Real-time sensing of enteropathogenic E. coli-induced effects on epithelial host cell height, cell-substrate interactions, and endocytic processes by infrared surface plasmon spectroscopy.

Enteropathogenic Escherichia coli (EPEC) is an important, generally non-invasive, bacterial pathogen that causes diarrhea in humans. The microbe infects mainly the enterocytes of the small intestine. Here we have applied our newly developed infrared surface plasmon resonance (IR-SPR) spectroscopy approach to study how EPEC infection affects epithelial host cells. The IR-SPR experiments showed that EPEC infection results in a robust reduction in the refractive index of the infected cells. Assisted by confocal and total internal reflection microscopy, we discovered that the microbe dilates the intercellular gaps and induces the appearance of fluid-phase-filled pinocytic vesicles in the lower basolateral regions of the host epithelial cells. Partial cell detachment from the underlying substratum was also observed. Finally, the waveguide mode observed by our IR-SPR analyses showed that EPEC infection decreases the host cell's height to some extent. Together, these observations reveal novel impacts of the pathogen on the host cell architecture and endocytic functions. We suggest that these changes may induce the infiltration of a watery environment into the host cell, and potentially lead to failure of the epithelium barrier functions. Our findings also indicate the great potential of the label-free IR-SPR approach to study the dynamics of host-pathogen interactions with high spatiotemporal sensitivity

Enteropathogenic Escherichia coli remodels host endosomes to promote endocytic turnover and breakdown of surface polarity.

Enteropathogenic E. coli (EPEC) is an extracellular diarrheagenic human pathogen which infects the apical plasma membrane of the small intestinal enterocytes. EPEC utilizes a type III secretion system to translocate bacterial effector proteins into its epithelial hosts. This activity, which subverts numerous signaling and membrane trafficking pathways in the infected cells, is thought to contribute to pathogen virulence. The molecular and cellular mechanisms underlying these events are not well understood. We investigated the mode by which EPEC effectors hijack endosomes to modulate endocytosis, recycling and transcytosis in epithelial host cells. To this end, we developed a flow cytometry-based assay and imaging techniques to track endosomal dynamics and membrane cargo trafficking in the infected cells. We show that type-III secreted components prompt the recruitment of clathrin (clathrin and AP2), early (Rab5a and EEA1) and recycling (Rab4a, Rab11a, Rab11b, FIP2, Myo5b) endocytic machineries to peripheral plasma membrane infection sites. Protein cargoes, e.g. transferrin receptors, β1 integrins and aquaporins, which exploit the endocytic pathways mediated by these machineries, were also found to be recruited to these sites. Moreover, the endosomes and cargo recruitment to infection sites correlated with an increase in cargo endocytic turnover (i.e. endocytosis and recycling) and transcytosis to the infected plasma membrane. The hijacking of endosomes and associated endocytic activities depended on the translocated EspF and Map effectors in non-polarized epithelial cells, and mostly on EspF in polarized epithelial cells. These data suggest a model whereby EPEC effectors hijack endosomal recycling mechanisms to mislocalize and concentrate host plasma membrane proteins in endosomes and in the apically infected plasma membrane. We hypothesize that these activities contribute to bacterial colonization and virulence

Schematic representation of the effects of EPEC infection on epithelial host cell architecture.

<p>In normal uninfected columnar epithelial cells (left panel), the cells maintain close intercellular contacts via tight junctions (TJ), and other junctional complexes (e.g., adherence junctions, gap junctions, and desmosomes). These junction complexes allow the regulation and exchange of different compounds between the underlying tissues and external body cavities, as well as among the connected cells. They are also responsible for maintaining physical contact between the cells and the underlying substrate. In particular, epithelial TJs, which are linked to actin fibers (the apical actin belt), interconnect individual cells into a continuous and rigid epithelial cell sheet. Proper TJ-actin interconnections are essential for developing tall and columnar epithelial cell morphology [42,43]. EPEC-<i>wt</i> (middle panel) attached to the apical cell surface of host epithelial cells injects a series of protein effectors into the host cells via T3SS (not shown), among which are effector proteins that subvert the TJs and the actin cytoskeleton, and thereby have broad effects on the epithelial host cell monolayer. Our novel findings suggest that some effector proteins exclusively induce the formation of large fluid-phase-filled endocytic vesicles, reminiscent of (macro)pinosomes. They also evoke substantial host cell detachment from the underlying substratum and a reduction in cell height. The latter could be contributed by a loss of the cortical actin tension maintained by the TJs and the associated cortical actin belt [43]. On the basis of these observations, and combined with other data implying that EPEC disrupts the TJ barrier functions, we can conclude that apically adhered EPEC impair the structural properties of the host in a way that causes a watery extracellular environment to infiltrate into the epithelial sheets, and possibly to the gut’s lumen, which contributes to the diarrheal effect. EPEC-<i>escV</i> (right panel) causes host cell basolateral membrane ruffling, expansion of intercellular spaces and some degree of host cell detachment, but does not initiate (macro)pinocytic vesicles formation. EPEC-<i>escV</i> has a much weaker effect on the disorganization of the epithelial cell monolayer and the actin cytoskeleton, which still allows the maintenance of the epithelium barrier function. Clearly, these type III secretion independent processes might be contributed by factors associated with the bacterial exterior. One interesting candidate is the EPEC bundle-forming pilus (BFP), which extends out from the bacteria and mediates the initial attachment of EPEC to its host [4]. Following attachment to the host, BFP retracts by a very powerful force generating machinery, bringing to a close apposition the bacterial and host cell surfaces [53]. The host cell may respond to this mechanical force by reorganizing its cortical actin cytoskeleton [54,55], signal transducing proteins [56] and proteins of the junction polarity complex [57]. An intriguing hypothesis is that at least some of these effects have contributed to the type III-independent changes observed upon EPEC-<i>escV</i> attachment to its host. </p

Confocal live cell imaging analysis of MDCK cells expressing LifeAct-GFP exposed to a fluid-phase fluorescent marker, SRB.

<p><b>A</b>. <b>Time series of confocal image sections taken from the lower basolateral regions of an MDCK cell monolayer</b>. A confluent MDCK cell monolayer, cultured on a multi-well plate (see Materials and Methods), was co-exposed to MEM medium containing the fluorescent fluid-phase marker, SRB (1µM), and the indicated bacterial strains, or to bacteria-free growth medium (uninfected). Note the pronounced enlargement of intercellular spaces in EPEC-<i>escV</i>- and EPEC-<i>wt</i>-infected cells compared with uninfected cells. These intercellular expanding spaces became visible ~80 min after cell exposure to the microbes. Also note the appearance of SRB-filled vesicles in the cytoplasm of EPEC-<i>wt-</i>infected cells. For clarity, the LifeAct-GFP labeling was omitted from the images. Scale bar: 20 µm. <b>B</b>. <b>Zoom of the intercellular interface of EPEC-<i>wt</i>-infected cells</b>. The image series clearly shows the gradual expansion of intercellular spaces filled with SRB and the emerging fluid-phase-filled endocytic vesicles over time. For clarity, the LifeAct-GFP labeling was omitted from the images. <b>C</b>. <b>Visualization of LifeAct-GFP and the extracellular SRB probe at XY and XZ resolutions</b>. An MDCK cell clone stably expressing LifeAct-GFP (green) was exposed to SRB (red) and EPEC-<i>wt</i>-containing medium. A confocal image at XY and XZ resolutions was taken 180 min after EPEC infection. The XY section, which was taken at a lower basal-lateral cell region (~1.5 µm above the cell-substrate interface), shows the actin-labeled tubular-ruffles (green) protruding into the SRB-labeled intercellular spaces (red, indicated by an arrow). Large intracellular SRB-containing vesicles are seen in these cells (indicated by an arrowhead). The XZ section shows a gap between the basal portion of the cell and the underlying substrate, which is filled with the fluid phase SRB marker (indicated by an arrow). Scale bar: 10 µm.</p

Temporal changes in the MDCK cell monolayer refractive indices and the average cell height.

<p><b>A</b>. <b>Time-dependent changes in the refractive index</b>. Confluent MDCK cell monolayers that formed after 6-7 days of culturing on an Au-coated prism were exposed to EPEC-<i>wt</i> (blue), EPEC-<i>escV</i> (red), EPEC-∆<i>espF</i> (purple), non-pathogenic HB101 laboratory bacteria (green), or just exposed to bacteria-free growth medium–uninfected (gray). <b>B</b>. <b>Time-dependent changes in the TM₀₁ waveguide mode</b>. The cell height was calculated from the waveguide (TM₀₁) resonant wavenumber. Since the initial cell height in these experiments varied from h₀=8.1 µm to h₀=11.2 µm, we show the relative cell height normalized to its initial value, i.e., h₀ is 100%.</p

Analysis of MDCK host cell basal membrane attachment to the substrate by TIRF microscopy.

<p>Confluent MDCK cell monolayers transiently expressing VSVG-YFP were grown on glass bottom dishes and imaged by TIRF and bright field (BF) microscopy. Images were acquired for at least 20 min prior to the addition of bacteria (not shown), immediately upon cell exposure to EPEC (t=0 min) and at subsequently at 1 min intervals. <b>A</b>. <b>Representative images</b>. Representative fluorescent and BF images of EPEC-<i>wt</i>- and EPEC-<i>escV</i>-infected cells are shown at the indicated times. Arrows in the BF images point towards cells that bulged out of the host cell monolayer. About 30% of the cells in the monolayer seemed to show this detaching behavior after 120 min of infection with EPEC-<i>wt</i>. In contrast, none of the host cells bulged out and detached from the cell monolayer upon EPEC-<i>escV</i> infection. The rounded cells seen in the BF images of the EPEC-<i>escV</i>-infected cells are cell-like floaters swept accidentally into the optical field. <b>B</b>. <b>Quantitative analysis</b>. Data plotted are a moving average of intensity measurement for at least 5 cells corrected for photobleaching and normalized to t=-5 min (pre-infection).</p

SPR and waveguide mode spectroscopy of infected MDCK cell monolayers.

<p><b>A</b>. <b>Experimental setup</b>. The lower panel illustrates an MDCK cell layer cultured on a ZnS prism coated with 20 nm Au film, as described in Materials and Methods. The prism with cells was mounted on a flow chamber and exposed to <i>E</i>. <i>coli</i> strains, which were continuously injected into the flow chamber. Cells and bacteria were imaged simultaneously with the IR-SPR measurements by light microscopy, as we previously described [14]. The upper panel shows images, obtained by microscope, of cells immediately upon (t=0 min) and 30 min after (t=30 min) EPEC-<i>wt</i> injection. Encircled are cell-adhering bacterial microcolonies following 30 min of MDCK cell exposure to EPEC. The cell layer was monitored by IR-SPR, in which a collimated and polarized infrared beam from the FTIR spectrometer was reflected from the cell/Au/ZnS prism assembly at an angle corresponding to the surface plasmon (SP) excitation in the Au/cell interface. The typical decay length of the SP field is 2 µm and 50 µm in the vertical and lateral directions, respectively. <b>B</b>. <b>Time evolution of the infrared reflectivity spectrum (R_p-polarized /R_s-polarized) upon host cell-bacteria interaction</b>. EPEC-<i>wt</i> injection was started at t=0 and continued for ~30 min (highlighted by a darkened box). The entire measurement lasted ~ 200 min. Note the gradual broadening and blue-shift of the SPR band and its merging with the TM₀₁ waveguide mode. <b>C</b>. <b>Representative infrared reflectivity spectra immediately upon, and after 120 or 180 min of bacterial injection</b>. Upon bacterial infection the sharp surface plasmon reflectivity dip broadens and becomes blue-shifted. Note the gradual disappearance of the additional reflectivity dip (4400 cm^-1) corresponding to the TM₀₁ waveguide mode propagating in the cell monolayer. <b>D</b>. <b>Time dependence of the SPR wavenumber (main y-axis, left) and the corresponding refractive index of the cell layer <i>Δn</i> (secondary blue-colored y-axis, left) during bacterial infection (blue line)</b>. <i>Δn</i> is the difference between the refractive indices of the cell monolayer and the buffer medium <i>Δn</i>=<i>n</i>_<i>cell</i>-<i>n</i>_{<i>medium</i>}. EPEC-<i>wt</i> infection results in a significant blue-shift of the SPR wavenumber, indicating a decrease in <i>Δn</i>. The <i>Δn</i>=0 at the end of the experiment has been measured after completely removing cells from the substrate by trypsinization. The red line shows the wavenumber of the TM₀₁ waveguide resonance in the course of EPEC-<i>wt</i> infection. Its change reveals a 1 µm reduction in the average cell monolayer height (secondary right y-axis) (from h=9 µm to h=8 µm) after ~160 min of infection. Thereafter, the TM₀₁ resonance becomes smeared and disappears (double zigzag line). This indicates disruption, specifically that the cell monolayer integrity is severely disrupted, and does no longer support the propagation of the waveguide mode. <i>The </i><i>inset</i> is a zoom-in of the boxed area in the main plot, essentially showing the <i>Δn</i> (t) and cell height dependences recorded for early infection times. </p

Bundle-forming pilus retraction enhances enteropathogenic Escherichia coli infectivity

Enteropathogenic Escherichia coli (EPEC) and other pathogenic bacteria use dynamic type IV pili to adhere to the host. Here we show that the capacity of the EPEC type IV pili to retract is required for the breakdown of the host epithelial tight-junction barrier, efficient actin-pedestal formation, and translocation of effectors via the type III secretion system