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. 2014 Jul;82(7):2860-71.
doi: 10.1128/IAI.01681-14. Epub 2014 Apr 28.

Epithelial microvilli establish an electrostatic barrier to microbial adhesion

Kaila M Bennett  1 , Sharon L Walker  2 , David D Lo  3
Affiliations

Epithelial microvilli establish an electrostatic barrier to microbial adhesion

Kaila M Bennett et al. Infect Immun. 2014 Jul.

Abstract

Microvilli are membrane extensions on the apical surface of polarized epithelia, such as intestinal enterocytes and tubule and duct epithelia. One notable exception in mucosal epithelia is M cells, which are specialized for capturing luminal microbial particles; M cells display a unique apical membrane lacking microvilli. Based on studies of M cell uptake under different ionic conditions, we hypothesized that microvilli may augment the mucosal barrier by providing an increased surface charge density from the increased membrane surface and associated glycoproteins. Thus, electrostatic charges may repel microbes from epithelial cells bearing microvilli, while M cells are more susceptible to microbial adhesion. To test the role of microvilli in bacterial adhesion and uptake, we developed polarized intestinal epithelial cells with reduced microvilli ("microvillus-minus," or MVM) but retaining normal tight junctions. When tested for interactions with microbial particles in suspension, MVM cells showed greatly enhanced adhesion and uptake of particles compared to microvillus-positive cells. This preference showed a linear relationship to bacterial surface charge, suggesting that microvilli resist binding of microbes by using electrostatic repulsion. Moreover, this predicts that pathogen modification of electrostatic forces may contribute directly to virulence. Accordingly, the effacement effector protein Tir from enterohemorrhagic Escherichia coli O157:H7 expressed in epithelial cells induced a loss of microvilli with consequent enhanced microbial binding. These results provide a new context for microvillus function in the host-pathogen relationship, based on electrostatic interactions.

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Figures

FIG 1
FIG 1
Development of microvillus-minus (MVM) cell lines by using Caco2BBe. (A) Flow cytometry histograms showing that WT Caco2BBe cells transfected with EPI64LA-DsRed have stable expression of DsRed protein (solid lines) compared to the WT unmanipulated control (dotted lines). (B) Confocal micrographs showing DsRed (red) expression and decreased phalloidin/F-actin staining (green) compared to WT cells. Phalloidin intensity was quantified, and significance was determined by unpaired Student's t test. ***, P < 0.0005. (C) Scanning electron micrographs confirm that MVM cells have few apical microvilli compared to WT cells.
FIG 2
FIG 2
Quantification of cell glycoprotein content in cells with or without microvilli. Cell lysate total glycoprotein levels were quantified by integrating the full scanned density of each lane, as indicated in the figure. MVM cells showed glycoprotein levels that were approximately 40% lower than those of WT cells. Error bars represent standard errors of the means (n = 4); significance was determined by using paired Student's t test (relative values were taken). MW, molecular weight marker. **, P < 0.005.
FIG 3
FIG 3
Epithelial cell uptake of live bacteria. Two-week monolayers of WT or MVM cells were assayed for endocytosis of bacteria (with measured zeta potentials indicated for each microbe). (A to D) Uptake of S. aureus (A), E. faecalis (B), Y. enterocolitica (C), and S. Typhimurium (D). Error bars represent data from two individual experiments run in triplicate; significance was determined by using unpaired Student's t test. *, P < 0.05; **, P < 0.005; NS, not significant. (E) Relationship between bacterial zeta potential and uptake. Ratios between the numbers of bacteria bound to MVM and WT cells were calculated and plotted against the bacterial zeta potential (values used were the means of three individual experiments run in triplicate). Bacterial uptake exhibits a linear relationship with bacterial zeta potential (R2 = 0.86). Error bars represent the standard errors of the means of duplicate experiments with two biological replicates (n = 4).
FIG 4
FIG 4
Adherence of fixed bacteria to epithelial cell monolayers. (A) S. aureus shows a preference for MVM cell compared to WT cells. Confocal images to the right show fluorescent bacteria (green) on monolayers, which were also stained for F-actin (phalloidin) (red). (B and C) Similar analyses of Y. enterocolitica (B) and S. Typhimurium (C). Error bars represent the standard errors of the means of three independent experiments with at least 10 images taken under each condition; significance was determined by using unpaired Student's t test. (D) Adherence ratios between the numbers of bacteria bound to MVM cells and those bound to WT cells plotted against bacterial zeta potential (values used were the means of three individual measurements). Bacterial adherence exhibits a linear relationship with bacterial zeta potential (R2 = 0.81). Error bars represent the sums of data from three individual experiments with two biological replicates each (n = 6). ***, P < 0.0005.
FIG 5
FIG 5
Epithelial cell adherence of synthetic nanoparticles. (A) SEM images of synthetic nanoparticles (left, negatively charged PLGA particles loaded with 5% DSS; right, particles loaded with 5% CTAB) showing uniformity of particle shape and size. Histograms show the size distribution, with medians calculated to be 0.74 μm for negatively charged particles and 0.81 μm for positively charged particles. Zeta potentials of synthetic nanoparticles (far right) were measured in triplicate. FITC, fluorescein isothiocyanate. (B and C) Nanoparticle adherence was assayed with fixed bacteria. Positively charged nanoparticles showed a higher preference for WT cells, while negatively charge nanoparticles showed a preference for MVM cells. ***, P < 0.0005. (D) Adhesion of fixed bacteria (from Fig. 4) and synthetic microparticles shown on same plot, showing a similar relationship between adhesion and charge. Error bars represent the standard errors of the means of three individual experiments with two biological replicates (n = 6); significance was determined by using unpaired Student's t tests.
FIG 6
FIG 6
Bacterial binding to epithelial cells under flow conditions. (A) Deposition kinetics for S. aureus. Each MVM clone had two replicates each (n = 2) compared to WT cells (n = 3). (B) Kinetics for Y. enterocolitica, with two replicates for MVM cells (n = 2) and two replicates for WT cells (n = 2).
FIG 7
FIG 7
Loss of microvilli in cells expressing the bacterial effacement effector Tir. (A) Caco2BBe cells were transfected with the E. coli O157:H7 effector Tir (TirBBe). F-actin (phalloidin) (red) staining (top row) suggests a loss of microvilli; SEM images (bottom row) confirm the absence of microvilli. (B) TirBBe cells show electrical resistances (TEER) similar to those of WT cells, indicating that tight junction formation is retained. Error bars represents the standard errors of the means of three individual experiments (n = 3). (C) qPCR showing that TirBBe cells had no induction of the M cell-related 41BB and gp2 genes but did show induction of RelB.
FIG 8
FIG 8
Bacterial adherence to TirBBe cells. (A to C) Adhesion of fixed S. aureus (A), Y. enterocolitica (B), and S. Typhimurium (C) bacteria. All assays were run in duplicate (two individual experiments). *, P < 0.05; **, P < 0.005; ***, P < 0.0005. (D) Adherence ratios plotted against bacterial surface charge, showing a linear relationship similar to that seen with MVM cells. Error bars represent the standard errors of the means of two individual experiments with three biological replicates (n = 6).
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