Medicine and Health Sciences Commons^™

Absence Of All Components Of The Flagellar Export And Synthesis Machinery Differentially Alters Virulence Of Salmonella Enterica Serovar Typhimurium In Models Of Typhoid Fever, Survival In Macrophages, Tissue Culture Invasiveness, And Calf Enterocolitis, Clare K. Schmitt, Jack S. Ikeda, Stephen C. Darnell, Patricia R. Watson, Jennifer Bispham, Timothy S. Wallis, Debra L. Weinstein, Eleanor S. Metcalf, Alison D. O'Brien

Uniformed Services University of the Health Sciences

In this study, we constructed an flhD (the master flagellar regulator gene) mutant of Salmonella enterica serovar Typhimurium and compared the virulence of the strain to that of the wild-type strain in a series of assays that included the mouse model of typhoid fever, the mouse macrophage survival assay, an intestinal epithelial cell adherence and invasion assay, and the calf model of enterocolitis. We found that the flhD mutant was more virulent than its parent in the mouse and displayed slightly faster net growth between 4 and 24 h of infection in mouse macrophages. Conversely, the flhD mutant exhibited diminished …

Predictors Of Patient Satisfaction, Jeffrey L. Jackson, Judith Chamberlin, Kurt Kroenke

Uniformed Services University of the Health Sciences

Correlates of patient satisfaction at varying points in time were assessed using a survey with 2-week and 3-month follow-up in a general medicine walk-in clinic, in USA. Five hundred adults presenting with a physical symptom, seen by one of 38 participating clinicians were surveyed and the following measurements were taken into account: patient symptom characteristics, symptom-related expectations, functional status (Medical Outcomes Study Short-Form Health Survey [SF-6]), mental disorders (PRIME-MD), symptom resolution, unmet expectations, satisfaction (RAND 9-item survey), visit costs and health utilization. Physician perception of difficulty (Difficult Doctor-Patient Relationship Questionnaire), and Physician Belief Scale. Immediately after the visit, 260 (52%) …

Tsc13p Is Required For Fatty Acid Elongation And Localizes To A Novel Structure At The Nuclear-Vacuolar Interface In Saccharomyces Cerevisiae, Sepp D. Kohlwein, Sandra Eder, Chan-Seok Oh, Charles E. Martin, Ken Gable, Dagmar Bacikova, Teresa Dunn

Uniformed Services University of the Health Sciences

The TSC13/YDL015c gene was identified in a screen for suppressors of the calcium sensitivity of csg2Δ mutants that are defective in sphingolipid synthesis. The fatty acid moiety of sphingolipids in Saccharomyces cerevisiae is a very long chain fatty acid (VLCFA) that is synthesized by a microsomal enzyme system that lengthens the palmitate produced by cytosolic fatty acid synthase by two carbon units in each cycle of elongation. The TSC13 gene encodes a protein required for elongation, possibly the enoyl reductase that catalyzes the last step in each cycle of elongation. The tsc13 mutant accumulates high levels of long-chain bases …

Mutation Of The Gene Encoding Cytotoxic Necrotizing Factor Type 1 (Cnf1) Attenuates The Virulence Of Uropathogenic Escherichia Coli, Karen E. Rippere-Lampe, Alison D. O'Brien, Richard Conran, Hank A. Lockman

Uniformed Services University of the Health Sciences

Cytotoxic necrotizing factor type 1 (CNF1) is a 115-kDa toxin that activates Rho GTPases and is produced by uropathogenic Escherichia coli (UPEC). While both epidemiological studies that link CNF1 production by E. coli with urinary tract disease and the cytopathic effects of CNF1 on cultured urinary tract cells are suggestive of a role for the toxin as a UPEC virulence factor, few in vivo studies to test this possibility have been reported. Therefore, in this investigation, we evaluated the importance of CNF1 in a murine model of urinary tract infection (UTI) by comparing the degree of colonization and damage induced …

Salmonella Typhimurium Translocates Flagellin Across Intestinal Epithelia, Inducing A Proinflammatory Response, Andrew T. Gewirtz, Peter O. Simon Jr., Clare K. Schmitt, Laura J. Taylor, Curt H. Hagedorn, Alison D. O'Brien, Andrew S. Neish, James L. Madara

Uniformed Services University of the Health Sciences

This study investigated whether soluble paracrine factors mediated Salmonella-induced IL-8 expression in polarized model intestinal epithelia. We found that the basolateral media of model epithelia that had been apically infected with Salmonella typhimurium for a short period (10 minutes) could activate IL-8 secretion in virgin model epithelia, demonstrating that a proinflammatory factor (PIF) was indeed present. Initial characterization found that PIF was a heat-stable protein with a molecular mass of about 50 kDa that acts on the basolateral, but not apical, surface of model intestinal epithelia to elicit IL-8 secretion. PIF was not present in the media of model …

Tsc3p Is An 80-Amino Acid Protein Associated With Serine Palmitoyltransferase And Required For Optimal Enzyme Activity, Ken Gable, Harry Slife, Dagmar Bacikova, Erin Monaghan, Teresa M. Dunn

Uniformed Services University of the Health Sciences

Serine palmitoyltransferase catalyzes the first step of sphingolipid synthesis, condensation of serine and palmitoyl CoA to form the long chain base 3-ketosphinganine. The LCB1/TSC2 and LCB2/TSC1 genes encode homologous proteins of the a-oxoamine synthase family required for serine palmitoyltransferase activity. The other a-oxoamine synthases are soluble homodimers, but serine palmitoyltransferase is a membrane-associated enzyme composed of at least two subunits, Lcb1p and Lcb2p. Here, we report the characterization of a third gene, TSC3, required for optimal 3-ketosphinganine synthesis in Saccharomyces cerevisiae. S. cerevisiae cells lacking the TSC3 gene have a temperature-sensitive lethal phenotype that is reversed by supplying …

Escherichia Coli O157:H7 In Beef Cattle Presented For Slaughter In The U.S.: Higher Prevalence Rates Than Previously Estimated, Lisa J. Gansheroff, Alison D. O'Brien

Uniformed Services University of the Health Sciences

Before 1982, the Centers for Disease Control and Prevention (CDC) had identified only one Escherichia coli isolate of the O157:H7 serotype; it had been isolated from a patient with bloody diarrhea. Then, in 1982, two outbreaks of severe bloody diarrhea occurred among people who had eaten hamburgers at a fast food chain. The CDC isolated E. coli O157:H7 from people who had become ill as well as from a hamburger patty. In 1983, Karmali et al. discovered an association between infection with E. coli that produce Shiga toxin (then called Vero toxin), including O157:H7 strains, and another severe and sometimes …

Bacterial Toxins: Friends Or Foes?, Clare K. Schmitt, Karen C. Meysick, Alison D. O'Brien

Uniformed Services University of the Health Sciences

Many emerging and reemerging bacterial pathogens synthesize toxins that serve as primary virulence factors. We highlight seven bacterial toxins produced by well-established or newly emergent pathogenic microbes. These toxins, which affect eukaryotic cells by a variety of means, include Staphylococcus aureus α-toxin, Shiga toxin, cytotoxic necrotizing factor type 1, Escherichia coli heat-stable toxin, botulinum and tetanus neurotoxins, and S. aureus toxic-shock syndrome toxin. For each, we discuss the information available on its synthesis and structure, mode of action, and contribution to virulence. We also review the role certain toxins have played in unraveling signal pathways in eukaryotic cells and summarize …

The Saccharomyces Cerevisiae Tsc10/Ybr265w Gene Encoding 3-Ketosphinganine Reductase Is Identified In A Screen For Temperature-Sensitive Suppressors Of The Ca2+-Sensitive Csg2Δ Mutant, Troy Beeler, Dagmar Bacikova, Ken Gable, Lisa Hopkins, Courtney Johnson, Harry Slife, Teresa Dunn

Uniformed Services University of the Health Sciences

Saccharomyces cerevisiae csg2Δ mutants accumulate the sphingolipid inositolphosphorylceramide, which renders the cells Ca²⁺-sensitive. Temperature-sensitive mutations that suppress the Ca²⁺ sensitivity of csg2Δ mutants were isolated and characterized to identify genes that encode sphingolipid synthesis enzymes. These temperature-sensitive csg2Δ suppressors (tsc) fall into 15 complementation groups. The TSC10/YBR265w gene was found to encode 3-ketosphinganine reductase, the enzyme that catalyzes the second step in the synthesis of phytosphingosine, the long chain base found in yeast sphingolipids. 3-Ketosphinganine reductase (Tsc10p) is essential for growth in the absence of exogenous dihydrosphingosine or phytosphingosine. Tsc10p is a member of the …

Hydroxylation Of Saccharomyces Cerevisiae Ceramides Requires Sur2p And Scs7p, Dale Haak, Ken Gable, Troy Beeler, Teresa Dunn

Uniformed Services University of the Health Sciences

The Saccharomyces cerevisiae SCS7 and SUR2 genes are members of a gene family that encodes enzymes that desaturate or hydroxylate lipids. Sur2p is required for the hydroxylation of C-4 of the sphingoid moiety of ceramide, and Scs7p is required for the hydroxylation of the very long chain fatty acid. Neither SCS7 nor SUR2 are essential for growth, and lack of the Scs7p- or Sur2p- dependent hydroxylation does not prevent the synthesis of mannosyldiinositolphosphorylceramide, the mature sphingolipid found in yeast. Deletion of either gene suppresses the Ca²⁺-sensitive phenotype of csg2Δ mutants, which arises from overaccumulation of inositolphosphorylceramide due to …

Activation Of Shiga-Like Toxins By Mouse And Human Intestinal Mucus Correlates With Virulence Of Enterohemorrhagic Escherichia Coli O91:H21 Isolates In Orally Infected, Streptomycin-Treated Mice, Angela R. Melton-Celsa, Stephen C. Darnell, Alison D. O'Brien

Uniformed Services University of the Health Sciences

The enterohemorrhagic Escherichia coli (EHEC) O91:H21 isolates B2F1 and H414-36/89 are virulent in an orally infected streptomycin-treated mouse model. Previous studies demonstrated that B2F1 and H414-36/89 grow to high levels in mucus isolated from the mouse small intestine and colon and that growth in smallintestinal mucus is related to virulence. We measured the levels of Shiga-like toxins (SLTs) SLT-IIvha and SLT-IIvhb produced by B2F1 after growth in Luria-Bertani (LB) broth supplemented with mouse intestinal mucus by assaying the cytotoxicity of culture supernatants on Vero cells. Culture supernatants from B2F1 grown in mouse intestinal mucus, but not EHEC strains that produce …

Truncated Enterohemorrhagic Escherichia Coli (Ehec) O157:H7 Intimin (Eaea) Fusion Proteins Promote Adherence Of Ehec Strains To Hep-2 Cells, Marian L. Mckee, Alison D. O'Brien

Uniformed Services University of the Health Sciences

Intimin, the product of the eaeA gene in enterohemorrhagic Escherichia coli O157:H7 (EHEC), is required for intimate adherence of these organisms to tissue culture cells and formation of the attaching and effacing lesion in the gnotobiotic pig. Because of the importance of intimin in the pathogenesis of EHEC O157:H7 infection in this animal model, we began a structure-function analysis of EaeA. For this purpose, we constructed amino-terminal fusions of the intimin protein with six histidine residues to form two independent fusions. The longer fusion, RIHisEae, contained 900 of the 935 predicted amino acids and included all but the extreme amino …

Investigation Of Enterohemorrhagic Escherichia Coli O157:H7 Adherence Characteristics And Invasion Potential Reveals A New Attachment Pattern Shared By Intestinal E. Coli, Marian L. Mckee, Alison D. O'Brien

Uniformed Services University of the Health Sciences

In this study, the interactions of enterohemorrhagic Escherichia coli (EHEC) O157 strains with human ileocecal (HCT-8) epithelial cells and HEp-2 cells were examined. EHEC adhered to, but did not invade, HCT-8 cells by the localized adherence mechanism and a heretofore unrecognized pattern which we called log jam. The log jam formation was (i) not observed on HEp-2 cells, (ii) independent of the EHEC eaeA gene required for localized adherence, and (iii) shared by pathogenic and nonpathogenic E. coli strains but not K-12 strains. The log jam phenotype may represent a basal means by which E. coli bacteria attach to the …

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, Susanne W. Lindgren, James E. Samuel, Clare K. Schmitt, Alison D. O'Brien

Uniformed Services University of the Health Sciences

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 091: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 DH5α(Str^r) strains that produced …

A Novel Protein, Csg2p, Is Required For Ca2+ Regulation In Saccharomyces Cerevisiae, Troy Beeler, Kenneth Gable, Chun Zhao, Teresa Dunn

Uniformed Services University of the Health Sciences

Nineteen mutants that lost the ability to grow in 100 mM Ca²⁺ (but remained insensitive to 50 mM Sr²⁺) were identified in a screen of approximately 60,000 mutagenized yeast colonies. Cells carrying mutations in the CSG2 gene grow normally in low Ca²⁺ medium but have decreased growth rates when the Ca²⁺ concentration is above 10 mM. The csg2 mutant cells accumulate much higher levels of Ca²⁺ in a compartment that is exchangeable with extracellular Ca²⁺ but the nonexchangeable Ca²⁺ pool which predominates in wild-type cells is not influenced. Sr²⁺ influx is …

Regulation Of Cellular Ca2+ By Yeast Vacuoles, Teresa Dunn, Kenneth Gable, Troy Beeler

Uniformed Services University of the Health Sciences

The role of vacuolar Ca²⁺ transport systems in regulating cellular Ca²⁺ was investigated by measuring the vacuolar Ca²⁺ transport rate, the free energy available to drive vacuolar Ca²⁺ transport, the ability of the vacuole to buffer lumenal Ca²⁺, and the vacuolar Ca²⁺ efflux rate. The magnitude of the Ca²⁺ gradient generated by the vacuolar H⁺gradient best supports a 1 Ca²⁺:2 H⁺ coupling ratio for the vacuolar Ca²⁺/H⁺ exchanger. This coupling ratio along with a cytosolic Ca²⁺ concentration of 125 nM would give a …

Cytotoxic Necrotizing Factor Type 2 Produced By Virulent Escherichia Coli Modifies The Small Gtp-Binding Proteins Rho Involved In Assembly Of Actin Stress Fibers, Eric Oswald, Motoyuki Sugai, Agnès Labigne, Henry C. Wu, Carla Fiorentini, Patrice Boquet, Alison D. O'Brien

Uniformed Services University of the Health Sciences

Cytotoxic necrotizing factor type 2 (CNF2)produced by Escherichia coli strains isolated from intestinal and extraintestinal infections is a dermonecrotic toxin of 110 kDa. We cloned the CNF2 gene from a large plasmid carried by an Escherichia coli strain isolated from a lamb with septicemica. Hydropathy analysis of the deduced amino acid sequence revealed a largely hydrophilic protein with two peotential hydrophobic transmembrane domains. The N-terminal half of CNF2 showed striking homology (27% identity and 80% conserved residues) to the N-terminal portion of Pasteurella multocida toxin. Methylamine protection experiments and immunofluorescence studies suggested that CNF2 enters the cytosol of the target …

Effect Of The General Anesthetic Halothane On The Activity Of The Transverse Tubule Ca2+-Activated K+ Channel, Troy Beeler, Kenneth Gable

Uniformed Services University of the Health Sciences

The effect of the general anesthetic halothane on the activity of the rat skeletal muscle Ca²⁺-activated K⁺ channel in planar lipid bilayers was investigated. Halothane concentrations in the clinical range (1.0-0.2 mM) alter the regulation of the channel by both Ca²⁺ and membrane potential. At Ca²⁺ concentrations between 10 and 250 µM and membrane potentials between 0 and -30 mV, halothane significantly decreases the open state probability without changing the channel conductance. The results demonstrate that halothane can act directly on the Ca²⁺-activated K⁺ channel or its lipid environment to alter the …

Mechanism Of Magainin 2a Induced Permeabilization Of Phospholipid Vesicles, Earl Grant, Troy J. Beeler, Kenneth M. P. Taylor, Kenneth Gable, Mark A. Roseman

Uniformed Services University of the Health Sciences

The magainins, peptide antibiotics secreted by the frog Xenopus laevis, have previously been shown to permeabilize phospholipid vesicles. To elucidate the mechanism of permeabilization, we have conducted detailed kinetic studies of magainin 2 amide (mgn2a)hduced release of 6-carboxyfluorescein from vesicles of phosphatidylserine. The results show that dye release occurs in (at least) two stages-an initial rapid phase, with t_1/2 ≈ 3 s, followed by a much slower phase that approaches zero leakage rate before all the dye is released. Light-scattering studies showed that mgn2a does not cause gross changes in vesicle structure. The peptide was found to rapidly …

Two Copies Of Shiga-Like Toxin Ii-Related Genes Common In Enterohemorrhagic Escherichia Coli Strains Are Responsible For The Antigenic Heterogeneity Of The 0157:H- Strain E32511, Clare K. Schmitt, Marian L. Mckee, Alison D. O'Brien

Uniformed Services University of the Health Sciences

Thirty-two clinical isolates of Shiga-like toxin (SLT)-producing Escherichia coli associated with single cases or outbreaks of bloody diarrhea, hemorrhagic colitis, the hemolytic uremic syndrome, or edema disease of swine were examined for multiple copies of genes belonging to the slt-I or slt-II toxin families. Five of 19 strains that were known to produce SLT-II or to hybridize to slt-II-specific probes by colony blot were found by Southern hybridization to contain two copies of toxin genes related to slt-II. The genes for two toxins closely related to slt-II were cloned from one of the isolates, Escherichia …

Evaluation Of The Role Of Shiga And Shiga-Like Toxins In Mediating Direct Damage To Human Vascular Endothelial Cells, Vernon L. Tesh, James E. Samuel, Liyanage P. Perera, John B. Sharefkin, Alison D. O'Brien

Uniformed Services University of the Health Sciences

Infection with Shinga toxin- and Shinga-like toxin -producing strains of Shigella dysenteriae and Escherichia coli, respectively, can progress to the hemolytic-uremic syndrome. It has been hypothesized that circulating Shinga toxin, Shinga-like toxins, and endotoxins may contribute to the disease by directly damaging glomerular endothelial cells. The effects of these toxins on HeLa, Vero, and human vascular endothelial cells (EC) were examined. Confluent EC were sensitive to Shinga toxin but were at least 10⁶-fold less sensitive to the toxins than were Vero cells. Shinga toxin was the predominant cytotoxic factor. Lipopolysaccharides were not cytotoxic and did not augment …

Raman Spectroscopy Of Synthetic Antimicrobial Frog Peptides Magainin 2a And Pgla, Robert W. Williams, Ruth Starman, Kenneth M. P. Taylor, Kenneth Gable, Troy Beeler, Michael Zasloff, David Covell

Uniformed Services University of the Health Sciences

Magainin and PGLa are 23- and 21-residue peptides isolated from the skin of the African clawed frog Xenopus lueuis. They protect the frog from infection and exhibit a broad-spectrum antimicrobial activity in vitro. The mechanism of this activity involves the interaction of magainin with microbial membranes. We have measured the secondary structure and membrane-perturbing ability of these peptides to obtain information about this mechanism. Our results show that mgn2a forms a helix with an average length of less than 20 Å upon binding to liposomes. At high concentrations (50 mg/mL) mgn2a spontaneously solubilizes phosphatidylcholine liposomes at temperatures above the …

In Vivo Formation Of Hybrid Toxins Comprising Shiga Toxin And The Shiga-Like Toxins And Role Of The B Subunit In Localization And Cytotoxic Activity, Debra L. Weinstein, Matthew P. Jackson, Liyanage P. Perera, Randall K. Holmes, Alison D. O'Brien

Uniformed Services University of the Health Sciences

Shiga toxin, Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT-H) are cell-associated cytotoxins that kill both Vero cells and HeLa cells, whereas Shiga-like toxin II variant (SLT-llv) is an extracellular cytotoxin that is more cytotoxic for Vero cells than for HeLa cells. The basis for these differences in cytotoxin localization and host cell specificity were examined in this study. The A and B subunit genes of Shiga toxin and the SLTs were recombined by two methods so that hybrid toxins would be formed in vivo. Complementation of heterologous subunits was accomplished by cloning the individual A and B subunit …

Shiga And Shiga-Like Toxins, Alison D. O'Brien, Randall K. Holmes

Uniformed Services University of the Health Sciences

Introduction ........................ 206

Shiga Toxin in Shigella Spp. ........................ 207

History ........................ 207

Purification and Structure ........................ 207

Mode of Action ........................ 208

Receptor binding and internalization ........................ 208

Inhibition of protein synthesis ........................ 209

Immunology and Immunochemistry ........................ 209

Genetics ........................ 210

Role of Toxin in Disease ........................ 210

Dysentery ........................ 210

Diarrhea ........................210

HUS ........................ 211

Shiga-like toxins in E. Coli ........................ 211

History and Nomenclature ........................ 211

Discovery that EPEC and EHEC make Shiga-like toxins ........................ 211

Relationship of Shiga-like toxins to Vero toxins ........................ 212

Purification and Structure ........................ 212

Mode of Action ........................ 212

Immunology …

Production Of Shiga-Like Toxin By Escherichia Coli, Lilian R. M. Marques, Marjorie A. Moore, Joy G. Wells, I. Kate Wachsmuth, Alison D. O'Brien

Uniformed Services University of the Health Sciences

Escherichia coli is one of several agents that cause intestinal disease in humans and animals. Four classes of E. coli have been recognized [1], Enterotoxigenic E. coli (ETEC) strains produce a heat-labile (LT) enterotoxin and/or a heat-stable( ST) enterotoxin. Enteroinvasive E . coli (EIEC) strains, like shigellae, penetrate and multiply within epithelial cells. Enteropathogenic E. coli (EPEC) strains belong to certain serogroups that have been incriminated as pathogens by epidemiological studies. Some EPEC strains have been shown to adhere to cells of the intestinal mucosa and to produce pathognomonic lesions at the site of attachment. Enterohemorrhagic E. coli (EHEC) strains …

The Diarrheal Response Of Humans To Some Classic Serotypes Of Enteropathogenic Escherichia Coli Is Dependent On A Plasmid Encoding An Enteroadhesiveness Factor, Myron M. Lavine, James P. Nataro, Helge Karch, Mary M. Baldini, James B. Kaper, Robert E. Black, Mary Lou Clements, Alison D. O'Brien

Uniformed Services University of the Health Sciences

Isolates of the most common 0 serogroups of enteropathogenic Escherichia coli (EPEC) associated with infant diarrhea (designated class I) adhere to Hep-2 cells; the genes for this adhesin, termed EPEC adherence factor (EAF), are located on plasmids 50-70 MDa in size. Volunteers ingested 10¹⁰ organisms of an 0127:H6 Hep-2-adhesive class I strain (E2348/69) or its plasmid-minus, nonadhesive derivative. Diarrhea occurred in nine of 10 volunteers who ingested the parent strain (mean, 1,178 ml) but in only two of nine who took the plasmid-minus variant (mean, 433 ml; P < .006). All volunteers ill from strain E2348/69 mounted serum IgA and IgG responses to a 94-kDa plasmid-associated outer membrane protein of E2348/69; this protein was found in other class I EPEC but not in enterotoxigenic or meningitic strains. The 50-70-MDa EAF plasmid seems neces- sary for full expression of pathogenicity in EPEC that exhibit Hep-2 adhesiveness. EPEC isolates of certain other, less common, 0 serogroups (044, 086, and 0114) are rarely Hep-2 adhesive. These EPEC, designated class II, possess distinct 50-70 MDa plasmids lacking EAF genes. Diarrhea was caused by 10⁸ or 10¹⁰ organisms of an 0114:H2 class II EPEC …

A Balb/C Congenic Strain Of Mice That Carries A Genetic Locus (ItyR) Controlling Resistance To Intracellular Parasites, Michael Potter, Alison D. O'Brien, Emil Skamene, Philippe Gros, Adrien Forget, Patricia A. L. Kongshavn, Judith S. Wax

Uniformed Services University of the Health Sciences

BALB/c.DBA/2 Idh-1^b-Ity^r-Pep-3^b congenic mice were developed by introgressively backcrossing the Idh-1^b and Pep-3^b markers of DBA/2 mice onto the BALB/cπ mice. This introduced a 30-centimorgan chromosome 1 segment of DBA/2 chromatin that contained the Ity^r gene. BALB/c.DBA/2 Idh-1^b-Ity^r-Pep-3^b mice were resistant to in vivo infections by Salmonella typhimurium, Mycobacterium bovis, and Leishmania donovani.

Purification And Characterization Of A Shigella Dysenteriae 1- Like Toxin Produced By Escherichia Coli, Alison D. O'Brien, Gerald D. Laveck

Uniformed Services University of the Health Sciences

A toxin from an enteropathogenic strain of Escherichia coli (E. coli H30) was purified to apparent homogeneity from cell lysates. The steps used to isolate the E. coli H30 toxin included French pressure-cell disruption of bacteria grown in iron-depleted media, Affi-Gel Blue chromatography, chromatofocusing, and anti- Shiga toxin affinity chromatography. The mobilities of the subunits of radioiodinated E. coli H30 toxin and Shiga toxin observed after the two toxins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis were identical. In the absence of 2-mercaptoethanol, a narrow band was seen at M_r 31,500 (±1,000), and a wide heavy band …

Production Of Shigella Dysenteriae Type 1-Like Cytotoxin By Escherichia Coli, Alison D. O'Brien, Gerald D. Laveck, Michael R. Thompson, Samuel B. Formal

Uniformed Services University of the Health Sciences

Strains of Escherichia coli previously implicated or proven to be causes of diarrhea were examined for production of a toxin similar to that of Shigella dysenteriae type 1 (Shiga). Organisms grown in an iron-depleted broth were lysed by pressure disruption followed by ultracentrifugation. Saline-dialyzed extracts were tested for cytotoxic effects on HeLa cells that were neutralizable with antiserum to Shiga toxin. Among the 13 E. coli strains so analyzed, 11 made a Shiga-like cytotoxin in levels ranging from trace (two avirulent isolates) to amounts equivalent to S. dysenteriae type 1 (two noninvasive strains that did not make E. coli heat-labile …