Phosphoethanolamine cellulose enhances curli-mediated adhesion of uropathogenic Escherichia coli to bladder epithelial cells

Evaluation of UPEC Adhesion to Bladder Cells Through Traditional Adhesion Assays.

A hallmark UPEC strain, UTI89 (39), and its isogenic mutants were used to consider the individual and combined contributions of type 1 pili, curli, and pEtN cellulose to adhesion of 5637 bladder cells. Distinct growth conditions have been defined for promoting the production of type 1 pili (static LB broth) (40) and curli [YESCA (0.5 g/L yeast extract, 10 g/L casamino acids) nutrient broth] and increased curli production (YESCA supplemented with DMSO, abbreviated Y + D in figures) (41). Type 1 pili and curli protein production was characterized by electron microscopy and Western blot analysis for the exact bacterial strains and conditions used in this work and confirmed expectations for the selected growth conditions (Fig. 1 A and B). Fluorescence microscopy of UTI89 grown shaking in YESCA nutrient broth supplemented with DMSO at 26 °C confirmed the presence of both curli (CsgA immunofluorescence) and pEtN cellulose (Calcofluor fluorescence) as shown in Fig. 1C, with colocalization of the two signals. Growth in YESCA nutrient broth or agar generally results in curli and pEtN cellulose production due to gene regulation by the global regulator, CsgD, which activates curli and cellulose gene transcription in stationary phase (42).

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Fig. 1.

Growth conditions modulate type 1 pili, curli, and pEtN cellulose production and influence adhesion to bladder cells in traditional adhesion assays. (A) Transmission electron microscopy (TEM) images of UTI89 grown to promote differential production of extracellular fibers. (B) Whole cell levels of CsgA and FimA determined by Western blot analysis. (C) Fluorescence images of UTI89 grown in YESCA broth supplemented with 4% DMSO and examined with the cellulose-binding dye Calcofluor White and immunofluorescent staining for the major curli subunit CsgA. (D) Bacterial adherence to bladder cells in the traditional tissue culture adhesion assay with UTI89 grown under different conditions. (E) Bacterial adherence comparing UTI89, UTI89∆csgA, and UTI89∆bcsA. Open and solid circles represent data from two separate experiments. Percent adherence was calculated as the number of adherent bacteria in one test well divided by the average number of bacteria from control wells used for total bacterial counts. Bars represent the median of each data set. Growth conditions were as follows: LB: LB broth, 37 °C, static culture; YESCA: YESCA broth, 26 °C, shaking culture; Y + D: YESCA broth supplemented with 4% DMSO, 26 °C, shaking culture.

Traditional tissue culture adhesion assays were employed to compare adhesion by UTI89 grown under the distinct type 1 pili and curli-promoting conditions. When grown under type 1 pili conditions (LB broth), UTI89 enumeration after a 10-min incubation revealed that ∼7% of bacteria remained adherent to bladder cells (Fig. 1D). Bacterial adhesion to bladder cells has been previously demonstrated to depend highly on type 1 pilus expression and the adhesin FimH (12, 43⇓–45), which was observed in our experiments as well (SI Appendix, Fig. S1). Assays with moderately curliated cells (YESCA nutrient broth) yielded a similar percent of adherent bacteria as the type 1 pili producing UTI89 (Fig. 1D). UTI89 with increased curliation (YESCA broth supplemented with DMSO) yielded an approximately fourfold increase in the percent of adherent bacteria over UTI89 grown in LB and YESCA (Fig. 1D). Adhesion of the double mutant UTI89ΔfimHΔcsgA was similar to UTI89ΔfimH and UTI89ΔcsgA in the LB and YESCA conditions, respectively, further supporting the specific roles of type 1 pili and curli for adhesion in those conditions and demonstrating that some minimal adhesion occurs even in the absence of type 1 pili and curli (SI Appendix, Fig. S1). Experiments were then performed to compare UTI89 with the curli mutant UTI89∆csgA (lacking the major curli subunit CsgA) and the cellulose mutant UTI89∆bcsA (lacking the major cellulose synthase protein BcsA) when grown in the increased curliation conditions of DMSO-supplemented YESCA broth. This comparison revealed that bacteria lacking curli were compromised in mediating adhesion to bladder cells. Loss of pEtN cellulose had a less dramatic effect, and UTI89∆bcsA was observed to exhibit a slight decrease or increase in different trials relative to UTI89, whereas adherence trends were always maintained for other strain and condition comparisons (Fig. 1E). Thus, these traditional tissue culture adhesion assays revealed that increased curli production by UTI89 yields an increased number of adherent bacteria associated with a bladder cell monolayer. However, by microscopy, clusters of bacteria were often observed associated with bladder cells (SI Appendix, Fig. S2), and adherence of UTI89∆bcsA was observed to be more variable than other strains (Fig. 1E). This presented the possibility that increased bacterial numbers in this adhesion assay could be attributed to aggregative interbacterial interactions rather than reflecting intrinsically more bacteria in specific contact with bladder cells. In addition, the traditional assay utilizes an uncontrolled, operator-dependent rinsing procedure to expel unattached bacteria from the bladder cells. This means that the procedure can provide a useful index of bacteria adherence but not a standardized quantification of adhesion. Thus, we sought to develop an approach for the quantitative evaluation of the strength of bacterial adhesion to mammalian cells.

The LCMR for Direct Measurement of Bacterial Adhesion to Mammalian Cells.

Several technologies enable measurement of bacterial adhesion to surfaces, including atomic force microscopy (AFM) and the quartz crystal microbalance (46). However, these approaches can require many trials to obtain statistically significant results and are not readily amenable to measuring bacterial adhesion to mammalian cells. In addition, techniques such as AFM only probe a small portion of a sample at a time. The LCMR (37, 38) was designed and built to enable quantitative measurements of adhesion involving mammalian cells. The LCMR enables the rapid determination of the average mechanical properties of an entire cell monolayer, while maintaining cell−cell contacts and the adherent geometry of living cells. We introduce the LCMR here as being uniquely suited to the examination of host−pathogen interfaces. As shown in Fig. 2A, the LCMR consists of a parallel glass plate assembly mounted over an inverted microscope that can be submerged in cell culture medium. In this work, bladder cells were cultured as a monolayer on the bottom plate. Either extracellular bacterial fibers or whole bacteria cells were attached to the top plate. The complete LCMR design and operation is provided in SI Appendix.

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Fig. 2.

LCMR instrument and adhesion measurements. (A) Diagram of the LCMR, illustrating its utility in measuring the adhesion of either isolated curli fibers or bacterial whole cells to a bladder cell monolayer. (B) Schematic representation of the covalent linking strategy implemented to attach whole bacterial cells to the top plate of the LCMR.

During experiments, the top plate was translated horizontally by movement of a piezoelectric stage connected to a force transducer, effectively shearing the plate across the bladder cells. This shearing motion, or step strain, will impart a greater stress in the bladder cell layer if there is adhesion between the top plate and bladder cells. This was quantified by recording the force on the top plate and distance of displacement, which was used to calculate a relaxation modulus and strain, providing a quantitative measure of adhesion. Following the step strain, stress in the bladder cell layer can be relieved via both internal structural rearrangements of the bladder cells and progressive detachment of the bladder cells from the top plate. As the bladder cell layer was of the same cell type and grown to confluency for every experiment, mechanics of the cell layer were assumed to be consistent from trial to trial. Differences in relaxation moduli were therefore due directly to differences in adhesion, with the initial magnitude and decay providing information on both the strength of adhesion and detachment dynamics.

Purified Curli Enhance Bladder Cell Adhesion.

To investigate the role of curli in bladder cell adhesion, we first performed experiments with isolated curli (47) attached to the top plate of the LCMR. We utilized the known fibronectin-binding property of curli to associate curli with a fibronectin-coated top plate. As shown in Fig. 3A, the presence of curli resulted in a significant increase of the relaxation modulus over the entire measurement time compared with control experiments with the top plate coated in fibronectin alone. It is therefore evident that curli can mediate physical adhesion to bladder cells. In addition, this phenomenon could be visualized through the inverted microscope as shown in Fig. 3B. In these images, the top plate is in focus and the upper surfaces of the bladder cells before and after shearing are outlined. In the absence of curli (fibronectin coating only), the upper cell surface is not significantly displaced by motion of the top plate. The top plate could essentially glide and slip across the cell layer, indicating low adhesion. On the other hand, with curli present on the top plate, the upper cell surface was significantly deformed and observed to translate in the direction of shear, revealing obvious and strong adhesive interactions (Movies S1 and S2).

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Fig. 3.

Interactions of isolated curli and bladder cells in the LCMR. (A) Relaxation moduli from LCMR experiments reveal the adhesiveness of curli to bladder cells compared with the fibronectin alone (control). Error bars represent the SD of three trials. The applied strain was 1.3 ± 0.3. (B) Images show the bladder cell deformation. White dashed lines outline bladder cells before top plate movement; colored lines outline cells directly after top plate motion. Solid circles identify the starting and final positions of a 1-μm tracking bead attached to the top plate.

The absolute magnitude of the moduli measured in these experiments, with maxima ranging from ∼50 Pa to ∼100 Pa, also indicates that the bladder cell layer is a relatively soft material. While cell moduli values generally can range from tens of pascals to tens of kilopascals depending on the cell type and measurement technique, the order of magnitude of our values agrees with previous studies on both bladder cells (48, 49) and other epithelial cell lines (50⇓⇓–53).

Experiments with Intact Bacteria Reveal Different Roles for Cell Surface Structures.

To utilize the LCMR instrument with intact bacteria, we developed a covalent attachment strategy to assemble a single compact layer of bacteria on the top plate (Fig. 2B). In addition, the amount of strain to be employed in each LCMR measurement was optimized and corresponded to strains of 1 to 1.5, i.e., a 5- to 7-µm movement of the top plate. This strain regime is larger than the diameter of individual bacteria, but not larger than the bladder cell diameter. We first used the LCMR to quantify adhesion of UTI89 to bladder cells when grown under type 1 pili (LB broth) and curli-promoting (YESCA and YESCA supplemented with DMSO) conditions, as in the traditional tissue culture assays presented in Fig. 1. As observed in Fig. 4A, adhesion of type 1 piliated and moderately curliated bacteria resulted in similar relaxation moduli, while bacteria with increased curliation had a higher relaxation modulus. We additionally demonstrated that reduced adhesion in the LCMR measurement was observed for UTI89ΔfimH grown in LB broth compared with UTI89, consistent with the contribution of type 1 pili to adhesion and without curli production in this growth condition (SI Appendix, Fig. S3). These measurements independently support the conclusion from the traditional adhesion assay: Strongly curliated bacteria exhibit enhanced bladder cell adhesion capacity compared with type 1 piliated bacteria.

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Fig. 4.

Interactions of whole bacteria and bladder epithelial cells in the LCMR. (A) Relaxation moduli comparing the adhesiveness of UTI89 producing type 1 pili (LB), curli (YESCA), and an increased level of curli (Y + D) to bladder cells. The applied strain in these experiments was 1.4 ± 0.5. (B) Decay constants were obtained from fitting data of the individual trials in A. The data were fit with a double exponential decay, and the decay constants shown are for the faster relaxation process. (C) Relaxation moduli comparing the adhesiveness of UTI89, UTI89∆bcsA, and UTI89∆csgA to bladder cells. The applied strain was 1.4 ± 0.5. (D) Decay constants were obtained and fit as in B. All error bars represent the SD of three trials.

We next sought to investigate the potential influence of pEtN cellulose in bladder cell adhesion using the LCMR platform. UTI89, UTI89∆csgA, and UTI89∆bcsA were each grown in YESCA supplemented with DMSO and prepared on top plates for the LCMR measurements. As shown in Fig. 4C, UTI89, expressing both curli and pEtN cellulose, exhibited the highest relaxation modulus. UTI89∆csgA, lacking curli, had a much lower relaxation modulus than UTI89 over the entire measurement time, indicating that loss of curli resulted in a reduction in adhesion. This contribution of curli to bladder cell adhesion was consistent with results from the traditional adhesion assay. For experiments with UTI89∆bcsA, lacking pEtN cellulose, the maximum relaxation modulus at time zero was intermediate between that of UTI89 and UTI89∆csgA. At later times (6 s to 10 s), the relaxation modulus of UTI89∆bcsA equaled that of UTI89∆csgA. This indicated that cells lacking pEtN cellulose exhibited some initial adhesion capacity (higher initial adhesion than UTI89∆csgA) but failed to maintain strong bladder cell adhesion after several seconds. These observations differed from the traditional adhesion assay, which was unable to differentiate bladder cell adhesion between UTI89 and UTI89∆bcsA.

Thus, we considered whether the LCMR data could provide further insight through examination of time-dependent processes occurring during the experiment. We discovered that the relaxation moduli for all experiments fit well to a double exponential decay model of the form y = Ae^−t/α+ Be^−t/β + C. The slower decay time, α, was similar for every bacterial strain and growth condition (∼15 s). In contrast, the faster decay time, β, was dependent on the bacterial strain. For experiments comparing type 1 piliated to curliated bacteria, values of β were 1.3 s to 1.5 s and did not vary significantly between conditions (Fig. 4B). However, for experiments examining the roles of curli and pEtN cellulose, the values of β were 0.5 s to 0.6 s for the two mutants (UTI89∆csgA and UTI89∆bcsA) and 1.5 s for UTI89 (Fig. 4D). We attribute the similar slow decay time, α, to the internal bladder cell mechanical response, which should be consistent between experiments. We attribute the different values for the faster decay time, β, to differences in detachment dynamics. This indicates that, although UTI89∆bcsA may have higher initial adhesion to the bladder cells than UTI89∆csgA, both detach more rapidly from the bladder cells than UTI89, which expresses both curli and pEtN cellulose.

pEtN Cellulose Enhances Bacterial Surface Association of Curli.

Based on the above results, we hypothesized that the role of pEtN cellulose in enhancing bladder cell adhesion could be to improve the association of curli with the bacterial cell surface. That is, a weaker association between curli and the bacterial cell surface could result in reduced curli-mediated adhesion to a bladder cell. This hypothesis was supported by the LCMR data: UTI89 and UTI89∆bcsA produce the same amount of curli per cell (Fig. 1B), yet UTI89∆bcsA exhibited rapid detachment after shearing.

To test this hypothesis, we designed an experiment to determine the extent to which curli are released from the bacterial cell surface when subjected to shearing forces in suspension. We mechanically perturbed bacterial cells through vortexing and, after pelleting the cells by centrifugation, performed a Western blot to detect curli CsgA protein in both the bacterial cell pellets and supernatants. Curli remained associated with UTI89 even after vortexing and were only detected in the cell pellet (Fig. 5A). In contrast, fewer curli were found in the UTI89∆bcsA cell pellet and more were detected in the supernatant as a function of increasing vortex time. Thus, curli remain cell-associated for pEtN cellulose-producing bacteria upon mechanical perturbation, but are less tightly cell-associated in the absence of cellulose. Electron microscopy confirmed the dramatic loss of curli from the bacterial cell surface in the absence of cellulose (Fig. 5B).

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Fig. 5.

Effect of pEtN cellulose production on curli association with bacterial cells. (A) Western blot analysis of curli abundance in bacterial cell pellets versus supernatant as evaluated by detection of the major curli subunit CsgA. Cellular suspensions of UTI89 and UTI89∆bcsA were vortexed for increasing times of 10 s, 1 min, or 5 min, and then separated into cell pellet and supernatant samples by centrifugation. Curli remained cell-associated in UTI89, in which pEtN cellulose is coproduced with curli. Curli were released from cells in the absence of pEtN cellulose production (UTI89∆bcsA). (B) TEM images of bacterial cells vortexed for either 0 min or 5 min. (C) Schematic summary of the potential roles of curli and pEtN cellulose in bladder cell adhesion under conditions of low and high shear.