Enteropathogenic E. coli, Salmonella, and Shigella: masters of host cell cytoskeletal exploitation.

Bacterial pathogens have evolved numerous strategies to exploit their host's cellular processes so that they can survive and persist. Often, a bacterium must adhere very tightly to the cells and mediate its effects extracellularly, or it must find a way to invade the host's cells and survive intracellularly. In either case, the pathogen hijacks the host's cytoskeleton. The cytoskeleton provides a flexible framework for the cell and is involved in mediating numerous cellular functions, from cell shape and structure to programmed cell death. Altering the host cytoskeleton is crucial for mediating pathogen adherence, invasion, and intracellular locomotion. We highlight recent advances in the pathogenesis of enteropathogenic Escherichia coli, Salmonella Typhimurium, and Shigella flexneri. Each illustrates how bacterial pathogens can exert dramatic effects on the host cytoskeleton

Status report on the Los Alamos National Laboratory Ion Beam Facility

The Ion Beam Facility operated for 6000 machine hours last year, ranging in energy from 300 Kev to 24 Mev. Improvements include cryopumps replacing diffusion pumps, a rebuilding of the tandem chopper electronics and the vertical's corona charging system. Methane molecules were successfully accelerated by the vertical in quantities of hundreds of nanoamperes. Two replacement magnet power supplies on the tandem and a completely new capacitor shell regulator on the vertical are soon to be installed

Lack of Adherence of Clinical Isolates of Pseudomonas aeruginosa to Asialo-GM(1) on Epithelial Cells

Numerous studies have reported that asialo-GM(1), gangliotetraosylceramide, or moieties serve as epithelial cell receptors for Pseudomonas aeruginosa. Usually this interaction is confirmed with antibodies to asialo-GM(1). However, few, if any, of these reports have evaluated the binding of fresh clinical isolates of P. aeruginosa to asialo-GM(1) or the specificity of the antibodies for the asialo-GM(1) antigen. We confirmed that asialo-GM(1) dissolved in dimethyl sulfoxide could be added to the apical membrane of Madin-Darby canine kidney cells growing as a polarized epithelium on Transwell membranes (J. C. Comolli, L. L. Waite, K. E. Mostov, and J. N. Engel, Infect. Immun. 67:3207–3214, 1999) and that such treatment enhanced the binding of P. aeruginosa strain PA103. However, no other P. aeruginosa strain, including eight different clinical isolates, exhibited enhanced binding to asialo-GM(1)-treated cells. Studies with commercially available antibodies to asialo-GM(1) showed that these preparations had high titers of antibody to P. aeruginosa antigens, including whole cells, purified lipopolysaccharide (LPS), and pili. Inhibition studies showed that adsorption of an antiserum to asialo-GM(1) with P. aeruginosa cells could remove the reactivity of antibodies to asialo-GM(1), and adsorption of this serum with asialo-GM(1) removed antibody binding to P. aeruginosa LPS. Antibodies in sera raised to asialo-GM(1) were observed to bind to P. aeruginosa cells by immunoelectron microscopy. Antibodies to asialo-GM(1) inhibited formation of a biofilm by P. aeruginosa in the absence of mammalian cells, indicating a direct inhibition of bacterial cell-cell interactions. These findings demonstrate that asialo-GM(1) is not a major cellular receptor for clinical isolates of P. aeruginosa and that commercially available antibodies raised to this antigen contain high titers of antibody to multiple P. aeruginosa antigens, which do not interfere with the binding of P. aeruginosa to mammalian cells but possibly interfere with the binding of P. aeruginosa cells to each other

Haemophilus ducreyi Inhibits Phagocytosis by U-937 Cells, a Human Macrophage-Like Cell Line

Haemophilus ducreyi is a gram-negative obligate human pathogen that causes the genital ulcer disease chancroid. Chancroid lesions are deep necrotic ulcers with an immune cell infiltrate that includes macrophages. Despite the presence of these phagocytic cells, chancroid ulcers can persist for months and live H. ducreyi can be isolated from these lesions. To analyze the interaction of H. ducreyi with macrophages, we investigated the ability of H. ducreyi strain 35000 to adhere to, invade, and survive within U-937 cells, a human macrophage-like cell line. We found that although H. ducreyi strain 35000 adhered efficiently to U-937 cells, few bacteria were internalized, suggesting that H. ducreyi avoids phagocytosis by human macrophages. The few bacteria that were phagocytosed in these experiments were rapidly killed. We also found that H. ducreyi inhibits the phagocytosis of a secondary target (opsonized sheep red blood cells). Antiphagocytic activity was found in logarithmic, stationary-phase, and plate-grown cultures and was associated with whole, live bacteria but not with heat-killed cultures, sonicates, or culture supernatants. Phagocytosis was significantly inhibited after a 15-min exposure to H. ducreyi, and a multiplicity of infection of approximately 1 CFU per macrophage was sufficient to cause a significant reduction in phagocytosis by U-937 cells. Finally, all of nine H. ducreyi strains tested were antiphagocytic, suggesting that this is a common virulence mechanism for this organism. This finding suggests a mechanism by which H. ducreyi avoids killing and clearance by macrophages in chancroid lesions and inguinal lymph nodes

Helicobacter pylori Resists Phagocytosis by Macrophages: Quantitative Assessment by Confocal Microscopy and Fluorescence-Activated Cell Sorting

Helicobacter pylori infection of the stomach epithelium is characterized by an infiltration of polymorphonuclear and mononuclear cells. These immune cells contribute to mucosal damage which may eventually lead to gastritis, peptic ulcer, gastric cancer, and/or MALT-associated gastric lymphoma. Here we show that H. pylori inhibits its own uptake, as well as in trans the phagocytosis of Neisseria gonorrhoeae, by human and murine macrophages. This antiphagocytic activity is dependent on the presence of the cag pathogenicity island in the H. pylori genome. We demonstrate that H. pylori also expresses its antiphagocytic activity towards the myelomonocytic cell line JOSKM, thus providing a potent model for the study of the interaction between H. pylori and phagocytes. Our data were obtained using laser confocal microscopy and flow cytometry after quenching the fluorescence of labeled extracellular bacteria. The antiphagocytic activity of H. pylori may explain the persistence of H. pylori and its pathological consequences. The use of cell lines and flow cytometry will hopefully facilitate progress in our understanding of the immune escape of these persistent bacteria

Adherent Invasive Escherichia coli Strains from Patients with Crohn's Disease Survive and Replicate within Macrophages without Inducing Host Cell Death

Escherichia coli strains recovered from Crohn's disease (CD) lesions are able to adhere to and invade cultured intestinal epithelial cells. We analyzed the behavior within macrophages of adherent invasive E. coli (AIEC) strains isolated from patients with CD. All the 15 AIEC strains tested were able to replicate extensively within J774-A1 cells: the numbers of intracellular bacteria increased 2.2- to 74.2-fold at 48 h over that at 1 h postinfection. By use of murine peritoneal macrophages and human monocyte-derived-macrophages, the reference AIEC strain LF82 was confirmed to be able to survive intracellularly. Transmission electron micrographs of AIEC LF82-infected macrophages showed that at 24 h postinfection, infected cells harbored large vacuoles containing numerous bacteria, as a result of the fusion of several vacuoles occurring after 8 h postinfection. No lactate dehydrogenase (LDH) release, no sign of DNA fragmentation or degradation, and no binding to fluorescein isothlocyanate-labeled annexin V were observed with LF82-infected J774-A1 cells, even after 24 h postinfection. LF82-infected J774-A1 cells secreted 2.7-fold more tumor necrosis factor alpha (TNF-α) than cells stimulated with 1 μg of lipopolysaccharide (LPS)/ml. No release of interleukin-1β was observed with LPS-prestimulated J774-A1 cells infected with AIEC LF82. These findings showed that (i) AIEC strains are able to survive and to replicate within macrophages, (ii) AIEC LF82 replication does not induce any cell death of the infected cells, and (iii) LF82-infected J774-A1 cells release high levels of TNF-α. These properties could be related to some features of CD and particularly to granuloma formation, one of the hallmarks of CD lesions