Rapid Loss of Motility of Helicobacter pylori in the Gastric Lumen In Vivo
http://www.100md.com
感染与免疫杂志 2005年第3期
Institut für Physiologie
Institut für Hygiene und Mikrobiologie, Ruhr-Universitt Bochum, Bochum
Institut für Medizinische Mikrobiologie und Krankenhaushygiene, Medizinische Hochschule Hannover, Hannover, Germany
ABSTRACT
The human pathogen Helicobacter pylori has infected more than half of the world's population. Nevertheless, the first step of infection, the acute colonization of the gastric mucus, is poorly understood. For successful colonization, H. pylori must retain active motility in the gastric lumen until it reaches the safety of the mucus layer. To identify the factors determining the acute colonization, we inserted bacteria into the stomach of anesthetized Mongolian gerbils. We adjusted the gastric juice to defined pH values of between 2.0 and 6.0 by using an autotitrator. Despite the fact that Helicobacter spp. are known to survive low pH values for a certain time in vitro, the length of time that H. pylori persisted under the assay conditions within the gastric juice in vivo was remarkably shorter. In the anesthetized animal we found H. pylori to be irreversibly immotile in less than 1 min at lumen pH values of 2 and 3. At pH 4 motility was lost after 2 min. However, the period of motility increased to more than 15 min at pH 6. Blocking pepsins in the gastric lumen in vivo by using pepstatin significantly increased the period of motility. It was possible to simulate the rapid in vivo immotilization in vitro by adding pepsins. We conclude that pepsin limits the persistence of H. pylori in the gastric chymus to only a few minutes by rapidly inhibiting active motility. It is therefore likely that this short period of resistance in the gastric lumen is one of the most critical phases of Helicobacter infection.
INTRODUCTION
The bacterium Helicobacter pylori causes chronic gastritis, which may progress to gastric or duodenal ulcers, gastric atrophy, mucosa-associated lymphoid tissue lymphoma, and adenocarcinoma (26).
This pathogen is unique among bacteria in its ability to persist in the harsh environment of the human stomach characterized by a combination of hydrochloric acid and different acidic aspartate proteases, historically referred to as pepsins (9). Although this bactericidal barrier protects the organism from microbes, H. pylori can colonize the gastric mucus.
Gastric proteases have been divided into three classes, namely, pepsin A and C, chymosin and the intracellular cathepsin E, each secreted as a proenzyme and activated at low pH values. Pepsin A (pepsin I) and pepsin C (pepsin II) are the dominant proteases in the human stomach. All of these isoenzymes exponentially lose proteolytic activity when pH values are above their pH optimum of between 2.0 and 3.8 (4, 5, 14, 22). The pH in the human stomach lumen varies between basal values of 1 to more neutral values after a meal (postprandial pH values). Although in adults the luminal pH rarely exceeds 5.5 (8), infants have a different pH profile. It was shown by 24-h pH-metry that the pH increases from basal values of 2 to pH 7 after breast feeding (17). Thus, the postprandial peptic activity in adults is higher than in children.
The specific conditions for a successful Helicobacter infection are still unknown. We do know, however, that motility is essential for H. pylori to initially colonize and chronically infect the gastric mucus (6, 7, 12, 19). The loss of motility of a bacterium in the lumen is therefore equivalent to its inability to colonize the mucus layer.
To simulate the process of colonization in the human stomach, we used the Mongolian gerbil, a well-established model for the study of H. pylori infection. Gerbils have luminal pH values similar to those in the human stomach (mean basal pH 1.4 [unpublished data]). In addition, the pathology that H. pylori induces in gerbils is very similar to that observed in humans (gastritis, ulcers, carcinoma, and lymphoma) (11, 28). Eradication of the bacterium can be achieved by the same treatment successfully used in humans (13).
In order to describe the phase of colonization from oral ingestion to invasion of the mucus, we inoculated highly motile bacteria into the stomach of anesthetized gerbils at defined postprandial pH values and measured bacterial motility. We found a rapid loss of motility under in vivo conditions. To investigate the possible role of pepsin in the observed immotilization, we conducted a second series of experiments in which the inhibitor pepstatin inactivated all pepsins present in the stomach of the anesthetized animal. Consequently, it was possible to distinguish between the bactericidal effect of a low pH and the additional effect of pepsin.
MATERIALS AND METHODS
Experimental techniques. (i) H. pylori culture. We chose H. pylori N6 wild type, which is a highly motile strain isolated from a gastritis patient in France (16). H. pylori frozen cultures were thawed and plated on 10% defibrinated horse blood agar containing H. pylori-selective supplement (Oxoid, Basingstoke, United Kingdom) and incubated at 37°C for 72 h in a microaerobic atmosphere (Anaerocult C; Merck, Darmstadt, Germany). For suspension of the bacteria, colonies from the plates were suspended in 2 ml of sterile brain heart infusion (BHI) broth. Subsequently, 10 ml of BHI broth, supplemented with 6% fetal calf serum and H. pylori-selective antibiotics (vancomycin, 10 mg/ml; polymyxin B, 3.2 mg/ml; trimethoprim, 5 mg/ml; amphotericin B, 4 mg/ml), was inoculated with the bacterial suspension. The broth was incubated in a microaerobic environment at 37°C for 24 h (12). For both in vivo and in vitro experiments, samples from this bacterial suspension with a density of 106 to 108 bacteria/ml were used. Before we introduced the bacteria into the gastric lumen, 5 ml of BHI in which the bacteria were cultured were replaced by 100 μl of brucella broth through centrifugation and resuspension. The density of the samples used was ca. 109 bacteria/ml.
(ii) Assessment of motility. Before an experiment was started, motility was measured by exposing a sample of the bacterial suspension for 30 min at a test pH of 5. Only bacterial suspensions in which more than 80% of the bacteria were motile after 30 min (pH 5) were used in the experiments. Here, as in all experiments, the motility was measured by counting the number of motile and immotile bacteria under the microscope (DMIRB, 100-fold apochromatic water-immersion lens; Leica, Microsystems, Wetzlar, Germany) at 1,000-fold magnification in the bright field. Portions (2 μl) of the bacterial suspension were enclosed in a small slide chamber (Menzel Glser, Braunschweig, Germany). The temperature of the sample was held at 37°C by using a small ring that heated the water between coverslip and lens (manufactured by the Institute of Physiology). The bacterial sample was never allowed to make contact with the ambient air. In the stomach the superfusing solution was saturated with a gas containing 5% O2, 10% CO2, and 85% N2. During the process of filling the slide chamber with the bacterial suspension, a gas flow of this mixture was applied over the slide. We kept the time between collecting the sample from the gastric juice and counting the bacteria under the microscope to less than 1 min. Motility was expressed as the percentage of motile bacteria from total bacteria, and at least 100 bacteria were counted in each sample.
(iii) Anesthesia and stomach preparation. We used specific-pathogen-free Mongolian gerbils (45 to 75 g, Crl:(MON)BR, Charles River, Germany, Sulzfeld, Germany) for in vivo experiments. All experiments were carried out under general inhalation anesthesia. The animals spontaneously breathed a mixture of halothane in 60% O2, 2% CO2, and 38% N2O in a semiclosed system. The anesthesia was performed with the aim of maintaining a stable blood supply to the stomach, which was monitored by observing the color of the gastric mucosa and the diameter of the vena gastrica dextra. Heart rate and respiratory frequency were also continuously monitored. The arterial oxygen saturation and acid/base status were measured at the end of the experiment as: sO2, 99% ± 3%; pCO2, 42 ± 9 mm Hg; HCO3–, 26 ± 1.4 mM; arterial blood pH, 7.39 ± 0.05 and hemoglobin concentration, 140 ± 1.4 g/liter (mean ± the standard deviation [SD]).
The animals were peritoneally dialyzed with a slightly hyposmolar solution to increase the total blood volume and the blood supply to the gastrointestinal tract (for a detailed description, see reference 24). The surgical procedure was a laparotomy using microsurgical equipment. With a radiofrequency electro-cautery device (Erbotom; Erbe, Tübingen, Germany), we made a subcostal incision in the skin. The abdominal wall was opened with a battery-driven high-temperature electro-cauterizer (small vessel cauterizer; Fine Science Tools, North Vancouver, Canada). The stomach was carefully lifted onto the platform of a micromanipulator. A metal pin on the platform, which perforated the ligamentum gastrohepaticum without touching the vena and arteria gastrica dextra, fixed the stomach onto the micromanipulator, exerting very little tension. Afterward, the stomach was opened at its ventral side by using again the radiofrequency electro-cauterizer with a small needle that produced a sharp spark. Small vessels that were cut were cauterized by using a small bipolar forceps. The gastric chymus was gently removed by using a suction catheter without touching the mucosal surface. The animals were left for at least half an hour until about half a milliliter of new gastric juice was secreted. The body temperature of the anesthetized Mongolian gerbil was held constant by using a heated chamber and an infrared lamp. All fluids were warmed to a temperature of 37°C before they reached the animal. The total observation time under continuous anesthesia was 2.5 h.
The luminal pH was continuously monitored by using a small pH electrode (InLab 423; Mettler-Toledo, Giessen, Germany). An autotitrator (ABU 80; Radiometer, Copenhagen, Denmark) was used to titrate and maintain the selected pH in the lumen. To ensure that the measured pH was the actual pH in the entire lumen, a small stirrer was inserted into the lumen to mix the luminal content (Fig. 1). The experimental protocol was approved by the institutional committee for the research involving animals (Bezirksregierung Arnsberg).
(iv) Chymus purification. To obtain pepsins from the gastric juice of the Mongolian gerbil, the chymus was extracted and purified. The gerbils were sacrificed, and the chymus immediately removed from the stomach and placed on ice. A sample of 500 μl of chymus was mixed with 1 ml of 20 mM sodium citrate buffer to a pH of 5.5. The samples were homogenized for 1 min and then centrifuged for 5 min at 5,000 x g. The supernatant was put through a Sephadex G-100 column, and the eluate (<50 kDa) was centrifuged for 5 min at 10,000 x g. The supernatant obtained was concentrated by using a Vivaspin centrifugal concentrator (Vivascience, Hannover, Germany) selective for particles of 10 kDa. All procedures were carried out on ice or at 4°C. This purified extract was tested by high-pressure liquid chromatography and showed peaks in the region of those of commercially available pepsins. The enzymatic activity of the extract was tested by using the Anson hemoglobin degradation test. We found most of the protein content of the extract to be enzymatically active pepsin. To measure pepsin activity in the purified chymus extract and in the gastric lumen, we used the definition that one unit of pepsin will produce an increase of 1.0 in photometric absorption (1-cm pathway) at a wavelength of 280 nm, measuring the trichloroacetic acid-soluble products of hemoglobin. The measurements were performed in accordance with the conditions determined by Anson (2) at the appropriate pH of 2 and at the higher pH of 5. Since the Anson test defines one unit of pepsin activity at the fixed pH value of 2, we refer to the activities determined at pH 5 as units (U).
To allow for the measurement of small amounts of pepsin, we modified the Anson test, with test volumes decreased 20-fold and the incubation times increased from the predefined time of 10 min up to 30 min. Extending incubation times had the disadvantage that the activity of pepsin decreased due to its autocatalytic activity. We measured pepsin activity at 10 and 30 min and found this decrease in activity to be constant. From these experiments, we obtained a multiplication factor of 1.2, which corrects the activity value measured at 30 min to the value at 10 min.
Experimental protocol. (i) Motility in vitro. The motility of H. pylori was tested in vitro at each pH value between 2.0 and 6.0. The test was performed in 100 μl of brucella medium (108 motile bacteria) incubated in a microaerobic atmosphere (5% O2, 10% CO2, 85% N2). The pH of the bacterial suspension was measured by using a small pH electrode (InLab 423; Mettler-Toledo) and titrated to the test pH values of 2.0, 3.0, 4.0, 4.5, 5.0, 5.5, and 6.0 by using an autotitrator. The titration solution contained 0.25 N HCl and 100 mM urea, producing an end concentration of ca. 10 mM urea in the brucella. A small gas lift was used to mix the bacterial suspension. For each pH value, the motility in a bacterial suspension was measured over a period of 15 min. After 1, 2, 5, 10, and 15 min, samples of 2 μl of the bacterial suspension were taken for measurement of motility. Figure 2 shows the setup for the measurement of bacterial motility in vitro.
(ii) Motility in vivo. A total of 50 μl of the bacterial suspension (108 motile bacteria) was inserted into the stomach lumen (final density of 106 to 108 bacteria/ml of gastric juice). The lumen was then titrated to test pH values of between 2.0 and 6.0 and held constant for 15 min. At the same time intervals of 1, 2, 5, 10, and 15 min, 2-μl samples of gastric juice were taken for measurement of motility.
(iii) Pepsin inactivation to determine whether loss of motility is reversible. To investigate whether bacteria that had become immotile by exposure to the conditions in the gastric juice could regain their motility, a second 2-μl sample, taken at the same time as the above samples, was introduced into brucella medium at pH 7.0. Motility was measured after 5 min in this solution. At this pH all pepsins are inactivated.
(iv) Test of viability. In an additional series of experiments, bacteria were cultured in vitro after being exposed to the gastric juice of the anesthetized animal at luminal pH values of 4 and 2. The bacteria remaining in the gastric lumen after the 15 min of exposure were counted. The amount of gastric juice containing 106 bacteria (100 to 500 μl) was used to culture the bacteria in vitro. The irreversibly immotile bacteria were plated on 10% defibrinated horse blood agar containing H. pylori-selective supplement (Oxoid) and incubated at 37°C for 4 days in a microaerobic atmosphere (Anaerocult C; Merck). The bacteria were diluted in decimal powers from 1:10 to 1:106 (agar dilution test). The number of bacteria counted in the gastric juice was compared to the number of colonies growing after plating (i.e., the number of CFU). The colonies were tested for urease activity, and only urease-positive colonies were counted.
(v) Motility in vivo after pepsins were blocked. To investigate the effect of pepsins on bacterial motility, similar experiments were performed, but the pepsin in the stomach was inactivated by rinsing the stomach with pepstatin (20) (100 μl of a 2-mg/ml stock solution). Samples were taken and analyzed for bacterial motility at the test pH values of 2.0, 3.0, 4.0, 4.5, and 5.0 and at the same periods after application of the bacterial suspension as in the experiments with active pepsins.
(vi) Effects of purified pepsins on motility in vitro. We incubated 100 μl of a bacterial suspension just as in the other tests for in vitro motility. The bacterial suspension was titrated down to a pH of 5.0 and held there by the autotitrator. To this suspension, 10 μl of purified pepsins from chymus was added, resulting in an activity of 100 U/ml. At intervals of 1, 2, 5, 10, and 15 min, 2-μl portions of the bacterial suspension were taken for measurement of the motility.
RESULTS
Motility in vitro. Between the pH values of 4 and 6 in the bacterial suspension, the motility of H. pylori remained constant over the 15-min test period. At least 80% of the bacteria were motile after 15 min of exposure to acid. At the pH values of 2 and 3, the motility decreased over the 15 min under the in vitro conditions. In Fig. 3 to 6 the motility in vitro at the given pH values is indicated by gray bars.
Motility in vivo. In the in vivo experiments (n = 5 for each pH) 2-μl samples were taken from the gastric juice after the insertion of the bacteria. At the pH values of 2.0 and 3.0, almost 100% of the bacteria were immotile within the first minute. Also at pH 4.0, no motile bacteria were detected after 2 min. The loss of bacterial motility at pH 4.5 was similar to that seen at pH 4.0 but at a reduced rate; 90% of the bacteria had already lost their motility after 1 min. At pH 5.0, the pattern of motility loss was similar again, but with an even longer delay. Even after 15 min of exposure, several bacteria remained motile. A continuous decrease in bacterial motility could also be observed at pH 5.5, but 10% of the bacteria were motile after 15 min of exposure. At pH 6.0, the loss of bacterial motility was minimal within the first 10 min, and after 15 min, more than 50% of the bacteria were still motile in the lumen (Fig. 3 and 4, black bars).
Irreversible loss of motility by pepsin and acid. A second sample was taken for all in vivo experiments at each respective time point and pH and was immediately mixed with buffered brucella broth (pH 7) to inactivate pepsins. Nevertheless, the immotile bacteria did not regain their motility; the loss of bacterial motility was irreversible.
Viability of immotile bacteria. The bacteria remaining in the gastric lumen after the 15 min of exposure were counted. The amount of gastric juice containing 106 bacteria was used to culture the bacteria in vitro. We tested whether bacteria were able to grow after exposure to the luminal pH 2 and 4 in the presence of pepsin (normal gastric juice) in an agar dilution test. Even though the bacteria had been shown to be irreversibly immobilized under these conditions, we could show that all bacteria exposed to pH 4 were still viable and formed colonies. The 106 immotile H. pylori collected from the gastric juice produced 106 colonies (n = 3). However, 15 min of exposure to the gastric juice at pH 2 reduced the number of CFU significantly from 106 to 103 (n = 3), and increasing the exposure time in the natural gastric juice at this pH to 30 min killed all of the bacteria (n = 3).
Motility in vivo after blocking of pepsins. We used pepstatin to block pepsins in the gastric lumen in vivo. The bacterial motility in the pepstatin-treated gastric juice was measured at the pH values of 2.0, 3.0, 4.0, 4.5, and 5.0 (n = 5 for each pH). The bacteria remained motile significantly longer than in the in vivo experiments without pepstatin (Fig. 5). Since the bacterial motility at the luminal pH of 6.0 was only slightly reduced under the natural conditions, we did not perform experiments with pepstatin at this pH.
Effects of purified pepsins on motility in vitro. Native gerbil chymus contained ca. 100 U of pepsins per milliliter at pH 5 (2,200 U at pH 2). Thus, pepsins extracted from chymus were concentrated to an activity of 100 U per ml of bacterial suspension in vitro. Bacteria mixed with extracted pepsins at pH 5.0 in vitro lost their motility as observed in vivo. Within the first minute, ca. 50% of the bacteria were immotilized and, after 15 min, the percentage of immotile bacteria had risen to >80% (Fig. 6).
DISCUSSION
The mechanism of H. pylori transmission and the factors that determine the transmissibility of this infection are largely unknown (10). Our data show that the period of time during which H. pylori stays motile in the gastric lumen in vivo—a prerequisite for active tactic colonization of deeper layers of the mucus (22a)—is much shorter than that extrapolated from in vitro data (25). The time to immotilization was strongly dependent on pepsin activity and pH. These findings suggest that it is the variability of the postprandial conditions in the gastric juice that determines the success of the Helicobacter colonization. The data presented here support a hypothesis that the susceptibility to acute Helicobacter infection is largely defined by the conditions in the stomach in the first few minutes after oral ingestion of the bacteria.
The gastric bactericidal barrier is formed by a combination of hydrochloric acid and different isoenzymes of the protease, pepsin. Pepsin isoenzymes show various pH optima between pH 2 and pH 4 (4, 3). At pH values above the optimum, pepsin activity decreases exponentially with neutralization. Therefore, the luminal pH defines the activity of each of these isoenzymes. As a result of the dependence of pepsin activity on pH, it is difficult to distinguish between the influences of the activated protease and the bactericidal effect of acidity. We used the pepsin inhibitor, pepstatin, to differentiate between these two factors. It was possible to reveal the importance of pepsin in protecting the stomach from being colonized by H. pylori. When pepsin was inactivated, the bacteria remained motile in the acidic environment of the stomach for a significantly longer period of time. Therefore, motile persistence in vivo of H. pylori is closely related to the activity of pepsin, which in turn is determined by the pH in the gastric lumen.
The interaction of pepsins and H. pylori in the stomach in vivo may occur in two phases: a first phase of rapid and irreversible loss of motility, after only a few minutes of exposure to active pepsins, which effectively hinders entry into the mucus and colonization, and a second phase at a lower pH (exposure of ca. 30 min and more) that will finally kill the bacteria remaining in the lumen. Since immotile bacteria are neither able to colonize the gastric mucus (6, 7, 12, 19) nor able to persist in the continuous flow of gastric mucus toward the lumen (23), rapid suppression of bacterial motility is a very effective strategy in mucosal defense.
Due to the high pepsin activity at pH values of less than 4, a colonization is hardly possible under these conditions. The situation is different for pH values greater than 6, at which the pepsin activity is nearly lost. At this pH, the bacteria remained motile significantly longer, making a colonization more likely.
In the stomach of the healthy adult, pH values range from 1 to 5 (8). Short neutralizations to a pH higher than 5.5 are occasional postprandial occurrences. Nevertheless, it is very unlikely that these bouts of high pH enable H. pylori to establish an infection, since these events only last for a few minutes. Our data provide a causal explanation for the concept established by multiple epidemiological studies that the risk of Helicobacter infection appears to be low for adults, since their gastric environment consists of large amounts of pepsin activated by low pH values. In young infants, however, neutral pH values persist for up to an hour after breast feeding (17), and pepsin secretion increases in the first months from low values until it reaches the adult values (1, 21). The decreased pepsin activity during this period of time might give H. pylori the opportunity to colonize.
This provides a basis for the hypothesis that a specific group of adults might have a higher risk of infection as well: the increasingly large group of patients treated by acid-suppressive therapy. It is known that long-term acid-suppressive therapy in H. pylori-infected patients causes a bacterial invasion into the corpus and fundus region (15, 18), and infection by other pathogens seems to be more likely (27).
Despite the advances made regarding the pathogenesis of H. pylori in recent years, the conditions allowing a successful colonization of the gastric mucus should be further explored to expand our knowledge of the mechanisms of Helicobacter transmission.
ACKNOWLEDGMENTS
We thank Jacqui Burton and Gabi Reimus for excellent technical assistance.
This study was supported by the grants Sche 46/14-1 and Su 133/4-1 from the Deutsche Forschungsgemeinschaft and a "Bennigsen-Foerder" award presented to S. Schreiber and C. Josenhans.
REFERENCES
1. Agunod, M., N. Yamaguchi, R. Lopez, A. L. Luhby, and G. B. Glass. 1969. Correlative study of hydrochloric acid, pepsin, and intrinsic factor secretion in newborns and infants. Am. J. Dig. Dis. 14:400-414.
2. Anson, M. L. 1938. The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J. Gen. Physiol. 22:79-89.
3. Athauda, S. B., T. Kageyama, T. Takahashi, H. Inoue, M. Ichinose, M. Ukai, and K. Takahashi. 1995. Isolation and characterization of human gastric procathepsin E and cathepsin E. Adv. Exp. Med. Biol. 362:201-210.
4. Becker, T., and W. Rapp. 1979. Characterization of human pepsin I obtained from purified gastric pepsinogen I. Klin. Wochenschr. 57:711-718.
5. Becker, T., and W. Rapp. 1979. Characterization of human pepsin II obtained from purified gastric pepsinogen II. Klin. Wochenschr. 57:719-724.
6. Eaton, K. A., D. R. Morgan, and S. Krakowka. 1992. Motility as a factor in the colonization of gnotobiotic piglets by Helicobacter pylori. J. Med. Microbiol. 37:123-127.
7. Eaton, K. A., S. Suerbaum, C. Josenhans, and S. Krakowka. 1996. Colonization of gnotobiotic piglets by Helicobacter pylori deficient in two flagellin genes. Infect. Immun. 64:2445-2448.
8. Fimmel, C. J., A. Etienne, T. Cilluffo, C. von Ritter, T. Gasser, J. P. Rey, P. Caradonna-Moscatelli, F. Sabbatini, F. Pace, and H. W. Buhler. 1985. Long-term ambulatory gastric pH monitoring: validation of a new method and effect of H2-antagonists. Gastroenterology 88:1842-1851.
9. Foltmann, B. 1981. Gastric proteinases: structure, function, evolution, and mechanism of action. Essays Biochem. 17:52-84.
10. Goodman, K. J., and P. Correa. 1995. The transmission of Helicobacter pylori: a critical review of the evidence. Int. J. Epidemiol. 24:875-887.
11. Ikeno, T., H. Ota, A. Sugiyama, K. Ishida, T. Katsuyama, R. M. Genta, and S. Kawasaki. 1999. Helicobacter pylori-induced chronic active gastritis, intestinal metaplasia, and gastric ulcer in Mongolian gerbils. Am. J. Pathol. 154:951-960.
12. Josenhans, C., and S. Suerbaum. 2002. The role of motility as a virulence factor in bacteria. Int. J. Med. Microbiol. 291:605-614.
13. Keto, Y., S. Takahashi, and S. Okabe. 1999. Healing of Helicobacter pylori-induced gastric ulcers in Mongolian gerbils: combined treatment with omeprazole and clarithromycin. Dig. Dis. Sci. 44:257-265.
14. Kotts, C., and R. Jenness. 1976. Rennin and pepsin in stomachs of rats (Rattus norvegicus). J. Dairy Sci. 59:1398-1400.
15. Kuipers, E. J., L. Lundell, E. C. Klinkenberg-Knol, N. Havu, H. P. Festen, B. Liedman, C. B. Lamers, J. B. Jansen, J. Dalenback, P. Snel, G. F. Nelis, and S. G. Meuwissen. 1996. Atrophic gastritis and Helicobacter pylori infection in patients with reflux esophagitis treated with omeprazole or fundoplication. N. Engl. J. Med. 334:1018-1022.
16. Labigne, A., V. Cussac, and P. Courcoux. 1991. Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity. J. Bacteriol. 173:1920-1931.
17. Mitchell, D. J., B. G. McClure, and T. R. Tubman. 2001. Simultaneous monitoring of gastric and oesophageal pH reveals limitations of conventional oesophageal pH monitoring in milk fed infants. Arch. Dis. Child. 84:273-276.
18. Moayyedi, P., C. Wason, R. Peacock, A. Walan, K. Bardhan, A. T. Axon, and M. F. Dixon. 2000. Changing patterns of Helicobacter pylori gastritis in long-standing acid suppression. Helicobacter 5:206-214.
19. Ottemann, K. M., and A. C. Lowenthal. 2002. Helicobacter pylori uses motility for initial colonization and to attain robust infection. Infect. Immun. 70:1984-1990.
20. Rich, D. H., and E. T. Sun. 1980. Mechanism of inhibition of pepsin by pepstatin. Effect of inhibitor structure on dissociation constant and time-dependent inhibition. Biochem. Pharmacol. 29:2205-2212.
21. Rdbro, P., P. Krasilnikoff, and V. Bitsch. 1967. Gastric secretion of pepsin in early childhood. Scand. J. Gastroenterol. 2:257-260.
22. Samloff, I. M., R. T. Taggart, T. Shiraishi, T. Branch, W. A. Reid, R. Heath, R. W. Lewis, M. J. Valler, and J. Kay. 1987. Slow moving proteinase: isolation, characterization, and immunohistochemical localization in gastric mucosa. Gastroenterology 93:77-84.
22. Schreiber, S., M. Konradt, C. Groll, P. Scheid, G. Hanauer, H. O. Werling, C. Josenhans, and S. Suerbaum. 2004. The spatial orientation of Helicobacter pylori in the gastric mucus. Proc. Natl. Acad. Sci. USA 101:5024-5029.
23. Schreiber, S., and P. Scheid. 1997. Gastric mucus of the guinea pig: proton carrier and diffusion barrier. Am. J. Physiol. 272:G63-G70.
24. Schreiber, S., M. Stüben, C. Josenhans, P. Scheid, and S. Suerbaum. 1999. In vivo distribution of Helicobacter felis in the gastric mucus of the mouse: experimental method and results. Infect. Immun. 67:5151-5156.
25. Stingl, K., E. M. Uhlemann, R. Schmid, K. Altendorf, and E. P. Bakker. 2002. Energetics of Helicobacter pylori and its implications for the mechanism of urease-dependent acid tolerance at pH 1. J. Bacteriol. 184:3053-3060.
26. Suerbaum, S., and P. Michetti. 2002. Helicobacter pylori infection. N. Engl. J. Med. 347:1175-1186.
27. Thorens, J., F. Froehlich, W. Schwizer, E. Saraga, J. Bille, K. Gyr, P. Duroux, M. Nicolet, B. Pignatelli, A. L. Blum, J. J. Gonvers, and M. Fried. 1996. Bacterial overgrowth during treatment with omeprazole compared with cimetidine: a prospective randomized double blind study. Gut 39:54-59.
28. Watanabe, T., M. Tada, H. Nagai, S. Sasaki, and M. Nakao. 1998. Helicobacter pylori infection induces gastric cancer in Mongolian gerbils. Gastroenterology 115:642-648.(Sren Schreiber, Roland Bü)
Institut für Hygiene und Mikrobiologie, Ruhr-Universitt Bochum, Bochum
Institut für Medizinische Mikrobiologie und Krankenhaushygiene, Medizinische Hochschule Hannover, Hannover, Germany
ABSTRACT
The human pathogen Helicobacter pylori has infected more than half of the world's population. Nevertheless, the first step of infection, the acute colonization of the gastric mucus, is poorly understood. For successful colonization, H. pylori must retain active motility in the gastric lumen until it reaches the safety of the mucus layer. To identify the factors determining the acute colonization, we inserted bacteria into the stomach of anesthetized Mongolian gerbils. We adjusted the gastric juice to defined pH values of between 2.0 and 6.0 by using an autotitrator. Despite the fact that Helicobacter spp. are known to survive low pH values for a certain time in vitro, the length of time that H. pylori persisted under the assay conditions within the gastric juice in vivo was remarkably shorter. In the anesthetized animal we found H. pylori to be irreversibly immotile in less than 1 min at lumen pH values of 2 and 3. At pH 4 motility was lost after 2 min. However, the period of motility increased to more than 15 min at pH 6. Blocking pepsins in the gastric lumen in vivo by using pepstatin significantly increased the period of motility. It was possible to simulate the rapid in vivo immotilization in vitro by adding pepsins. We conclude that pepsin limits the persistence of H. pylori in the gastric chymus to only a few minutes by rapidly inhibiting active motility. It is therefore likely that this short period of resistance in the gastric lumen is one of the most critical phases of Helicobacter infection.
INTRODUCTION
The bacterium Helicobacter pylori causes chronic gastritis, which may progress to gastric or duodenal ulcers, gastric atrophy, mucosa-associated lymphoid tissue lymphoma, and adenocarcinoma (26).
This pathogen is unique among bacteria in its ability to persist in the harsh environment of the human stomach characterized by a combination of hydrochloric acid and different acidic aspartate proteases, historically referred to as pepsins (9). Although this bactericidal barrier protects the organism from microbes, H. pylori can colonize the gastric mucus.
Gastric proteases have been divided into three classes, namely, pepsin A and C, chymosin and the intracellular cathepsin E, each secreted as a proenzyme and activated at low pH values. Pepsin A (pepsin I) and pepsin C (pepsin II) are the dominant proteases in the human stomach. All of these isoenzymes exponentially lose proteolytic activity when pH values are above their pH optimum of between 2.0 and 3.8 (4, 5, 14, 22). The pH in the human stomach lumen varies between basal values of 1 to more neutral values after a meal (postprandial pH values). Although in adults the luminal pH rarely exceeds 5.5 (8), infants have a different pH profile. It was shown by 24-h pH-metry that the pH increases from basal values of 2 to pH 7 after breast feeding (17). Thus, the postprandial peptic activity in adults is higher than in children.
The specific conditions for a successful Helicobacter infection are still unknown. We do know, however, that motility is essential for H. pylori to initially colonize and chronically infect the gastric mucus (6, 7, 12, 19). The loss of motility of a bacterium in the lumen is therefore equivalent to its inability to colonize the mucus layer.
To simulate the process of colonization in the human stomach, we used the Mongolian gerbil, a well-established model for the study of H. pylori infection. Gerbils have luminal pH values similar to those in the human stomach (mean basal pH 1.4 [unpublished data]). In addition, the pathology that H. pylori induces in gerbils is very similar to that observed in humans (gastritis, ulcers, carcinoma, and lymphoma) (11, 28). Eradication of the bacterium can be achieved by the same treatment successfully used in humans (13).
In order to describe the phase of colonization from oral ingestion to invasion of the mucus, we inoculated highly motile bacteria into the stomach of anesthetized gerbils at defined postprandial pH values and measured bacterial motility. We found a rapid loss of motility under in vivo conditions. To investigate the possible role of pepsin in the observed immotilization, we conducted a second series of experiments in which the inhibitor pepstatin inactivated all pepsins present in the stomach of the anesthetized animal. Consequently, it was possible to distinguish between the bactericidal effect of a low pH and the additional effect of pepsin.
MATERIALS AND METHODS
Experimental techniques. (i) H. pylori culture. We chose H. pylori N6 wild type, which is a highly motile strain isolated from a gastritis patient in France (16). H. pylori frozen cultures were thawed and plated on 10% defibrinated horse blood agar containing H. pylori-selective supplement (Oxoid, Basingstoke, United Kingdom) and incubated at 37°C for 72 h in a microaerobic atmosphere (Anaerocult C; Merck, Darmstadt, Germany). For suspension of the bacteria, colonies from the plates were suspended in 2 ml of sterile brain heart infusion (BHI) broth. Subsequently, 10 ml of BHI broth, supplemented with 6% fetal calf serum and H. pylori-selective antibiotics (vancomycin, 10 mg/ml; polymyxin B, 3.2 mg/ml; trimethoprim, 5 mg/ml; amphotericin B, 4 mg/ml), was inoculated with the bacterial suspension. The broth was incubated in a microaerobic environment at 37°C for 24 h (12). For both in vivo and in vitro experiments, samples from this bacterial suspension with a density of 106 to 108 bacteria/ml were used. Before we introduced the bacteria into the gastric lumen, 5 ml of BHI in which the bacteria were cultured were replaced by 100 μl of brucella broth through centrifugation and resuspension. The density of the samples used was ca. 109 bacteria/ml.
(ii) Assessment of motility. Before an experiment was started, motility was measured by exposing a sample of the bacterial suspension for 30 min at a test pH of 5. Only bacterial suspensions in which more than 80% of the bacteria were motile after 30 min (pH 5) were used in the experiments. Here, as in all experiments, the motility was measured by counting the number of motile and immotile bacteria under the microscope (DMIRB, 100-fold apochromatic water-immersion lens; Leica, Microsystems, Wetzlar, Germany) at 1,000-fold magnification in the bright field. Portions (2 μl) of the bacterial suspension were enclosed in a small slide chamber (Menzel Glser, Braunschweig, Germany). The temperature of the sample was held at 37°C by using a small ring that heated the water between coverslip and lens (manufactured by the Institute of Physiology). The bacterial sample was never allowed to make contact with the ambient air. In the stomach the superfusing solution was saturated with a gas containing 5% O2, 10% CO2, and 85% N2. During the process of filling the slide chamber with the bacterial suspension, a gas flow of this mixture was applied over the slide. We kept the time between collecting the sample from the gastric juice and counting the bacteria under the microscope to less than 1 min. Motility was expressed as the percentage of motile bacteria from total bacteria, and at least 100 bacteria were counted in each sample.
(iii) Anesthesia and stomach preparation. We used specific-pathogen-free Mongolian gerbils (45 to 75 g, Crl:(MON)BR, Charles River, Germany, Sulzfeld, Germany) for in vivo experiments. All experiments were carried out under general inhalation anesthesia. The animals spontaneously breathed a mixture of halothane in 60% O2, 2% CO2, and 38% N2O in a semiclosed system. The anesthesia was performed with the aim of maintaining a stable blood supply to the stomach, which was monitored by observing the color of the gastric mucosa and the diameter of the vena gastrica dextra. Heart rate and respiratory frequency were also continuously monitored. The arterial oxygen saturation and acid/base status were measured at the end of the experiment as: sO2, 99% ± 3%; pCO2, 42 ± 9 mm Hg; HCO3–, 26 ± 1.4 mM; arterial blood pH, 7.39 ± 0.05 and hemoglobin concentration, 140 ± 1.4 g/liter (mean ± the standard deviation [SD]).
The animals were peritoneally dialyzed with a slightly hyposmolar solution to increase the total blood volume and the blood supply to the gastrointestinal tract (for a detailed description, see reference 24). The surgical procedure was a laparotomy using microsurgical equipment. With a radiofrequency electro-cautery device (Erbotom; Erbe, Tübingen, Germany), we made a subcostal incision in the skin. The abdominal wall was opened with a battery-driven high-temperature electro-cauterizer (small vessel cauterizer; Fine Science Tools, North Vancouver, Canada). The stomach was carefully lifted onto the platform of a micromanipulator. A metal pin on the platform, which perforated the ligamentum gastrohepaticum without touching the vena and arteria gastrica dextra, fixed the stomach onto the micromanipulator, exerting very little tension. Afterward, the stomach was opened at its ventral side by using again the radiofrequency electro-cauterizer with a small needle that produced a sharp spark. Small vessels that were cut were cauterized by using a small bipolar forceps. The gastric chymus was gently removed by using a suction catheter without touching the mucosal surface. The animals were left for at least half an hour until about half a milliliter of new gastric juice was secreted. The body temperature of the anesthetized Mongolian gerbil was held constant by using a heated chamber and an infrared lamp. All fluids were warmed to a temperature of 37°C before they reached the animal. The total observation time under continuous anesthesia was 2.5 h.
The luminal pH was continuously monitored by using a small pH electrode (InLab 423; Mettler-Toledo, Giessen, Germany). An autotitrator (ABU 80; Radiometer, Copenhagen, Denmark) was used to titrate and maintain the selected pH in the lumen. To ensure that the measured pH was the actual pH in the entire lumen, a small stirrer was inserted into the lumen to mix the luminal content (Fig. 1). The experimental protocol was approved by the institutional committee for the research involving animals (Bezirksregierung Arnsberg).
(iv) Chymus purification. To obtain pepsins from the gastric juice of the Mongolian gerbil, the chymus was extracted and purified. The gerbils were sacrificed, and the chymus immediately removed from the stomach and placed on ice. A sample of 500 μl of chymus was mixed with 1 ml of 20 mM sodium citrate buffer to a pH of 5.5. The samples were homogenized for 1 min and then centrifuged for 5 min at 5,000 x g. The supernatant was put through a Sephadex G-100 column, and the eluate (<50 kDa) was centrifuged for 5 min at 10,000 x g. The supernatant obtained was concentrated by using a Vivaspin centrifugal concentrator (Vivascience, Hannover, Germany) selective for particles of 10 kDa. All procedures were carried out on ice or at 4°C. This purified extract was tested by high-pressure liquid chromatography and showed peaks in the region of those of commercially available pepsins. The enzymatic activity of the extract was tested by using the Anson hemoglobin degradation test. We found most of the protein content of the extract to be enzymatically active pepsin. To measure pepsin activity in the purified chymus extract and in the gastric lumen, we used the definition that one unit of pepsin will produce an increase of 1.0 in photometric absorption (1-cm pathway) at a wavelength of 280 nm, measuring the trichloroacetic acid-soluble products of hemoglobin. The measurements were performed in accordance with the conditions determined by Anson (2) at the appropriate pH of 2 and at the higher pH of 5. Since the Anson test defines one unit of pepsin activity at the fixed pH value of 2, we refer to the activities determined at pH 5 as units (U).
To allow for the measurement of small amounts of pepsin, we modified the Anson test, with test volumes decreased 20-fold and the incubation times increased from the predefined time of 10 min up to 30 min. Extending incubation times had the disadvantage that the activity of pepsin decreased due to its autocatalytic activity. We measured pepsin activity at 10 and 30 min and found this decrease in activity to be constant. From these experiments, we obtained a multiplication factor of 1.2, which corrects the activity value measured at 30 min to the value at 10 min.
Experimental protocol. (i) Motility in vitro. The motility of H. pylori was tested in vitro at each pH value between 2.0 and 6.0. The test was performed in 100 μl of brucella medium (108 motile bacteria) incubated in a microaerobic atmosphere (5% O2, 10% CO2, 85% N2). The pH of the bacterial suspension was measured by using a small pH electrode (InLab 423; Mettler-Toledo) and titrated to the test pH values of 2.0, 3.0, 4.0, 4.5, 5.0, 5.5, and 6.0 by using an autotitrator. The titration solution contained 0.25 N HCl and 100 mM urea, producing an end concentration of ca. 10 mM urea in the brucella. A small gas lift was used to mix the bacterial suspension. For each pH value, the motility in a bacterial suspension was measured over a period of 15 min. After 1, 2, 5, 10, and 15 min, samples of 2 μl of the bacterial suspension were taken for measurement of motility. Figure 2 shows the setup for the measurement of bacterial motility in vitro.
(ii) Motility in vivo. A total of 50 μl of the bacterial suspension (108 motile bacteria) was inserted into the stomach lumen (final density of 106 to 108 bacteria/ml of gastric juice). The lumen was then titrated to test pH values of between 2.0 and 6.0 and held constant for 15 min. At the same time intervals of 1, 2, 5, 10, and 15 min, 2-μl samples of gastric juice were taken for measurement of motility.
(iii) Pepsin inactivation to determine whether loss of motility is reversible. To investigate whether bacteria that had become immotile by exposure to the conditions in the gastric juice could regain their motility, a second 2-μl sample, taken at the same time as the above samples, was introduced into brucella medium at pH 7.0. Motility was measured after 5 min in this solution. At this pH all pepsins are inactivated.
(iv) Test of viability. In an additional series of experiments, bacteria were cultured in vitro after being exposed to the gastric juice of the anesthetized animal at luminal pH values of 4 and 2. The bacteria remaining in the gastric lumen after the 15 min of exposure were counted. The amount of gastric juice containing 106 bacteria (100 to 500 μl) was used to culture the bacteria in vitro. The irreversibly immotile bacteria were plated on 10% defibrinated horse blood agar containing H. pylori-selective supplement (Oxoid) and incubated at 37°C for 4 days in a microaerobic atmosphere (Anaerocult C; Merck). The bacteria were diluted in decimal powers from 1:10 to 1:106 (agar dilution test). The number of bacteria counted in the gastric juice was compared to the number of colonies growing after plating (i.e., the number of CFU). The colonies were tested for urease activity, and only urease-positive colonies were counted.
(v) Motility in vivo after pepsins were blocked. To investigate the effect of pepsins on bacterial motility, similar experiments were performed, but the pepsin in the stomach was inactivated by rinsing the stomach with pepstatin (20) (100 μl of a 2-mg/ml stock solution). Samples were taken and analyzed for bacterial motility at the test pH values of 2.0, 3.0, 4.0, 4.5, and 5.0 and at the same periods after application of the bacterial suspension as in the experiments with active pepsins.
(vi) Effects of purified pepsins on motility in vitro. We incubated 100 μl of a bacterial suspension just as in the other tests for in vitro motility. The bacterial suspension was titrated down to a pH of 5.0 and held there by the autotitrator. To this suspension, 10 μl of purified pepsins from chymus was added, resulting in an activity of 100 U/ml. At intervals of 1, 2, 5, 10, and 15 min, 2-μl portions of the bacterial suspension were taken for measurement of the motility.
RESULTS
Motility in vitro. Between the pH values of 4 and 6 in the bacterial suspension, the motility of H. pylori remained constant over the 15-min test period. At least 80% of the bacteria were motile after 15 min of exposure to acid. At the pH values of 2 and 3, the motility decreased over the 15 min under the in vitro conditions. In Fig. 3 to 6 the motility in vitro at the given pH values is indicated by gray bars.
Motility in vivo. In the in vivo experiments (n = 5 for each pH) 2-μl samples were taken from the gastric juice after the insertion of the bacteria. At the pH values of 2.0 and 3.0, almost 100% of the bacteria were immotile within the first minute. Also at pH 4.0, no motile bacteria were detected after 2 min. The loss of bacterial motility at pH 4.5 was similar to that seen at pH 4.0 but at a reduced rate; 90% of the bacteria had already lost their motility after 1 min. At pH 5.0, the pattern of motility loss was similar again, but with an even longer delay. Even after 15 min of exposure, several bacteria remained motile. A continuous decrease in bacterial motility could also be observed at pH 5.5, but 10% of the bacteria were motile after 15 min of exposure. At pH 6.0, the loss of bacterial motility was minimal within the first 10 min, and after 15 min, more than 50% of the bacteria were still motile in the lumen (Fig. 3 and 4, black bars).
Irreversible loss of motility by pepsin and acid. A second sample was taken for all in vivo experiments at each respective time point and pH and was immediately mixed with buffered brucella broth (pH 7) to inactivate pepsins. Nevertheless, the immotile bacteria did not regain their motility; the loss of bacterial motility was irreversible.
Viability of immotile bacteria. The bacteria remaining in the gastric lumen after the 15 min of exposure were counted. The amount of gastric juice containing 106 bacteria was used to culture the bacteria in vitro. We tested whether bacteria were able to grow after exposure to the luminal pH 2 and 4 in the presence of pepsin (normal gastric juice) in an agar dilution test. Even though the bacteria had been shown to be irreversibly immobilized under these conditions, we could show that all bacteria exposed to pH 4 were still viable and formed colonies. The 106 immotile H. pylori collected from the gastric juice produced 106 colonies (n = 3). However, 15 min of exposure to the gastric juice at pH 2 reduced the number of CFU significantly from 106 to 103 (n = 3), and increasing the exposure time in the natural gastric juice at this pH to 30 min killed all of the bacteria (n = 3).
Motility in vivo after blocking of pepsins. We used pepstatin to block pepsins in the gastric lumen in vivo. The bacterial motility in the pepstatin-treated gastric juice was measured at the pH values of 2.0, 3.0, 4.0, 4.5, and 5.0 (n = 5 for each pH). The bacteria remained motile significantly longer than in the in vivo experiments without pepstatin (Fig. 5). Since the bacterial motility at the luminal pH of 6.0 was only slightly reduced under the natural conditions, we did not perform experiments with pepstatin at this pH.
Effects of purified pepsins on motility in vitro. Native gerbil chymus contained ca. 100 U of pepsins per milliliter at pH 5 (2,200 U at pH 2). Thus, pepsins extracted from chymus were concentrated to an activity of 100 U per ml of bacterial suspension in vitro. Bacteria mixed with extracted pepsins at pH 5.0 in vitro lost their motility as observed in vivo. Within the first minute, ca. 50% of the bacteria were immotilized and, after 15 min, the percentage of immotile bacteria had risen to >80% (Fig. 6).
DISCUSSION
The mechanism of H. pylori transmission and the factors that determine the transmissibility of this infection are largely unknown (10). Our data show that the period of time during which H. pylori stays motile in the gastric lumen in vivo—a prerequisite for active tactic colonization of deeper layers of the mucus (22a)—is much shorter than that extrapolated from in vitro data (25). The time to immotilization was strongly dependent on pepsin activity and pH. These findings suggest that it is the variability of the postprandial conditions in the gastric juice that determines the success of the Helicobacter colonization. The data presented here support a hypothesis that the susceptibility to acute Helicobacter infection is largely defined by the conditions in the stomach in the first few minutes after oral ingestion of the bacteria.
The gastric bactericidal barrier is formed by a combination of hydrochloric acid and different isoenzymes of the protease, pepsin. Pepsin isoenzymes show various pH optima between pH 2 and pH 4 (4, 3). At pH values above the optimum, pepsin activity decreases exponentially with neutralization. Therefore, the luminal pH defines the activity of each of these isoenzymes. As a result of the dependence of pepsin activity on pH, it is difficult to distinguish between the influences of the activated protease and the bactericidal effect of acidity. We used the pepsin inhibitor, pepstatin, to differentiate between these two factors. It was possible to reveal the importance of pepsin in protecting the stomach from being colonized by H. pylori. When pepsin was inactivated, the bacteria remained motile in the acidic environment of the stomach for a significantly longer period of time. Therefore, motile persistence in vivo of H. pylori is closely related to the activity of pepsin, which in turn is determined by the pH in the gastric lumen.
The interaction of pepsins and H. pylori in the stomach in vivo may occur in two phases: a first phase of rapid and irreversible loss of motility, after only a few minutes of exposure to active pepsins, which effectively hinders entry into the mucus and colonization, and a second phase at a lower pH (exposure of ca. 30 min and more) that will finally kill the bacteria remaining in the lumen. Since immotile bacteria are neither able to colonize the gastric mucus (6, 7, 12, 19) nor able to persist in the continuous flow of gastric mucus toward the lumen (23), rapid suppression of bacterial motility is a very effective strategy in mucosal defense.
Due to the high pepsin activity at pH values of less than 4, a colonization is hardly possible under these conditions. The situation is different for pH values greater than 6, at which the pepsin activity is nearly lost. At this pH, the bacteria remained motile significantly longer, making a colonization more likely.
In the stomach of the healthy adult, pH values range from 1 to 5 (8). Short neutralizations to a pH higher than 5.5 are occasional postprandial occurrences. Nevertheless, it is very unlikely that these bouts of high pH enable H. pylori to establish an infection, since these events only last for a few minutes. Our data provide a causal explanation for the concept established by multiple epidemiological studies that the risk of Helicobacter infection appears to be low for adults, since their gastric environment consists of large amounts of pepsin activated by low pH values. In young infants, however, neutral pH values persist for up to an hour after breast feeding (17), and pepsin secretion increases in the first months from low values until it reaches the adult values (1, 21). The decreased pepsin activity during this period of time might give H. pylori the opportunity to colonize.
This provides a basis for the hypothesis that a specific group of adults might have a higher risk of infection as well: the increasingly large group of patients treated by acid-suppressive therapy. It is known that long-term acid-suppressive therapy in H. pylori-infected patients causes a bacterial invasion into the corpus and fundus region (15, 18), and infection by other pathogens seems to be more likely (27).
Despite the advances made regarding the pathogenesis of H. pylori in recent years, the conditions allowing a successful colonization of the gastric mucus should be further explored to expand our knowledge of the mechanisms of Helicobacter transmission.
ACKNOWLEDGMENTS
We thank Jacqui Burton and Gabi Reimus for excellent technical assistance.
This study was supported by the grants Sche 46/14-1 and Su 133/4-1 from the Deutsche Forschungsgemeinschaft and a "Bennigsen-Foerder" award presented to S. Schreiber and C. Josenhans.
REFERENCES
1. Agunod, M., N. Yamaguchi, R. Lopez, A. L. Luhby, and G. B. Glass. 1969. Correlative study of hydrochloric acid, pepsin, and intrinsic factor secretion in newborns and infants. Am. J. Dig. Dis. 14:400-414.
2. Anson, M. L. 1938. The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. J. Gen. Physiol. 22:79-89.
3. Athauda, S. B., T. Kageyama, T. Takahashi, H. Inoue, M. Ichinose, M. Ukai, and K. Takahashi. 1995. Isolation and characterization of human gastric procathepsin E and cathepsin E. Adv. Exp. Med. Biol. 362:201-210.
4. Becker, T., and W. Rapp. 1979. Characterization of human pepsin I obtained from purified gastric pepsinogen I. Klin. Wochenschr. 57:711-718.
5. Becker, T., and W. Rapp. 1979. Characterization of human pepsin II obtained from purified gastric pepsinogen II. Klin. Wochenschr. 57:719-724.
6. Eaton, K. A., D. R. Morgan, and S. Krakowka. 1992. Motility as a factor in the colonization of gnotobiotic piglets by Helicobacter pylori. J. Med. Microbiol. 37:123-127.
7. Eaton, K. A., S. Suerbaum, C. Josenhans, and S. Krakowka. 1996. Colonization of gnotobiotic piglets by Helicobacter pylori deficient in two flagellin genes. Infect. Immun. 64:2445-2448.
8. Fimmel, C. J., A. Etienne, T. Cilluffo, C. von Ritter, T. Gasser, J. P. Rey, P. Caradonna-Moscatelli, F. Sabbatini, F. Pace, and H. W. Buhler. 1985. Long-term ambulatory gastric pH monitoring: validation of a new method and effect of H2-antagonists. Gastroenterology 88:1842-1851.
9. Foltmann, B. 1981. Gastric proteinases: structure, function, evolution, and mechanism of action. Essays Biochem. 17:52-84.
10. Goodman, K. J., and P. Correa. 1995. The transmission of Helicobacter pylori: a critical review of the evidence. Int. J. Epidemiol. 24:875-887.
11. Ikeno, T., H. Ota, A. Sugiyama, K. Ishida, T. Katsuyama, R. M. Genta, and S. Kawasaki. 1999. Helicobacter pylori-induced chronic active gastritis, intestinal metaplasia, and gastric ulcer in Mongolian gerbils. Am. J. Pathol. 154:951-960.
12. Josenhans, C., and S. Suerbaum. 2002. The role of motility as a virulence factor in bacteria. Int. J. Med. Microbiol. 291:605-614.
13. Keto, Y., S. Takahashi, and S. Okabe. 1999. Healing of Helicobacter pylori-induced gastric ulcers in Mongolian gerbils: combined treatment with omeprazole and clarithromycin. Dig. Dis. Sci. 44:257-265.
14. Kotts, C., and R. Jenness. 1976. Rennin and pepsin in stomachs of rats (Rattus norvegicus). J. Dairy Sci. 59:1398-1400.
15. Kuipers, E. J., L. Lundell, E. C. Klinkenberg-Knol, N. Havu, H. P. Festen, B. Liedman, C. B. Lamers, J. B. Jansen, J. Dalenback, P. Snel, G. F. Nelis, and S. G. Meuwissen. 1996. Atrophic gastritis and Helicobacter pylori infection in patients with reflux esophagitis treated with omeprazole or fundoplication. N. Engl. J. Med. 334:1018-1022.
16. Labigne, A., V. Cussac, and P. Courcoux. 1991. Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity. J. Bacteriol. 173:1920-1931.
17. Mitchell, D. J., B. G. McClure, and T. R. Tubman. 2001. Simultaneous monitoring of gastric and oesophageal pH reveals limitations of conventional oesophageal pH monitoring in milk fed infants. Arch. Dis. Child. 84:273-276.
18. Moayyedi, P., C. Wason, R. Peacock, A. Walan, K. Bardhan, A. T. Axon, and M. F. Dixon. 2000. Changing patterns of Helicobacter pylori gastritis in long-standing acid suppression. Helicobacter 5:206-214.
19. Ottemann, K. M., and A. C. Lowenthal. 2002. Helicobacter pylori uses motility for initial colonization and to attain robust infection. Infect. Immun. 70:1984-1990.
20. Rich, D. H., and E. T. Sun. 1980. Mechanism of inhibition of pepsin by pepstatin. Effect of inhibitor structure on dissociation constant and time-dependent inhibition. Biochem. Pharmacol. 29:2205-2212.
21. Rdbro, P., P. Krasilnikoff, and V. Bitsch. 1967. Gastric secretion of pepsin in early childhood. Scand. J. Gastroenterol. 2:257-260.
22. Samloff, I. M., R. T. Taggart, T. Shiraishi, T. Branch, W. A. Reid, R. Heath, R. W. Lewis, M. J. Valler, and J. Kay. 1987. Slow moving proteinase: isolation, characterization, and immunohistochemical localization in gastric mucosa. Gastroenterology 93:77-84.
22. Schreiber, S., M. Konradt, C. Groll, P. Scheid, G. Hanauer, H. O. Werling, C. Josenhans, and S. Suerbaum. 2004. The spatial orientation of Helicobacter pylori in the gastric mucus. Proc. Natl. Acad. Sci. USA 101:5024-5029.
23. Schreiber, S., and P. Scheid. 1997. Gastric mucus of the guinea pig: proton carrier and diffusion barrier. Am. J. Physiol. 272:G63-G70.
24. Schreiber, S., M. Stüben, C. Josenhans, P. Scheid, and S. Suerbaum. 1999. In vivo distribution of Helicobacter felis in the gastric mucus of the mouse: experimental method and results. Infect. Immun. 67:5151-5156.
25. Stingl, K., E. M. Uhlemann, R. Schmid, K. Altendorf, and E. P. Bakker. 2002. Energetics of Helicobacter pylori and its implications for the mechanism of urease-dependent acid tolerance at pH 1. J. Bacteriol. 184:3053-3060.
26. Suerbaum, S., and P. Michetti. 2002. Helicobacter pylori infection. N. Engl. J. Med. 347:1175-1186.
27. Thorens, J., F. Froehlich, W. Schwizer, E. Saraga, J. Bille, K. Gyr, P. Duroux, M. Nicolet, B. Pignatelli, A. L. Blum, J. J. Gonvers, and M. Fried. 1996. Bacterial overgrowth during treatment with omeprazole compared with cimetidine: a prospective randomized double blind study. Gut 39:54-59.
28. Watanabe, T., M. Tada, H. Nagai, S. Sasaki, and M. Nakao. 1998. Helicobacter pylori infection induces gastric cancer in Mongolian gerbils. Gastroenterology 115:642-648.(Sren Schreiber, Roland Bü)