Showing papers by "Alison D. O'Brien published in 2008"

Hemolysin of Uropathogenic Escherichia coli Evokes Extensive Shedding of the Uroepithelium and Hemorrhage in Bladder Tissue within the First 24 Hours after Intraurethral Inoculation of Mice

Abstract: Many uropathogenic Escherichia coli (UPEC) strains produce both hemolysin (Hly) and cytotoxic necrotizing factor type 1 (CNF1), and the loci for these toxins are often linked. The conclusion that Hly and CNF1 contribute to urovirulence is supported by the results of epidemiological studies associating the severity of urinary tract infections (UTIs) with toxin production by UPEC isolates. Additionally, we previously reported that mouse bladders and rat prostates infected with UPEC strain CP9 exhibit a more profound inflammatory response than the organs from animals challenged with CP9cnf(1) and that CNF1 decreases the antimicrobial activities of polymorphonuclear leukocytes. More recently, we created an Hly mutant, CP9Delta hlyA(1)::cat, and showed that it was less hemolytic and destructive for cultured bladder cells than CP9 was. Here we evaluated the relative effects of mutations in hlyA(1) or cnf(1) alone or together on the pathogenicity of CP9 in a mouse model of ascending UTI. To do this, we constructed an hlyA(1)-complemented clone of CP9Delta hlyA(1)::cat and an hlyA(1) cnf(1) CP9 double mutant. We found that Hly had no influence on bacterial colonization of the bladder or kidneys in single or mixed infections with the wild type and CP9Delta hlyA(1)::cat but that it did provoke sloughing of the uroepithelium and bladder hemorrhage within the first 24 h after challenge. Finally, we confirmed that CNF1 expression induces bladder inflammation and, in particular, as shown in this study, submucosal edema. From these data, we speculate that Hly and CNF1 may be largely responsible for the signs and symptoms of cystitis in humans infected with toxigenic UPEC.

Mouse Model of Hemolytic-Uremic Syndrome Caused by Endotoxin-Free Shiga Toxin 2 (Stx2) and Protection from Lethal Outcome by Anti-Stx2 Antibody

Abstract: Hemolytic-uremic syndrome (HUS) results from infection by Shiga toxin (Stx)-producing Escherichia coli and is the most common cause of acute renal failure in children. We have developed a mouse model of HUS by administering endotoxin-free Stx2 in multiple doses over 7 to 8 days. At sacrifice, moribund animals demonstrated signs of HUS: increased blood urea nitrogen and serum creatinine levels, proteinuria, deposition of fibrin(ogen), glomerular endothelial damage, hemolysis, leukocytopenia, and neutrophilia. Increased expression of proinflammatory chemokines and cytokines in the sera of Stx2-treated mice indicated a systemic inflammatory response. Currently, specific therapeutics for HUS are lacking, and therapy for patients is primarily supportive. Mice that received 11E10, a monoclonal anti-Stx2 antibody, 4 days after starting injections of Stx2 recovered fully, displaying normal renal function and normal levels of neutrophils and lymphocytes. In addition, these mice showed decreased fibrin(ogen) deposition and expression of proinflammatory mediators compared to those of Stx2-treated mice in the absence of antibody. These results indicate that, when performed during progression of HUS, passive immunization of mice with anti-Stx2 antibody prevented the lethal effects of Stx2.

Detection of Epithelial-Cell Injury, and Quantification of Infection, in the HCT-8 Organoid Model of Cryptosporidiosis

Abstract: Background. Intestinal cells grown in microgravity produce a three-dimensional tissue assembly, or "organoid," similar to the human intestinal mucosa, making it an ideal model for enteric infections such as cryptosporidiosis. Methods. HCT-8 cells were grown in a reduced-gravity, low-shear, rotating-wall vessel (RWV) and were infected with Cryptosporidium parvum oocysts. Routine and electron microscopy (EM), immunolabeling with fluorescein-labeled Vicia villosa lectin and phycoerythrin-labeled monoclonal antibody to a 15-kD surface-membrane protein, and quantitative polymerase chain reaction (qPCR) using probes for 18s rRNA of C. parvum and HCT-8 cells were performed. Results. The RWV allowed development of columnar epithelium-like structures. Higher magnification revealed well-developed brush borders at the apical side of the tissue. Incubation with C. parvum resulted in patchy disruption of the epithelium and, at the surface of several epithelial cells, in localized infection with the organism. EM revealed irregular stunting of microvilli, foci of indistinct tight junctions, and areas of loose paracellular spaces. qPCR showed a 1.85-log (i.e., 70-fold) progression of infection from 6 h to 48 h of incubation. Conclusion. The HCT-8 organoid displayed morphologic changes indicative of successful and quantifiable infection with C. parvum. The HCT-8 organoid-culture system may have application in interventional in vitro studies of cryptosporidiosis.

Detection of Bacillus anthracis Spore Germination In Vivo by Bioluminescence Imaging

Abstract: We sought to visualize the site of Bacillus anthracis spore germination in vivo. For that purpose, we constructed a reporter plasmid with the lux operon under control of the spore small acid-soluble protein B (sspB) promoter. In B. subtilis, sspB-driven synthesis of luciferase during sporulation results in incorporation of the enzyme in spores. We observed that B. anthracis Sterne transformed with our sspBp::lux plasmid was only luminescent during germination. In contrast, Sterne transformed with a similarly constructed plasmid with lux expression under control of the protective antigen promoter displayed luminescence only during vegetative growth. We then infected A/J mice intranasally with spores that harbored the germination reporter. Mice were monitored for up to 14 days with the Xenogen In Vivo Imaging System. While luminescence only became evident in live animals at 18 h, dissection after sacrificing infected mice at earlier time points revealed luminescence in lung tissue at 30 min after intranasal infection. Microscopic histochemical and immunofluorescence studies on luminescent lung sections and imprints revealed that macrophages were the first cells in contact with the B. anthracis spores. By 6 h after infection, polymorphonuclear leukocytes with intracellular spores were evident in the alveolar spaces. After 24 h, few free spores were observed in the alveolar spaces; most of the spores detected by immunofluorescence were in the cytoplasm of interstitial macrophages. In contrast, mediastinal lymph nodes remained nonluminescent throughout the infection. We conclude that in this animal system, the primary site of B. anthracis spore germination is the lungs.

Identification and characterization of Shiga toxin type 2 variants in Escherichia coli isolates from animals, food, and humans.

Abstract: There is considerable heterogeneity among the Shiga toxin type 2 (Stx2) toxins elaborated by Shiga toxin-producing Escherichia coli (STEC). One such Stx2 variant, the Stx2d mucus-activatable toxin (Stx2dact), is rendered more toxic by the action of elastase present in intestinal mucus, which cleaves the last two amino acids of the A2 portion of the toxin A subunit. We screened 153 STEC isolates from food, animals, and humans for the gene encoding Stx2dact by using a novel one-step PCR procedure. This method targeted the region of stx(2dact) that encodes the elastase recognition site. The presence of stx(2dact) was confirmed by DNA sequencing of the complete toxin genes. Seven STEC isolates from cows (four isolates), meat (two isolates), and a human (one isolate) that carried the putative stx(2dact) gene were identified; all were eae negative, and none was the O157:H7 serotype. Three of the isolates (CVM9322, CVM9557, and CVM9584) also carried stx(1), two (P1332 and P1334) carried stx(1) and stx(2c), and one (CL-15) carried stx(2c). One isolate, P1130, harbored only stx(2dact). The Vero cell cytotoxicities of supernatants from P1130 and stx(1) deletion mutants of CVM9322, CVM9557, and CVM9584 were increased 13- to 30-fold after treatment with porcine elastase. Thus, Stx2dact-producing strains, as detected by our one-step PCR method, can be isolated not only from humans, as previously documented, but also from food and animals. The latter finding has important public health implications based on a recent report from Europe of a link between disease severity and infection with STEC isolates that produce Stx2dact.