Helicobacter pylori containing only cytoplasmic urease is susceptible to acid

Abstract

Helicobacter pylori, an important etiologic agent in a variety of gastroduodenal diseases, produces large amounts of urease as an essential colonization factor. We have demonstrated previously that urease is located within the cytoplasm and on the surface of H. pylori both in vivo and in stationary-phase culture. The purpose of the present study was to assess the relative contributions of cytoplasmic and surface-localized urease to the ability of H. pylori to survive exposure to acid in the presence of urea. Toward this end, we compared the acid resistance in vitro of H. pylori cells which possessed only cytoplasmic urease to that of bacteria which possessed both cytoplasmic and surface-localized or extracellular urease. Bacteria with only cytoplasmic urease activity were generated by using freshly subcultured bacteria or by treating repeatedly subcultured H. pylori with flurofamide (1 microM), a potent, but poorly diffusible urease inhibitor. H. pylori with cytoplasmic and surface-located urease activity survived in an acid environment when 5 mM urea was present. In contrast, H. pylori with only cytoplasmic urease shows significantly reduced survival when exposed to acid in the presence of 5 mM urea. Similarly, Escherichia coli SE5000 expressing H. pylori urease and the Ni2+ transport protein NixA, which expresses cytoplasmic urease activity at levels similar to those in wild-type H. pylori, survived minimally when exposed to acid in the presence of 5 to 50 mM urea. We conclude that cytoplasmic urease activity alone is not sufficient (although cytoplasmic urease activity is likely to be necessary) to allow survival of H. pylori in acid; the activity of surface-localized urease is essential for resistance of H. pylori to acid under the assay conditions used. Therefore, the mechanism whereby urease becomes associated with the surface of H. pylori, which involves release of the enzyme from bacteria due to autolysis followed by adsorption of the enzyme to the surface of intact bacteria ("altruistic autolysis"), is essential for survival of H. pylori in an acid environment. The ability of H. pylori to survive exposure to low pH is likely to depend on a combination of both cytoplasmic and surface-associated urease activities.

FIG. 1

Effects of exposure to acid (pH 2, 30 min) on survival of H. pylori N6 (urease positive; 72-h repeat sub), H. pylori N6ureB::Km (urease-negative mutant; 72-h repeat sub), and P. mirabilis ATCC 7002 (urease positive) in the presence and absence of 5 mM urea. Solid bars represent H. pylori N6, shaded bars represent H. pylori N6ureB::Km (urease-negative mutant), and open bars represent P. mirabilis ATCC 7002. In this and subsequent graphs, bacterial survival is expressed as CFU per milliliter ± standard error.

FIG. 2

Effects of the urease inhibitors flurofamide (FF [1 μM]) and acetohydroxamic acid (AHA [7 mM]) on the time course of urease activity in whole-cell suspensions of H. pylori 84-183 (72-h fresh sub).

FIG. 3

Effects of the urease inhibitors flurofamide and acetohydroxamic acid on resistance of H. pylori 84-183 (72-h fresh sub) to acid in the presence or absence of 5 mM urea. S, saline (pH 7.2); A, acid (pH 2.0); A+U, acid containing 5 mM urea; FF, bacteria preincubated in flurofamide (1 μM) for 30 min and then exposed to acid containing 5 mM urea for 30 min; AHA, bacteria preincubated in acetohydroxamic acid (7 mM) for 30 min then exposed to acid containing 5 mM urea for 30 min.

FIG. 4

Effects of culture age (hence urease distribution) on survival of H. pylori 84-183 in saline (pH 7.2) and in acid (pH 2.0) containing 5 mM urea. Solid bars represent survival of 24-h fresh sub bacteria (which contain cytoplasmic urease almost exclusively), while open bars represent survival of 72-h fresh sub bacteria (which possess both cytoplasmic and/or surface-associated or extracellular urease).

FIG. 5

Immunolocalization of H. pylori urease in E. coli SE5000(pHP808/pUEF202) and H. pylori 84-183. (A) Colloidal gold particles representing localization of H. pylori urease in E. coli SE5000(pHP808/pUEF202) are located exclusively within the cytoplasmic compartment (arrow). (B) Colloidal gold particles representing localization of urease in H. pylori 84-183 (24-h fresh sub) are located exclusively within the cytoplasmic compartment (arrow). (C) Immunolabeling was not observed when preimmune ascites fluid was substituted for primary antibody during immunolabelling of E. coli SE5000(pHP808/pUEF202). In all experiments, the concentration of primary antibody was 1:250. Bars, 120 nm.

FIG. 5

Immunolocalization of H. pylori urease in E. coli SE5000(pHP808/pUEF202) and H. pylori 84-183. (A) Colloidal gold particles representing localization of H. pylori urease in E. coli SE5000(pHP808/pUEF202) are located exclusively within the cytoplasmic compartment (arrow). (B) Colloidal gold particles representing localization of urease in H. pylori 84-183 (24-h fresh sub) are located exclusively within the cytoplasmic compartment (arrow). (C) Immunolabeling was not observed when preimmune ascites fluid was substituted for primary antibody during immunolabelling of E. coli SE5000(pHP808/pUEF202). In all experiments, the concentration of primary antibody was 1:250. Bars, 120 nm.

FIG. 5

Immunolocalization of H. pylori urease in E. coli SE5000(pHP808/pUEF202) and H. pylori 84-183. (A) Colloidal gold particles representing localization of H. pylori urease in E. coli SE5000(pHP808/pUEF202) are located exclusively within the cytoplasmic compartment (arrow). (B) Colloidal gold particles representing localization of urease in H. pylori 84-183 (24-h fresh sub) are located exclusively within the cytoplasmic compartment (arrow). (C) Immunolabeling was not observed when preimmune ascites fluid was substituted for primary antibody during immunolabelling of E. coli SE5000(pHP808/pUEF202). In all experiments, the concentration of primary antibody was 1:250. Bars, 120 nm.

FIG. 6

Effects of urea concentration on survival of E. coli SE5000(pHP808/pUEF202) in acid (pH 2, 30 min). The concentrations of urea used were as follows: no urea present (U-0), 5 mM (U-5), 15 mM (U-15), and 50 mM (U-50).

FIG. 7

Model describing the roles of H. pylori urease activity in the cytoplasmic and surface-associated compartments. H. pylori urease activity exhibits a pH optimum of 8.3. Free urease is rapidly inactivated by exposure to pH <5 (2, 39) and likely does not contribute to acid resistance. (A) Most of the early-logarithmic-phase and some late-logarithmic-phase H. pylori cells contain cytoplasmic urease exclusively, with no surface-associated urease (38). H. pylori cells exhibiting cytoplasmic urease activity only are also generated by inhibition of surface-associated urease with 1 μM flurofamide (38). The cytoplasmic urease degrades urea to produce ammonia, which may be exported but is not sufficient to permit survival at pH 2 for 30 min. (B) In the late logarithmic phase of growth, H. pylori possesses both cytoplasmic and surface-associated urease activity (38), allowing quantitatively more urease activity per bacterium. The surface-associated urease activity is sensitive to flurofamide (38), a poorly diffusible urease inhibitor, and is protected from external low pH due to its association with the molecular chaperonin HspB (heat shock protein B, a groEL homolog) at the cell surface. Rapid external hydrolysis of urea helps to prevent entry of H⁺ into the bacterial cytoplasm, therefore maintaining neutral-to-alkaline cytoplasmic pH and allowing full urease activity in the cytoplasmic compartment. Internal urease in the absence of external urease is unable to maintain a neutral-to-alkaline cytoplasmic pH for optimum urease activity. It is important to note that the observations presented above are valid for an external pH of 2.0. It remains to be determined whether cytoplasmic urease alone is sufficient to allow survival of H. pylori at a higher external pH (3 to 5).