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

To determine whether flagella, chemotaxis, and motility of Salmonella typhimurium are virulence factors in infected C57BL/6J mice, we constructed isogenic pairs of derivatives of the nonfimbriated virulent strain SL3201. Of each pair, one member contained a mutation in a single gene that is required for expression of normal chemotactically directed motility, whereas the other member contained the wild-type form of the gene. No additional differences between the members of a pair were evident. The phenotypic parameters examined for all derivatives included in vitro growth rate, sensitivity to P22 phage, amino acid auxotrophy, and biotype. For a flagellated and nonflagellated pair, the electron microscopic appearance of each member was examined as well as its lipopolysaccharide and outer membrane profiles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The virulence of the various derivatives was then assessed in mice challenged orally, intraperitoneally, or intravenously. The results established that flagella, whether functional or nonfunctional as organelles of motility, were S. typhimurium virulence factors and that neither chemotaxis nor motility was required for virulence. Images

Flagella of Salmonella typhimurium are a virulence factor in infected C57BL/6J mice.

The role of macrophages in the innate immunity of (CBA/N female X DBA/2N male)F1 female mice to Salmonella typhimurium was assessed with silica, an agent which has been reported to selectively inactivate macrophages. Silica, administered intravenously to mice, markedly decreased the phagocytic capacity of splenic macrophages but had no effect on splenic responsiveness to the B-cell mitogen lipopolysaccharidide or the T-cell mitogen phytohemagglutinin, nor did it affect the frequency of surface immunoglobulin-positive cells (B cells). Silica given to mice 1 day before intraperitoneal challenge decreased the 50% lethal dose of S. typhimurium 100-fold. The incidence of survival of mice given silica up to 14 days before infection with a sublethal dose of organisms was also decreased. This susceptibility could also be demonstrated when silica was given 10 days, but not 20 days, after S. typhimurium infection. Poly-2-vinylpyridine-N-oxide, a lysosomal stabilizing agent, abrogated the silica effect. Deaths among silica-treated mice followed uncontrolled multiplication of the organism in the spleen. These results provide direct evidence that macrophages play an essential role in natural immunity to murine typhoid and demonstrate the efficacy of silica as a tool to analyze macrophage function.

https://iai.asm.org/content/iai/25/2/513.full.pdf

Effect of silica on the innate resistance of inbred mice to Salmonella typhimurium infection.

CBA/N mice have an X-linked immune defect that is associated with the absence of a subpopulation of mature or late developing B lymphocytes. The effect of this B cell defect on susceptibility of mice to Salmonella typhimurium was investigated. The 50% lethal dose (LD50) of S. typhimurium for CBA/N mice was 1000-fold less than for the immunologically normal, histocompatible CBA/CaHN strain. Genetic analysis revealed that the susceptibility of the CBA/N strain to S. typhimurium, like the immune defect, was X-linked since immune-defective F1 male mice derived from crosses between CBA/N and immunologically normal strains were susceptible to S. typhimurium (LD50 ≤ 20), whereas immunologically normal F1 males and females derived from these matings were resistant (LD50 ≥ 5 × 103). By contrast, both immune-defective and normal F1 mice were resistant to another facultative intracellular parasite, Listeria monocytogenes. Deaths among the immune-defective mice occurred primarily late in the course of infection (>10 days) whereas deaths of immunologically normal, S. typhimurium-susceptible strains (e.g., C57BL/6 and BALB/c) occurred early in the infectious process. To determine if the increased susceptibility of B cell-defective mice to S. typhimurium and the expression of the X-linked immune-deficiency gene (xid) were actually linked, F2 and backcross mice were phenotyped for the immune defect by measuring serum IgM levels and then challenged with the organism. Although no formal linkage was established, there was a close correlation between low serum IgM levels and susceptibility, since 93% of such mice succumbed to infection. These studies suggest that B cells play an important role in those events that govern the development of resistance to S. typhimurium late in the course of infection.

Susceptibility of CBA/N mice to infection with Salmonella typhimurium: influence of the X-linked gene controlling B lymphocyte function.

Characteristically, enterohemorrhagic Escherichia coli (EHEC) strains produce Shiga-like toxin type I (SLT-I), SLT-II, or both of these immunologically distinct cytotoxins. No antigenic or receptor-binding variants of SLT-I have been identified, but a number of SLT-II-related toxins have been described. Because EHEC O91:H21 strain B2F1, which produces two SLT-II-related toxins, is exquisitely virulent in an orally infected, streptomycin-treated mouse model (oral 50% lethal dose [LD50], < 10 organisms), we asked whether the pathogenicity of strain B2F1 was a consequence of SLT-II-related toxin production. For this purpose, we compared the lethality of orally administered E. coli DH5 alpha (Strr) strains that produced different cytotoxic levels of SLT-II, SLT-IIvha (cloned from B2F1), SLT-IIvhb (also cloned from B2F1), or SLT-IIc (cloned from EHEC O157:H7 strain E32511) on Vero cells. We also calculated the specific activities of purified SLT-IIvhb and SLT-II in intraperitoneally injected mice and on Vero cells. The two purified toxins were equally toxic for mice, but SLT-IIvhb was approximately 100-fold less active than SLT-II on Vero cells and bound to the glycolipid receptor Gb3 with lower affinity than did SLT-II. In addition, characterization of SLT-II-related toxin-binding (B) subunit mutants generated in this study revealed that the reduced in vitro cytotoxic levels of the SLT-II-related toxins were due to Asn-16 in the B subunit. Taken together, these findings do not support the idea that B2F1 is uniquely virulent because of the in vivo toxicity of SLT-II-related toxins but do demonstrate differences in in vitro cytotoxic activity among the SLT-II group produced by human EHEC isolates.

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The specific activities of Shiga-like toxin type II (SLT-II) and SLT-II-related toxins of enterohemorrhagic Escherichia coli differ when measured by Vero cell cytotoxicity but not by mouse lethality.

The Bacillus anthracis genome encodes four superoxide dismutases (SODs), enzymes capable of detoxifying oxygen radicals. That two of these SODs, SOD15 and SODA1, are present in the outermost layers of the B. anthracis spore is indicated by previous proteomic analyses of the exosporium. Given the requirement that spores must survive interactions with reactive oxygen species generated by cells such as macrophages during infection, we hypothesized that SOD15 and SODA1 protect the spore from oxidative stress and contribute to the pathogenicity of B. anthracis. To test these theories, we constructed a double-knockout (Δsod15 ΔsodA1) mutant of B. anthracis Sterne strain 34F2 and assessed its lethality in an A/J mouse intranasal infection model. The 50% lethal dose of the Δsod15 ΔsodA1 strain was similar to that of the wild type (34F2), but surprisingly, measurable whole-spore SOD activity was greater than that in 34F2. A quadruple-knockout strain (Δsod15 ΔsodA1 ΔsodC ΔsodA2) was then generated, and as anticipated, spore-associated SOD activity was diminished. Moreover, the quadruple-knockout strain, compared to the wild type, was attenuated more than 40-fold upon intranasal challenge of mice. Spore resistance to exogenously generated oxidative stress and to macrophage-mediated killing correlated with virulence in A/J mice. Allelic exchange that restored sod15 and sodA1 to their wild-type state restored wild-type characteristics. We conclude that SOD molecules within the spore afford B. anthracis protection against oxidative stress and enhance the pathogenicity of B. anthracis in the lung. We also surmise that the presence of four SOD alleles within the genome provides functional redundancy for this key enzyme.

https://iai.asm.org/content/iai/77/1/274.full.pdf

Alison D. O'Brien

Papers

Flagella of Salmonella typhimurium are a virulence factor in infected C57BL/6J mice.

Effect of silica on the innate resistance of inbred mice to Salmonella typhimurium infection.

Susceptibility of CBA/N mice to infection with Salmonella typhimurium: influence of the X-linked gene controlling B lymphocyte function.

The specific activities of Shiga-like toxin type II (SLT-II) and SLT-II-related toxins of enterohemorrhagic Escherichia coli differ when measured by Vero cell cytotoxicity but not by mouse lethality.

Four Superoxide Dismutases Contribute to Bacillus anthracis Virulence and Provide Spores with Redundant Protection from Oxidative Stress