phalloidine and Escherichia-coli-Infections

Enterohaemorrhagic E. coli (EHEC) is a type of human pathogenic bacteria. The main virulence characteristics of EHEC include the formation of attaching and effacing lesions (A/E lesions) and the production of one or more Shiga-like toxins, which may induce human uremic complications. When EHEC infects host cells, it releases translocated intimin receptor (Tir) and effector proteins inside the host cells, inducing the rearrangement and accumulation of the F-actin cytoskeleton, a phenotype leading to the formation of pedestals in the apical cell surface, and the growth of stress fibers at the base of the cells. To examine the effect of EHEC infection on cell mechanics, we carried out a series of experiments to examine HeLa cells with and without EHEC infection to quantify the changes in (1) focal adhesion area, visualized by anti-vinculin staining; (2) the distribution and orientation of stress fibers; and (3) the intracellular viscoelasticity, via directional video particle tracking microrheology. Our results indicated that in EHEC-infected HeLa cells, the focal adhesion area increased and the actin stress fibers became thicker and more aligned. The cytoskeletal reorganization induced by EHEC infection mediated a dramatic increase in the cytoplasmic elastic shear modulus of the infected cells, and a transition in the viscoelastic behavior of the cells from viscous-like to elastic-like. These changes in mechanobiological characteristics might modulate the attachments between EHEC and the host cell to withstand exfoliation, and between the host cell and the extracellular matrix, and might also alter epithelial integrity.

Topics: Actin Cytoskeleton; Elasticity; Enterohemorrhagic Escherichia coli; Escherichia coli Infections; Fluorescence Polarization; Focal Adhesions; HeLa Cells; Host-Pathogen Interactions; Humans; Phalloidine

Escherichia coli D2 (serotype 07:H-) that was isolated from a child with diarrhea hybridized with an F1845 DNA probe used to detect diffuse adherence. Strain D2 adhered to tissue culture cells (HeLa and HEp-2 cells) in a clustered pattern but did not autoagglutinate on the cell surface and induced the elongation of microvilli after 3 h of incubation. After 6 h of incubation, the infected cells were positive for fluorescent-actin staining at the site of clustered adherence. When analyzed with a confocal laser scanning microscope, each D2 cell was surrounded by accumulated actin in a capsule-like formation. Capsule-like, accumulated actin was also observed with enteropathogenic E. coli (EPEC), although in this case, actin accumulation was associated with EPEC microcolonies in a localized pattern. Four other strains of F1845 DNA probe-positive, diffusely adhering E. coli were negative for actin accumulation. Strain D2 did not hybridize with EPEC attaching and effacing DNA or EPEC adherence factor DNA probes. In addition, clustered D2 cells were found inside tissue culture cells. The data suggest a novel infectious mechanism as well as genetic heterogeneity of F1845 DNA probe-positive E. coli. Capsule-like, accumulated actin may protect the bacteria from host defense mechanisms.

Topics: Actins; Bacterial Adhesion; Diarrhea; DNA Probes; Escherichia coli; Escherichia coli Infections; Female; Fluorescein-5-isothiocyanate; Humans; Infant; Microscopy, Electron; Microscopy, Fluorescence; Microvilli; Phalloidine; Plasmids; Thailand; Transformation, Genetic; Tumor Cells, Cultured; Virulence