The influence of pH on the specific adhesion of P piliated Escherichia coli

Abstract

Adhesion to host tissues is an initiating step in a majority of bacterial infections. In the case of Gram-negative bacteria this adhesion is often mediated by a specific interaction between an adhesin, positioned at the distal end of bacterial pili, and its receptor on the surface of the host tissue. Furthermore, the rod of the pilus, and particularly its biomechanical properties, is believed to be crucial for the ability of bacteria to withstand external forces caused by, for example, (in the case of urinary tract infections) urinary rinsing flows by redistributing the force to several pili. In this work, the adhesion properties of P-piliated E. coli and their dependence of pH have been investigated in a broad pH range by both the surface plasmon resonance technique and force measuring optical tweezers. We demonstrate that P piliated bacteria have an adhesion ability throughout the entire physiologically relevant pH range (pH 4.5 - 8). We also show that pH has a higher impact on the binding rate than on the binding stability or the biomechanical properties of pili; the binding rate was found to have a maximum around pH 5 while the binding stability was found to have a broader distribution over pH and be significant over the entire physiologically relevant pH range. Force measurements on a single organelle level show that the biomechanical properties of P pili are not significantly affected by pH.

Figure 1. AFM micrographs of HB101/pPAP5 cells expressing P pili.

Figure 2. Binding of E. coli expressing P pili and type 1 pili in SPR assay.

Each type of bacteria (P pili-black line and type 1 pili-gray line) was injected into the flow chambers five times and washed with buffer at pH 7.0 between each injection. P-piliated bacteria were thereafter exposed to three injections of low pH buffer (3.5). The sensogram shows the difference in response between the galabiose-coated and the BSA-coated cells. Large arrows indicate start of injection and short arrows indicate end of injections. The alternating levels during injections of buffer with pH 3.5 originate from different index of refractions for the two buffer solutions (pH 3.5 and 7, respectively).

Figure 3. Binding of P pili in SPR assay at four different pH.

Black lines show pili binding to galabiose-coated surface and gray line – to BSA-coated surface.

Figure 4. Calculated binding rates of pili and bacteria at different pH.

Panels A and B show the binding to galabiose-BSA-coated surface (black bars) and BSA-coated surface (gray bars) for pili and bacteria respectively. Panels C and D show the specific binding rates of pili and bacteria respectively, calculated as difference between binding to galabiose-BSA-coated surfaces and BSA-coated surfaces obtained in SPR experiments.

Figure 5. Relative binding stability of pili (A) and bacteria (B).

Black bars represent the remaining amount of pili and bacteria on galabiose-coated surfaces and gray bars on BSA-coated surfaces at different pH measured after a wash of the surfaces with a buffer at pH 7.0. 100% corresponds to the amount of bacteria on the surface before the wash.

Figure 6. Relative binding of P piliated E. coli in artificial urine medium when compared to binding in buffer at pH 7.0.

Figure 7. Force-vs.-elongation response of a single P pilus at different pH.

Panels A-D show the elongation at pH 7.4, pH 5.0, pH 3.0 and pH 8.0 respectively. The plateaus of the black curves illustrate uncoiling forces of a single pilus, and the gray curve the recoiling forces. The three regions of force-vs.-elongation response are marked with Roman numerals (I–III), and the characteristic force plateau (region II) is indicated with a double arrow in (A).

Figure 8. Uncoiling forces at different pH in FMOT experiments.