The effect of enterohemorrhagic E. coli infection on the cell mechanics of host cells.

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.

Intracellular viscoelasticity of HeLa cells during cell division studied by video particle-tracking microrheology.

Cell division plays an important role in regulating cell proliferation and differentiation. It is managed by a complex sequence of cytoskeleton alteration that induces dividing cells to change their morphology to facilitate their division. The change in cytoskeleton structure is expected to affect the intracellular viscoelasticity, which may also contribute to cellular dynamic deformation during cell division. However, the intracellular viscoelasticity during cell division is not yet well understood. In this study, we injected 100-nm (diameter) carboxylated polystyrene beads into the cytoplasm of HeLa cells and applied video particle tracking microrheology to measure their intracellular viscoelasticity at different phases during cell division. The Brownian motion of the intracellular nanoprobes was analyzed to compute the viscoelasticity of HeLa cells in terms of the elastic modulus and viscous modulus as a function of frequency. Our experimental results indicate that during the course of cell division, both intracellular elasticity and viscosity increase in the transition from the metaphase to the anaphase, plausibly due to the remodeling of cytoskeleton and redistributions of molecular motors, but remain approximately the same from the anaphase to the telophase.

Metabolism of HeLa cells revealed through autofluorescence lifetime upon infection with enterohemorrhagic Escherichia coli.

Fluorescence lifetime imaging microscopy (FLIM) is a sensitive technique in monitoring functional and conformational states of nicotinamide adenine dinucleotide reduced (NADH) and flavin adenine dinucleotide (FAD),main compounds participating in oxidative phosphorylation in cells. In this study, we have applied FLIM to characterize the metabolic changes in HeLa cells upon bacterial infection and made comparison with the results from the cells treated with staurosporine (STS), a well-known apoptosis inducer. The evolving of NADH's average autofluorescence lifetime during the 3 h after infection with enterohemorragic Escherichia coli (EHEC) or STS treatment has been observed. The ratio of the short and the long lifetime components' relative contributions of NADH increases with time, a fact indicating cellular metabolic activity, such as a decrease of oxidative phosphorylation over the course of infection, while opposite dynamics is observed in FAD. Being associated with mitochondria, FAD lifetimes and redox ratio could indicate heterogeneous mitochondrial function, microenvironment with bacterial infection, and further pathway to cell death. The redox ratios for both EHEC-infected and STS-treated HeLa cells have been observed and these observations also indicate possible apoptosis induced by bacterial infection.

Bacterial colony from two-dimensional division to three-dimensional development.

On agar surface, bacterial daughter cells form a 4-cell array after the first two rounds of division, and this phenomenon has been previously attributed to a balancing of interactions among the daughter bacteria and the underneath agar. We studied further the organization and development of colony after additional generations. By confocal laser scanning microscopy and real-time imaging, we observed that bacterial cells were able to self-organize and resulted in a near circular micro-colony consisting of monolayer cells. After continuous dividing, bacteria transited from two-dimensional expansion into three-dimensional growth and formed two to multi-layers in the center but retained a monolayer in the outer ring of the circular colony. The transverse width of this outer ring appeared to be approximately constant once the micro-colony reached a certain age. This observation supports the notion that balanced interplays of the forces involved lead to a gross morphology as the bacteria divide into offspring on agar surface. In this case, the result is due to a balance between the expansion force of the dividing bacteria, the non-covalent force among bacterial offspring and that between bacteria and substratum.

Factors affecting daughter cells' arrangement during the early bacterial divisions.

On agar plates, daughter cells of Escherichia coli mutually slide and align side-by-side in parallel during the first round of binary fission. This phenomenon has been previously attributed to an elastic material that restricts apparently separated bacteria from being in string. We hypothesize that the interaction between bacteria and the underneath substratum may affect the arrangement of the daughter bacteria. To test this hypothesis, bacterial division on hyaluronic acid (HA) gel, as an alternative substratum, was examined. Consistent with our proposition, the HA gel differs from agar by suppressing the typical side-by-side alignments to a rare population. Examination of bacterial surface molecules that may contribute to the daughter cells' arrangement yielded an observation that, with disrupted lpp, the E. coli daughter cells increasingly formed non-typical patterns, i.e. neither sliding side-by-side in parallel nor forming elongated strings. Therefore, our results suggest strongly that the early cell patterning is affected by multiple interaction factors. With oscillatory optical tweezers, we further demonstrated that the interaction force decreased in bacteria without Lpp, a result substantiating our notion that the side-by-side sliding phenomenon directly reflects the strength of in-situ interaction between bacteria and substratum.