mammals, E. coli has also been widely exploited as a cloning host in recombinant DNA technology. But E. coli is more than just a laboratory workhorse or harmless intestinal inhabitant; it can also be a highly versatile, and frequently deadly, pathogen. Several different E. coli strains cause diverse intestinal and extraintestinal diseases by means of virulence factors that affect a wide range of cellular processes.

Pathogenic Escherichia coli

Unlike eukaryotes, which evolve principally through the modification of existing genetic information, bacteria have obtained a significant proportion of their genetic diversity through the acquisition of sequences from distantly related organisms. Horizontal gene transfer produces extremely dynamic genomes in which substantial amounts of DNA are introduced into and deleted from the chromosome. These lateral transfers have effectively changed the ecological and pathogenic character of bacterial species.

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Lateral gene transfer and the nature of bacterial innovation

The invention relates to transgenic non-human animals capable of producing heterologous antibodies and methods for producing human sequence antibodies which bind to human antigens with substantial affinity.

Transgenic non-human animals capable of producing heterologous antibodies

This invention satisfies needs in the art by providing intimin, the Enterohemorrhagic Escherichia coli (EHEC) adherence protein, alone or as a fusion protein with one or more other antigens, expressed by transgenic plants and the use of those plants as vehicles for stimulating a protective immune response against EHEC and the one or more other antigens. Various plant species are transformed to protect various animal species and also humans against EHEC, against pathogens expressing intimin-like proteins, and against pathogens expressing any of the one or more other antigens to which intimin may be fused. The eae gene encoding intimin, a functional portion thereof, or a recombination that encodes a fusion protein is put under the control of a constitutive plant promoter in a plasmid and the plasmid is introduced into plants by the type of transformation appropriate for the particular plant species. The engineered plants expressing intimin or the intimin fusion protein are then fed to animals and/or humans to elicit the production of antibodies, which protect the animals/humans against EHEC colonization and infection, and against pathogens expressing the one or more other antigens and any cross-reactive antigens. The invention may also be practiced by expressing the intimin or intimin fusion protein in other host organisms such as bacteria, yeast, and fungi.

Plants and plant cells expressing histidine tagged intimin

Along with identification of the enzymatic function of ribosome-inactivating proteins (RIPs) came the recognition that certain bacterial toxins, called Shiga toxins (Stxs), fit into the category of plant proteins. We now know that these plant and bacterial poisons have the broader specificity of polynucleotide:adenosine glycosidases, at least in vitro. This chapter refers to the enzymes interchangeably as RIPs or RNA N-glycosidases. Recently, additional enzymatic activities have been attributed to the RIPs. However, when four different RIPs were highly purified, the only enzymatic activity they retained was the capacity to remove adenine from rRNA or DNA. This observation indicates that the RIPs, originally defined as RNA N-glycosidases, are actually polynucleotide:adenosine glycosidases, at least in vitro. One unusual feature of the RIPs is that, although they share the same enzymatic activity and their preferred target is intact mammalian ribosomes, they are not equally active on target ribosomes in vitro. There appear to be at least two explanations for this phenomenon. One reason that some RNA N-glycosidases do not appear to be as active as others in cell-free assays is that they require cofactors such as ATP. The second reason that some RIPs have varying specificities is that they appear to interact with ribosomal proteins. Consistent with a protective function for the plant RNA N-glycosidases is that the RIPs are both lethal and often stored in large quantities.

Plant and Bacterial Toxins as RNA N-Glycosidases

Shiga toxin type 2

Murine typhoid, a naturally occurring disease in mice caused by Salmonella typhimurium, is a good experimental model with which to study the interactions between host and parasite and is especially important since the pathogenesis of this disease is similar to typhoid fever in man (1). Although the role of the immune system in the regulation of this disease has not been studied extensively, previous studies have suggested that the murine immune response to S. typhimurium was primarily regulated by T cells and macrophages (2). However, recent observations suggest that B cells, hence protective antibodies, also play an important role in resistance (3–5). Nevertheless, the identification of the salmonella cell surface antigens towards which these protective antibodies are directed remains controversial. One approach to the identification of these antigenic determinants would be to generate large quantities of antibody directed against a specific bacterial cell surface determinant and to use these antibodies in passive transfer experiments to determine their role in the protection of the host from murine typhoid.

Characterization of Monoclonal Antibodies Which Recognize Specific Cell Surface Determinants on Salmonella Typhimurium

The Shiga toxins (Stxs) (also known as Vero toxins and formerly called Shiga-like toxins) constitute a family of toxins that features Stx from Shigella dysenteriae type 1 as the prototype. Among bacterial toxins, the Stxs are particularly potent poisons that are estimated to be as toxic per unit of weight as tetanus toxin and second only to botulinum toxin in potency. The major difference among the Stxs is reflected in the division of the Stx family into two groups, Stx/Stx1 and Stx2, based on the capacity of antitoxin to neutralize the cytotoxic activity of each member of the homologous but not the heterologous group. The Stx-producing Escherichia coli (STEC) bacteria grow within the colon and elaborate Stx1 and/or Stx2. The toxin crosses the intestinal barrier and enters the bloodstream. Once in the bloodstream, the toxin may bind to polymorphonuclear cells and circulate. The major target organ of the Stxs is the kidney, although sites within the central nervous system may also be affected. The interaction of Stx and bacterial lipopolysaccharide with cells of the immune system and locally within the kidney may lead to increased cytokine and chemokine expression. Some epidemiological evidence suggests that STEC strains that produce Stx2 cause more severe disease than STEC strains that make Stx1 or both Stx1 and Stx2.

Alison D. O'Brien

Papers

Plants and plant cells expressing histidine tagged intimin

Plant and Bacterial Toxins as RNA N-Glycosidases

Shiga toxin type 2

Characterization of Monoclonal Antibodies Which Recognize Specific Cell Surface Determinants on Salmonella Typhimurium

Shiga Toxins (Stxs): Multifaceted Pathogenicity Determinants