Medium pH-dependent redistribution of the urease of Helicobacter pylori

pH change of urease under different pH conditions.
H. pylori cultured on agar was collected and resuspended in Brucella broth supplemented with 5 % horse serum and the turbidity was adjusted to McFarland standard 0.5. Two ml of this H. pylori suspension, and 1.0 ml of pH-adjusted urease assay buffer, which contained 1.3 M urea, 0.06 M NaN₃, 0.005 M phenol red and McIlvaine buffer (pH 5.0 and 7.0), were mixed in a 10 ml tube. Measurement of pH was performed in the tube at room temperature using a pH meter (Checker, HANNA).

Antibodies.
The mouse monoclonal antibody against H. pylori urease subunit A (Austral Biologicals) and rabbit polyclonal anti-UreI antibody (Weeks et al., 2000) were used as primary antibodies, and 5 nm colloidal gold-labelled anti-mouse IgG and 10 nm gold-labelled anti-rabbit IgG-labelled goat antibodies (Amersham) were used as secondary antibodies.

Fixation and staining.
H. pylori colonies were fixed with 1 % glutaraldehyde in 0.05 M cacodylate buffer (pH 7.2) at 4 °C for 60 min. Fixed colonies were washed five times with 0.05 M cacodylate buffer and dehydrated in serially graded ethanol. Samples were then embedded in Lowicryl K4M resin (Electron Microscopy Science). Polymerization was allowed to occur in an ultraviolet irradiator (Dosaka EM) at -30 °C for 2 days and then at room temperature for 2 days. Ultrathin sections were cut using a Porter Blum Ultramicrotome (Sorvall MT-5000, Du Pont) and mounted on a nickel grid (300 mesh) supported by a carbon-coated collodion film.

Ultrathin sections on the grid were treated with 5 % normal goat serum in 0.15 M PBS (pH 7.2) to block nonspecific reactions. Sections were then double-stained with mouse antibody against H. pylori UreA and rabbit antibody against UreI (primary antibodies) and then 5 nm colloidal gold-labelled anti-mouse IgG and 10 nm gold-labelled anti-rabbit IgG-labelled goat antibodies (secondary antibodies). Sections were first reacted by adding drops of the primary antibodies at room temperature for 120 min and washed five times in PBS. Sections were then allowed to react with the secondary antibodies at room temperature for 60 min and were subsequently washed five times in PBS.

Immunostained sections were fixed with 1 % glutaraldehyde in 0.05 M cacodylate buffer for 15 min and washed five times in distilled water. Finally, sections were contrast-enhanced with a mixture of alcian blue and osmium tetroxide solution and were stained with uranyl acetate aqueous solution as described previously (Hong et al., 2000). All procedures for contrast enhancement were performed at room temperature.

All sections were observed under a transmission electron microscope (H-300 and H-7100, Hitachi). Photomicrographs were taken at a magnification of x15 000 and enlarged to a magnification of x30 000.

Morphometric analysis.
The number of immunogold particles associated with H. pylori cells was determined using the immunoelectron photomicrographs. Areas of bacterial cells in the photomicrographs were measured using an image analyser (MCID system, Imaging Res.) and the numbers of gold particles per unit area were compared.

In order to quantify the distribution of gold particles, the densities of the particles per square micrometer in three portions of a bacterial cell were determined. The bacterial cell was divided into three portions: the outer portion is the area extending from a depth of one-sixth of the diameter to the surface of the cell; the medium portion is the area between the outer and inner portions extending from one- to two-sixths of the diameter; and the inner portion is the central area of the cell extending beyond two-sixths of the diameter to the centre of the cell. The principle of dividing portions of H. pylori is explained in Fig. 1. As shown, the outer portion that is between the surface and the depth of one-sixth of the shorter diameter (approx. 85 nm) is equal to the sum of the cell wall (approx. 30 nm) width, length of the antibody (approx. 25 nm) and x (approx. 30 nm).

To clarify whether the number of gold particles associated with UreA and the distribution of urease are influenced by environmental factors, the localization and the number of gold particles associated with urease were determined in cells incubated for 15 min at different pHs. The overall densities of gold particles in the bacterial cells incubated at neutral pH in the presence and absence of urea were 105.1 ± 14.8 µm^-2 and 117.4 ± 35.7 µm^-2, respectively. These values were not found to be significantly different by Student's t-test (P < 0.01). At acidic pH, the overall densities in the cell in the presence and absence of urea were 151.2 ± 36.4 µm^-2 and 161.1 ± 56.8 µm^-2, respectively. These values at the same pH were found to be statistically the same by Student's t-test (P < 0.01). The distribution of gold particles seemed to be uniform in bacterial cells incubated in the presence of urea at neutral pH in wild-type (Fig. 3a) but eccentric in those incubated at acidic pH (Fig. 3b).

Although the presence of urea in the buffer did not influence the densities of gold particles in any portion of the bacterial cells, a definite change in the distribution of gold particles was observed in cells incubated at different pHs. At neutral pH, the density was highest in the inner portion and lowest in the outer portion (Student's t-test paired analysis, P < 0.05). In contrast, at acidic pH, the density was highest in the outer portion and lowest in the inner portion (Student's t-test paired analysis, P < 0.05) (Table 1).

Table 1.Distribution of urease immunoreactivity in H. pylori after a 15 min incubation Double indirect immunolabelling was performed using mouse monoclonal anti-urease antiserum, rabbit polyclonal anti-UreI antiserum and 5 and 10 nm gold particle-labelled goat anti-mouse IgG and anti-rabbit IgG.

To determine whether the eccentric distribution of urease at acidic pH is UreI dependent, the ureI-deletion mutant (Weeks et al., 2000) was also observed under the same experimental conditions (Table 1). The densities at each portion were similar to those of wild-type cells incubated at neutral pH (Fig. 3c, d); i.e. the eccentric shift of the distribution of urease molecules did not occur in the ureI-deletion mutant at acidic pH (Student's t-test paired analysis, P < 0.05).

In order to determine whether the shift of urease distribution was due to nonspecific effects of the acidic condition, the localization of UreI was determined by similar methods (Fig. 3a, b). The difference in the density of gold particles for UreI between neutral pH (Fig. 3a) and acidic pH (Fig. 3b) was similar to that reported previously (Hong et al., 2000) and was not found to be statistically significant by Student's t-test (P < 0.01). The densities of the gold particles in the outer portion of wild-type cells incubated at neutral and acidic pH were similar to those reported previously (Hong et al., 2000).

To determine whether the shift occurs in proliferating bacteria, the bacteria incubated for 2 days at reduced pH were observed in the experiments (Table 2). The urease immunogold particles were distributed in a similar manner to those incubated for 15 min at neutral pH (Fig. 4a) and acidic pH (Fig. 4b). It was also observed by immunoelectron microscopy that the urease gold particles in H. pylori incubated for 2 days at acidic pH were gathered and associated with UreI gold particles (Fig. 4c).

Table 2.Distribution of urease immunoreactivity in cells of H. pylori after 2 days of incubation Double indirect immunolabelling was performed using mouse monoclonal anti-urease antiserum, rabbit polyclonal anti-UreI antiserum and 5 and 10 nm gold particle-labelled goat anti-mouse IgG and anti-rabbit IgG.

Recent reports have shown that it is cytoplasmic rather than surface-associated urease that is required for the acid resistance of H. pylori (Rektorschek et al., 1998; Scott et al., 1998; Weeks & Sachs, 2001). It is unclear where cytoplasmic urease neutralizes pH in a bacterial cell under acidic conditions. Urea enters the bacterial cytoplasm through the proton-gated urea channel, UreI, after exposure of the cell to a medium of pH 6.0 or less (Marcus & Scott, 2001; Rektorschek et al., 1998; Scott et al., 1998; Athmann et al., 2000). The urease of H. pylori catalyses the degradation of urea to ammonia to protect the bacterium from harmful effects of acid. However, if urea in large amounts is transported into the cytoplasm and ammonia is generated equally throughout the cytoplasm, there could be lethal protein degeneration due to alkalinization of the cytoplasm. The bacterium does not survive at pH_in>8.5. (Meyer-Rosberg et al., 1996).

The catalytic expression of cytoplasmic urease is regulated by a mechanism that is closely associated with extracellular pH. Thus, we hypothesized that cytoplasmic urease molecules shift toward the inner membrane and assemble with UreI molecules at acidic pH. Assembly of urease and UreI has been demonstrated biochemically (Voland et al., 2003). To establish this hypothesis in intact bacteria using quantitative contrast-enhanced immunoelectron microscopy (Hong et al., 2000), we examined whether urease is redistributed to the bacterial inner membrane at low pH. The redistribution of urease was observed with a change in medium pH and was associated with the presence of UreI molecules. This was not observed in the ureI-deletion mutant and was independent of the presence of urea.

UreI is required for an acidic pH-induced increase in cytoplasmic urease activity in H. pylori (Scott et al., 2000). The data presented here show that when UreI is activated by an acidic medium pH, urease moves closer to the source of urea and presumably ammonia production occurs at or near the inner membrane. This eliminates the necessity of generalized cytoplasmic alkalinization due to intra-bacterial urease.

In the proteinprotein interaction map of H. pylori urease, the urease operon that encodes UreI may be associated with the transmission of proton motive force to outer membrane receptors (Rain et al., 2001). When UreI is present, acidic medium pH activation of cytoplasmic urease is observed and urease activity in intact bacteria at neutral pH depends on the presence of UreI, indicating that urease activity is membrane limited (Scott et al., 2000). In this study, we confirmed that the ureI-deletion mutant does not induce neutralization of an acidic medium. It was also observed that the urease gold particles in H. pylori were associated with UreI gold particles in immunoelectron microscopy. Urease activity was associated with the presence of UreI, and urease and UreI may assemble a supramolecular complex in an acidic medium.

This is the first report that cytoplasmic molecules in bacteria are trafficked to the membrane with a change in environmental conditions outside the cell. We propose that H. pylori possesses a proton-regulated urease redistribution mechanism, which may be a new target for anti-H. pylori therapy.