cytochalasin-d and Escherichia-coli-Infections

Haemolytic uraemic syndrome caused by Shiga toxin-producing E. coli (STEC) is dependent on release of Shiga toxins (Stxs) during intestinal infection and subsequent absorption into the bloodstream. An understanding of Stx-related events in the human gut is limited due to lack of suitable experimental models. In this study, we have used a vertical diffusion chamber system with polarized human colon carcinoma cells to simulate the microaerobic (MA) environment in the human intestine and investigate its influence on Stx release and translocation during STEC O157:H7 and O104:H4 infection. Stx2 was the major toxin type released during infection. Whereas microaerobiosis significantly reduced bacterial growth as well as Stx production and release into the medium, Stx translocation across the epithelial monolayer was enhanced under MA versus aerobic conditions. Increased Stx transport was dependent on STEC infection and occurred via a transcellular pathway other than macropinocytosis. While MA conditions had a similar general effect on Stx release and absorption during infection with STEC O157:H7 and O104:H4, both serotypes showed considerable differences in colonization, Stx production, and Stx translocation which suggest alternative virulence strategies. Taken together, our study suggests that the MA environment in the human colon may modulate Stx-related events and enhance Stx absorption during STEC infection.

Topics: Anaerobiosis; Animals; Cell Line, Tumor; Chlorocebus aethiops; Colonic Diseases; Cytochalasin D; Escherichia coli Infections; Gastrointestinal Tract; Hemolytic-Uremic Syndrome; Heterocyclic Compounds, 4 or More Rings; Humans; Oxygen; Pinocytosis; Shiga Toxin; Shiga-Toxigenic Escherichia coli; Vero Cells

Extra-intestinal pathogenic Escherichia coli (ExPEC) strains are responsible for a wide range of diseases in humans and animals. Using in vitro invasion assays and transmission electron microscopy, we showed that BEN2908, an ExPEC strain of avian origin (also termed APEC for Avian Pathogenic E. coli), is able to usurp cellular endocytic pathways to invade A549 human type II pneumocytes and LMH avian hepatocytes where it is able to survive over several days. Although type 1 fimbriae are the major adhesin of BEN2908, proportions of adherent fimbriated or afimbriated bacteria that entered cells were comparable. Internalization of BEN2908 into human pneumocytes reinforces previous studies indicating that APEC strains could represent a zoonotic risk.

Topics: Alveolar Epithelial Cells; Animals; Anti-Bacterial Agents; Birds; Cell Line; Chlorpromazine; Cytochalasin D; Endocytosis; Epithelial Cells; Escherichia coli; Escherichia coli Infections; Escherichia coli Proteins; Filipin; Fimbriae, Bacterial; Hepatocytes; Humans; Microbial Viability; Microscopy, Electron, Transmission; Nystatin; Time Factors

Bovine mastitis caused by Escherichia coli has traditionally been viewed as a transient infection. However, E. coli can also cause clonal persistent intramammary infection (IMI) in dairy cows. In this study, we explored the possibility that E. coli strains associated with persistent IMI are better able to adhere to, invade, survive and replicate in cultured mammary epithelial cells (MAC-T) than transient strains, and examined their serotype, overall genotype, phylogenetic group, and the presence of known virulence genes. Both transient and persistent E. coli strains adhered to MAC-T cells, but persistent strains invaded MAC-T cells 2.6-63.5 times more than transient strains. Blocking the adhesin/invasin FimH with mannose diminished but did not eliminate adhesion and invasion of any strain. Cytoskeletal and protein kinase inhibitors cytochalasin D, colchicine, genistein and wortmannin dramatically reduced invasion of MAC-T cells by both strains. All of the persistent strains, but only one transient strain, were able to survive and replicate intracellularly in MAC-T cells over 48 h. Transient and persistent strains displayed heterogeneous serotypes and overall genotypes, but similar phylogeny (group A), and lacked virulence genes of invasive E. coli. We have found that E. coli strains associated with persistent IMI are better able to invade and replicate within cultured mammary epithelial cells than transient strains. The invasion process involves the host cytoskeleton and signaling cascades and is not FimH dependent. Our findings suggest that the invasion of mammary epithelial cells and intracellular survival play an important role in the pathogenesis of persistent E. coli mastitis.

Topics: Analysis of Variance; Animals; Bacterial Adhesion; Cattle; Cells, Cultured; Colchicine; Cytochalasin D; Enzyme Inhibitors; Epithelial Cells; Escherichia coli; Escherichia coli Infections; Female; Genistein; Genotype; In Situ Hybridization, Fluorescence; Mammary Glands, Animal; Mastitis, Bovine; Phylogeny; Serotyping; Virulence

One mechanism of initiating innate host defenses against uropathogenic Escherichia coli (UPEC) is the production of cytokines by bladder epithelial cells; however, the means by which these cells recognize bacterial pathogens is poorly understood. Type 1 pili, expressed by the majority of UPEC, have been shown to have a critical role in inducing the expression of IL-6 in bladder epithelial cells after exposure to E. coli. In this study, we demonstrate that type 1 pili are not sufficient to activate IL-6 production by bladder epithelial cells. Instead, it was shown that bacterial invasion mediated by type 1 pili augments bladder epithelial responses to E. coli via an LPS-dependent mechanism, leading to the production of IL-6. RNA transcripts for the LPSR Toll-like receptor 4 (TLR4) was detected in cultured bladder epithelial cells. The in vivo role of TLR4 was assessed using C3H/HeJ mice, which express a dominant negative form of TLR4. After infection with UPEC, C3H/HeJ mice have large foci of intracellular bacteria that persist within the bladder epithelium in the absence of any notable inflammatory response. These results indicate that LPS is required for bacterial invasion to enhance host responses to E. coli within the bladder.

Topics: Adhesins, Bacterial; Adhesins, Escherichia coli; Adjuvants, Immunologic; Animals; Bacteriuria; Cytochalasin D; Cytokines; Drosophila Proteins; Escherichia coli; Escherichia coli Infections; Female; Fimbriae Proteins; Fimbriae, Bacterial; Humans; Immunosuppressive Agents; Interleukin-6; Lipopolysaccharides; Membrane Glycoproteins; Mice; Mice, Inbred C3H; Receptors, Cell Surface; Toll-Like Receptor 4; Toll-Like Receptors; Tumor Cells, Cultured; Urinary Bladder; Urothelium

The purpose of this study was to investigate the interaction between Escherichia coli and primary mammary epithelial cell cultures derived from cows with persistent intramammary infection (IMI). Two strains of E. coli, isolated from the milk of two different cows suffering from persistent E. coli IMI were tested for adhesion to and invasion of three primary mammary epithelial cell cultures derived from mammary biopsies of the two infected cows. Intracellular E. coli were detected during five days post infection in vitro. Both strains of E. coli adhered to and invaded monolayers of all three primary mammary epithelial cell cultures. One strain adhered less but invaded more than the other. Comparison with other mammary pathogens indicated that E. coli invaded the cells less efficiently than Staphylococcus aureus, about as efficiently as Streptococcus dysgalactiae and more efficiently than Streptococcus uberis. The mechanism of E. coli invasion was studied using the cytoskeleton disrupting agents colchicine and cytochalasin D. These compounds inhibited the invasion of E. coli. Invasion of E. coli could also be inhibited by the phosphokinase inhibitors genistein and staurosporin in a dose-dependent fashion. Phorbol-myristyl-acetate (PMA) had no effect on the invasion of E. coli. Histology of mammary tissue revealed chronic inflammatory changes in quarters that were persistently infected by E. coli. Intracellular bacteria were not detected in mammary tissue sections. Polymerase chain reaction (PCR) analysis suggested that the two strains of E. coli lacked genes encoding for bundle-forming pili (bfpA), intimin (eae) and translocated intimin receptor (tir), which are characteristic for enteropathogenic E. coli (EPEC).

Topics: Animals; Bacterial Adhesion; Cattle; Cells, Cultured; Colchicine; Cytochalasin D; Enzyme Inhibitors; Epithelial Cells; Escherichia coli; Escherichia coli Infections; Female; Genistein; Histocytochemistry; Mammary Glands, Animal; Mastitis, Bovine; Polymerase Chain Reaction; Staphylococcus aureus; Streptokinase

Escherichia coli isolates that cause meningitis in newborns are able to invade the circulation and subsequently cross the blood-brain barrier. One mechanism for traversing the blood-brain barrier might involve transcytosis through the endothelial cells. The ability of the meningitis isolate E. coli IHE3034, of serotype 018:K1:H7, to invade epithelial (T24) and endothelial (EA-hy926) cells was investigated by the standard gentamicin survival assay and by electron microscopy. Human bladder epithelial and endothelial cells were efficiently invaded by strain IHE3034, whereas epithelial human colon Caco-2 cells, canine kidney MDCK cells, and the opossum [correction of opposum] epithelial kidney cell line OK were not invaded. The ability to invade human epithelial cells of the bladder could also be demonstrated for several other newborn meningitis E. coli strains and one septicemic E. coli strain. Studies utilizing inhibitors which act on eukaryotic cells revealed a dependence on microfilaments as well as on microtubules in the process of E. coli IHE3034 entry into T24 and EA-hy926 cells. These results indicated that cell cytoskeletal rearrangements are involved in bacterial uptake and suggest that there are either two pathways (microtubule dependent and microfilament dependent) or one complex pathway involving both microtubules and microfilaments. The intracellular IHE3034 organisms were contained in a host-membrane-confined compartment mainly as single microorganisms. Intracellular replication of 1HE3034 was not detected, nor did the number of intracellular bacteria decrease significantly during a 48-h period. The ability of E. coli O18:K1 to invade and survive within certain eukaryotic cells may be another virulence factor of meningitis-associated E. coli.

Topics: Actin Cytoskeleton; Animals; Cell Line; Colchicine; Cytochalasin D; Dogs; Endothelium; Epithelium; Escherichia coli; Escherichia coli Infections; Humans; Infant, Newborn; Meningitis, Bacterial; Microscopy, Electron; Microtubules; Nocodazole; Penicillins; Streptomycin; Virulence

Diffusely adherent Escherichia coli (DAEC) are diarrheagenic E. coli whose pathogenetic mechanisms are largely unknown. DAEC have been shown to induce an unusual phenotype upon adherence to HEp-2 cells in culture characterized by the induction of long thin membrane processes extending from the cell surface. In addition, DAEC have been shown to be protected from the bactericidal effects of gentamicin when incubated with HEp-2 cells. In our studies, we found that three DAEC strains induced formation of eukaryotic cell processes and were protected from gentamicin killing after a 3 h incubation. Preincubation of HEp-2 cells with colchicine or cytochalasin D prior to infection with DAEC strain C1845 resulted in decreased projection formation, suggesting that the effect was dependent upon microfilament and microtubule rearrangement. When the standard gentamicin protection assay was extended for an additional 3 h incubation in the presence of gentamicin, a greater number of DAEC survived gentamicin treatment, more eukaryotic projections were seen in association with the bacteria and the bacteria were actually observed to be "embedded' within these projections. Projection formation was not observed when the bacteria were separated from the cells by a permeable membrane or when the inoculum was inactivated by ultraviolet irradiation. Transposon TnphoA mutants of C1845 were screened for decreased gentamicin protection. All three mutants which were deficient in gentamicin protection demonstrated less projection formation. Insertion mutations affecting gentamicin protection were localized to both the chromosome (two) and a plasmid (one). Eukaryotic projections are a novel interaction of DAEC with epithelial cells, may play a role of the survival of the bacteria against host defenses and may contribute to DAEC pathogenesis. The effect is dependent upon epithelial cell contact and requires multiple bacterial genes.

Topics: Actin Cytoskeleton; Anti-Bacterial Agents; Bacterial Adhesion; Caco-2 Cells; Cells, Cultured; Colchicine; Cytochalasin D; DNA Transposable Elements; Escherichia coli; Escherichia coli Infections; Gentamicins; Humans; Microscopy, Electron; Microtubules; Mutagenesis, Insertional; Nucleic Acid Synthesis Inhibitors; Plasmids; Pseudopodia

A gentamicin survival assay, using primary human renal epithelial cells and Escherichia coli strains isolated from the feces of asymptomatic individuals and from the urine or blood of patients with acute pyelonephritis, was used to investigate bacterial internalization as a model for renal parenchymal invasion in pyelonephritis. E. coli strains, regardless of their origin, efficiently entered into human renal epithelial cells, a process inhibited by cytochalasin D. While the percentage of survival of nonhemolytic pyelonephritis isolates did not differ from that of fecal isolates, survival of hemolytic pyelonephritis strains was lower than that of nonhemolytic strains, perhaps as a consequence of the greater cytotoxicity of hemolytic strains. There was no evidence of intracellular multiplication of E. coli. These results demonstrate that human renal epithelial cells are capable of efficient uptake of E. coli regardless of the source of the bacteria.

Topics: Cells, Cultured; Cytochalasin D; Dose-Response Relationship, Drug; Epithelial Cells; Epithelium; Escherichia coli; Escherichia coli Infections; Feces; Female; Gentamicins; Humans; Kidney; Kidney Tubules, Collecting; Kidney Tubules, Proximal; Microbial Sensitivity Tests; Pyelonephritis