Gastroenterology

Gastroenterology

Volume 114, Issue 1, January 1998, Pages 58-70
Gastroenterology

Alimentary Tract
The role of internal urease in acid resistance of Helicobacter pylori☆,☆☆

https://doi.org/10.1016/S0016-5085(98)70633-XGet rights and content

Abstract

Background & Aims: The relative role of internal urease for acid protection of Helicobacter pylori is unknown. The aim of this study was to determine the comparative importance of internal and external urease under acidic conditions. Methods: The pH optimum and measured Michaelis constant for urea of external urease and urease in intact bacteria at different medium pH (pHout) were measured using 14CO2 release from 14C-urea. The effect of urea on membrane potential and bacterial cytoplasmic pH was measured at different fixed pHout. 35S-methionine labeling and sodium dodecyl sulfate–polyacrylamide gel electrophoresis of labeled proteins in the organism and medium measured protein synthesis at different pHout and mechanisms of urease externalization. Results: External urease had activity between pH 5.0 and 8.5 and internal urease between pHout 2.5 and 6.5, and its Michaelis constant at pHout 7.5 was 300 mmol/L but at pHout 4.5 was 0.5 mmol/L, similar to free urease. The addition of 5 mmol/L urea to bacteria at fixed pHout from 3.0 to 6.0 elevated potential to about −105 mV and periplasmic pH to about pH 6.2. Protein synthesis occurred mainly between pH 6.5 and 8.0, and urease activity resulted in increased protein synthesis at acidic pH. The labeling pattern of intrabacterial and released protein was similar. Conclusions: Intracellular urease activity is regulated by external pH, defends against gastric acidity by increasing periplasmic pH and membrane potential, and stimulates protein synthesis at acidic pH. External urease is produced mostly by cell lysis.

GASTROENTEROLOGY 1998;114:58-70

Section snippets

Growth of H. pylori

H. pylori was grown as described previously.24 H. pylori strain ATCC 43504, a clinical isolate, was used. Bacteria were grown on blood agar plates (Baltimore Biological Laboratories trypticase soy agar 5% sheep blood; Becton Dickinson, Cockeysville, MD) in a microaerophilic atmosphere (5% O2, 10% CO2, and 85% N2) at 37°C for 24 hours. Cells from one plate were harvested in 300 μL brain-heart infusion supplemented with 5% fetal calf serum (Difco Laboratories, Detroit, MI), from which aliquots

pH optimum of urease

The experimental system for measurement of urease activity was calibrated to be in a linear range at 30-minute incubation. The 100 mmol/L buffer at each pH and low protein or bacterial numbers resulted in a stable pH (± 0.1 units) throughout the time of measurement of enzyme activity. The pH optimum curve of urease activity in HP buffer in a bacterial homogenate was about 7.5, similar to that of external urease (data not shown). Activity was maintained down to a pH of about 5.0, and then a

Discussion

The colonization by H. pylori of the human stomach and its relationship to peptic ulcer disease is well documented.33, 34, 35 The organism is absent in the pernicious anemia stomach where the pH is neutral.36 The pH range over which H. pylori survives and generates a proton motive force in the absence of urea is between pH 4.0 and 8.0,24 and the organisms grow in vitro between pH 6.0 and 8.0.3, 6 H. pylori is therefore a neutrophile without the membrane properties of an acidophile.37

A large

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    ☆

    Address requests for reprints to: George Sachs, D.Sc., Building 113, Room 324, VA Medical Center West Los Angeles, 11301 Wilshire Boulevard, Los Angeles, California 90073. Fax: (310) 312-9478.

    ☆☆

    Supported by United States Veterans Administration Senior Medical Investigator Funds, grants DK 41301 and DK 40615 from the National Institutes of Health, and a fellowship from Chemgenics, Cambridge, Massachusetts.

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    Copyright © 1998 American Gastroenterological Association. Published by Elsevier Inc. All rights reserved.