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Unique mechanism of Helicobacter pylori for colonizing the gastric mucus
Microbes and Infection, 2, 2000, 55−60 © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
Review
Unique mechanism of Helicobacter pylori for colonizing the gastric mucus Hironori Yoshiyama, Teruko Nakazawa* Department of Microbiology, Yamaguchi University School of Medicine, Minamikogushi 1 1–1, Ube, Yamaguchi 755–8505, Japan
ABSTRACT – Helicobacter pylori is a human gastric pathogen causing chronic infection. Urease and motility using flagella are essential factors for its colonization. Urease of H. pylori exists both on the surface and in the cytoplasm, and is involved in neutralizing gastric acid and in chemotactic motility. H. pylori senses the concentration gradients of urea in the gastric mucus layer, then moves toward the epithelial surface by chemotactic movement. The energy source for the flagella movement is the proton motive force. The hydrolysis of urea by the cytoplasmic urease possibly generates additional energy for the flagellar rotation in the mucus gel layer. © 2000 Éditions scientifiques et médicales Elsevier SAS Helicobacter pylori / colonization / chemotaxis / motility / urease / mucous layer
1. Helicobacter pylori as a causative agent of upper gastrointestinal diseases Helicobacter pylori is a Gram-negative microaerophilic human-specific bacterium which was first isolated by Warren and Marshal in 1983 from a patient with chronic active gastritis [1]. The fact that this gastric pathogen infects more than half of the human population has had a strong impact on health care, since it had been believed no bacteria can colonize the stomach because of its high acidity. It is now recognized that H. pylori is a primary causative agent of chronic gastritis and peptic ulcer diseases [1, 2]. Recently, a strong association of H. pylori with gastric lymphoma [3] and gastric cancer [4-6] was reported. For the development of more effective therapeutic strategies, it is important to understand the mechanism by which H. pylori colonizes and persists in this highly specialized niche, namely the viscous and acidic mucus layer of the human stomach.
2. Attachment of H. pylori in the stomach of infected patients Both microbial and host factors for attachment of H. pylori are thought responsible for the variety of pathogenic outcomes of infection. The genetic diversity not only of humans, but of H. pylori may provide various combinations of adhesins and receptors [7, 8]. It is predicted by genome sequencing that H. pylori possesses around 1 600 * Correspondence and reprints Microbes and Infection 2000, 55-60
coding genes, in which a supergene family of 32 genes encoding putative outer membrane proteins (OMPs) is included [9]. Among these OMPs, the bacterial adhesin, BabA2 has been identified to bind human blood group antigen Lewis b [10]. Two related genes, babB, having a central region divergent from babA2 and another copy of babA lacking an initiation codon, have also been identified. There is extensive amino acid sequence homology between these three genes, and recombination between duplicate segments would allow adhesin synthesis to be readily switched on or off [10]. Two other OMP members, Alp A and B, are necessary for H. pylori to attach to human gastric tissue: the binding pattern being different from that for the BabA2-mediated adherence to Lewis b [11]. Different receptors may be involved for AlpA, B and for BabA2 [11]. Some strains of H. pylori have Lewis X and Y antigens as components of the lipopolysaccharide O-antigen and thus, mimic the host [12]. Phase variant expression of Lewis X and Y antigens as well as OMPs by slippage or frameshift are possible mechanisms by which this bacterium can escape elimination by the host immune system [13, 14]. H. pylori catalase, HpaA, heat shock protein 60, and a 19.6-kDa conserved protein are also proposed adhesins [15, 16]. For cellular receptors, phosphatidylethanolamine, laminin, and sialic acid-containing molecules are regarded as potential receptors other than Lewis b [17].
3. Urease and chemotaxis of H. pylori H. pylori has a right-handed spiral shaped body with a bundle of sheathed flagella (figure 1). Several factors may 55
Review
Figure 1. Electron micrograph of H. pylori. Spiral bacterial bodies with a bundle of sheathed flagella. Bar = 1 µm.
be involved in the colonization by H. pylori of the gastric mucus [18]. Among them, urease activity and chemotactic motility using flagella are established as essential factors for colonization [19–21]. Urease hydrolyzes urea to produce ammonia and carbon dioxide. The biosynthesis of urease is controlled by two gene clusters, one for structural genes consisting of ureAB, and the other for accessory genes consisting of ureIEFGH for nickel incorporation to form the active center for the enzyme [22], although the role of ureI is not known [23]. The main role of urease is thought to be the neutralization of the acidic microenvironment by producing ammonia. In fact, a stable urease-negative (ureB–) mutant constructed by genetic engineering could not colonize the stomach of nude mice [19]. Furthermore, co-inoculation of a urease-negative (ureG–) strain with a urease-positive strain resulted in preferential colonization of the urease positive bacteria in the stomach of gnotobiotic piglets [20], suggesting that neutralization of the microenvironment is not the sole role of urease for colonization. Motile flagellated bacteria swim toward chemical attractants and away from repellents. Chemotaxis is a response to microenvironmental alteration and is regulated by a concerted regulatory system. When attractants such as amino acids and sugars are added, bacteria sense the concentration gradient of attractants and swim toward them. H. pylori has the ability to sense and move toward urea (ureataxis), bicarbonate ion, and sodium ion at the concentration of 1 to 10 mM [24]. Urea is synthesized in liver, circulated by the blood stream at an average concentration of 5 mM [25], and secreted into the gastric juice through capillary networks beneath the gastric epithelial surface. Thus, a concentration gradient of urea is formed in the gastric mucus layer, which should be sensed by H. 56
Yoshiyama and Nakazawa
pylori. The concentration gradient of urea must be maintained throughout the infection, since bacterial urease is continuously hydrolyzing urea to produce ammonia and protect the bacteria from attack by gastric acid. Bicarbonate is also secreted to the gastric mucosa by chloride bicarbonate exchangers localized in parietal cells and Na+-H+ exchangers distributed in mucous neck, chief, and surface mucous cells, respectively [26]. Since these concentrations of urea and bicarbonate are in the effective range to attract H. pylori in vitro, the chemotactic motility may operate in vivo. The bacterial flagella rotate by a motor that converts electrochemical energy into mechanical energy. The energy source for the motor is the ion motive force, not the force produced by ATP hydrolysis [27]. Bacterial motors including those of Escherichia coli use proton motive force, except the polar flagellar motor of Vibrio parahaemoliticus, which uses sodium ion motive force [28]. The flagella motor of H. pylori also uses proton motive force, as evidenced by the inhibition of motility by a proton carrier, m-chlorophenylhydrazone (CCCP), but not by a sodium pump inhibitor, amiloride [29, 30].
4. Motility of H. pylori in a viscous environment The velocity at which a bacterium moves in a viscous solution increases with the viscosity to a point, and thereafter decreases [31]. In a viscous solution of 10 centipoise (cp) or more, H. pylori moves faster than E. coli [32]. The extent to which the production of proinflammatory cytokines in human gastric epithelial cells was induced by H. pylori was increased by overlaying epithelial cells with methylcellulose [33], suggesting that high viscosity enhances the bacterial contact with epithelia by increasing the bacterial motility. Most populations of H. pylori reside within the gastric mucus layer, but a few (2%) associate with the gastric epithelia, and the former population is present to replenish the latter [34]. The surface mucous gel layer of the stomach is composed of alternate multilaminae of two types of mucinous layers; the surface mucous cell type layer consisting of MUC5 and the gland mucous cell type layer consisting of MUC6 [35, 36]. According to Shimizu et al. [35], H. pylori is exclusively detected in the surface mucous layer, which becomes thinner in the infected stomach. Since the gel layers turn over rapidly and move to the highly acidic lumen and to the duodenum, bacteria in the mucous layer must move towards the epithelial cell surface against the flow of mucus, crossing the gland mucous gel layer which may contain gastric acid and trypsin (figure 2). For such movement, chemotaxis toward urea and sodium bicarbonate may play an important role.
5. Urease activity is required for the motility in the viscous environment The ureB-disrupted mutant does not show any motility on soft agar, though the wild-type H. pylori strain shows Microbes and Infection 2000, 55-60
Helicobacter pylori colonization of the gastric mucus
Review
Figure 2. Localization of H. pylori in the multilamelar gastric mucus. H. pylori colonizing the stomach mostly (98%) resides in the gastric mucus layer, but a small (2%) population is attached to the gastric epithelia [34]. The gastric mucus is composed of multilaminae of two types of mucinous layers, the surface and gland cell-type mucins. H. pylori is exclusively detected in the surface mucous gel layer [35]. Since the mucus turns over rapidly due to peristalsis and peptic digestion, H. pylori must cross the viscous gel layer towards the epithelial cell surface. Chemotactic movement toward urea and sodium bicarbonate, which can be secreted from epithelia to form a concentration gradient in the mucus, is important.
good motility [29]. In addition, the chemotactic motility of urease-negative bacteria is far less than that of the ureasepositive bacteria, though the chemotaxis itself is independent of the urease activity [24]. It appears that urease is needed for the chemotactic motility in a high-viscous environment. To determine the role of urease in chemotactic motility, we assayed the chemotactic activity of urease-positive and -negative strains toward urea and sodium bicarbonate in the absence or presence of 3% polyvinyl pyrrolidone (PVP), which gives a viscosity of 5.6 cp. To our surprise, the chemotactic motility was markedly stimulated by PVP in urease-positive bacteria, but not in the isogenic ureasenegative mutant. [29]. Two urease inhibitors, flurofamide and acetohydroxamic acid [37], were useful for examining the role of urease activity in a viscous environment. Acetohydroxamic acid is a small molecule (molecular weight, 75.07) which can easily enter the cytoplasm, whereas flurofamide is a large hydrophobic molecule (molecular weight, 217.14) which has difficulty in passing through the membrane [38]. Treatment of the bacterial cells with acetohydroxamic acid prior to chemotaxis assay completely abrogated the chemotactic activity toward sodium bicarbonate concomitant with the loss of the urease activity. In contrast, the inhibitory effect of flurofamide on the chemotactic and urease activities was only partial [29]. It should be noted that the chemotaxis assay was carried out without urea, suggesting that urea is supplied internally from L-arginine by the action of arginase (RocF). Although the presence of complete urea cycle enzymes has been suggested [40], two of the enzymes involved cannot be identified as a result of sequencing [9]. The importance of cytoplasmic urease has been reported in Ureaplasma urealyticum, a small, wall-less Microbes and Infection 2000, 55-60
prokaryote associated with urinary infection [39]. U. urealyticum has a potent cytoplasmic urease and internally hydrolyzes urea from the environment to produce ammonium chemical potential and simultaneously, to increase the proton motive force with resultant de novo ATP synthesis [39]. If urea hydrolysis in H. pylori also generates ammonium chemical potential, urea hydrolysis might provide additional energy for the flagellar motor to rotate in a high-viscosity environment. Based on the above results and considerations, we propose two roles for urease in H. pylori (figure 3). H. pylori is characterized as having potent urease activity, which is essential for colonization in the stomach [19, 20]. It was found that urease was located exclusively within the cytoplasm in fresh log-phase cultures, whereas a significant amount of it was associated with the outer membrane in older preparations [38]. Both extracellular and intracellular urease hydrolyze urea to generate ammonia for neutralizing gastric acidity, which gives bacteria a neutral microenvironment for their survival. The cytoplasmic urease may have an additional function in the motility, possibly through generating membrane potential of proton (figure 4).
6. Adaptation of H. pylori to an ecological niche H. pylori exhibits a right-handed helical morphology with a bundle of unipolar flagella. The flagellar filaments have a sheath, a membrane structure contiguous to the outer membrane that protects the filaments against mechanical and enzymatic digestion. This morphology enables the bacterium to move across the viscous gastric 57
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Yoshiyama and Nakazawa
Figure 3. Two roles of urease in H. pylori. Urease of H. pylori is localizing on the cell surface and in the cytoplasm [38]. Urease localizing on the outer membrane hydrolyzes urea only to neutralize the gastric acid. Cytoplasmic urease hydrolyzes intracellular urea, which is derived extracellularly or intracellularly from arginine by arginase (RocF). Ammonia produced in the cytoplasm is utilized to synthesize glutamine and also converted to ammonium ion coupled with the consumption of proton. Thus, cytoplasmic urease may serve to produce a proton gradient in addition to redox potential (respiration) and provide more proton motive force to the flagellar motor. Ammonium ion, when overproduced, might be exported by a membrane protein, UreI, as has been suggested [23]. It is also possible that UreI functions as a transporter to incorporate urea. AH2 and A; reduced and oxidized metabolite, RocE; basic amino acid permease, RocF; arginase. mucus, so that it can penetrate the gastric mucous layer like a corkscrew and swim fast in the viscous mucus. Because the flagellar motor is powered by a proton motive force, H. pylori might be able to swim fast at low pH. Thus,
orally uptaken H. pylori can promptly evade the acidic periphery of the mucous layer and move toward the epithelial surface and further into the gastric pits by chemoattraction of substances, such as urea and bicarbonate, which diffuse out from the gastric epithelial surface. While urease acts to neutralize the gastric acid to which the bacterium is frequently exposed, it may also act to facilitate the rotation of the flagellar motor. The spiralshaped, strong urease, ureataxis, as well as the complex motility control, should enable this bacterium to adapt to the ecological niche provided by the gastric mucus.
7. Perspectives
Figure 4. A model for the colonization by H. pylori of the gastric mucosa. Details are described in the text. 58
Recent publications highlighting the involvement of H. pylori in gastric carcinogenesis have aroused public interest in the eradication of H. pylori [5, 6]. Unfortunately however, reports of antibiotic-resistant clinical isolates are increasing [41, 42]. Therefore, specific drugs targeting those factors important for bacterial colonization, such as urease and chemotaxis, may be useful to minimize generation of drug-resistant bacteria. Lansoprazole and omeprazole, which are proton pump inhibitors (PPIs) for the H+/K+ ATPase of parietal cells and inhibit the secretion of gastric acid, have been used to eradicate H. pylori in combination with antibiotics [43]. The PPIs are known to have antimicrobial activity and also antiurease activity against H. pylori [44]. The two activities seem to be indeMicrobes and Infection 2000, 55-60
Helicobacter pylori colonization of the gastric mucus
pendent, because both urease-positive and ureasenegative strains of H. pylori have similar minimum inhibitory concentrations of the PPIs. In our recent studies, the PPIs were found to inhibit the chemotactic motility of H. pylori in a high-viscous environment at concentrations similar to those inhibiting urease activity, but much lower for inhibiting growth [45]. It is tempting to speculate that one of the roles of PPIs in the combination therapy for eradication of H. pylori is to inhibit the chemotactic motility of the bacteria in the mucus layer of the stomach. Further studies to elucidate the role of urease in bacterial colonization should facilitate the development of new therapeutic strategies to control H. pylori infection.
Acknowledgments The authors are grateful to Dr H. Konishi for providing the electronmicrograph and to the Ministry of Education, Science, Culture and Sports of Japan for supporting the research with grants (BA08307004, BB08457089).
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[26] Stuart-Tilley A.C., Sardet J., Pouyssegur M.A. et al., Immunolocalization of anion exchanger AE2 and cation exchanger NHE-1 in distinct adjacent cells of gastric mucosa, Am. J. Physiol. 266 (1994) C 559–568. [27] Khan S., MacNab R.M., Proton chemical potential, proton electrical potential and bacterial motility, J. Mol. Biol. 138 (1980) 599–614. [28] Atsumi T., McCarterand L., Imae Y., Polar and lateral flagellar motors of marine Vibrio are driven by different ion-motive forces, Nature 355 (1992) 182–184. [29] Nakamura H., Yoshiyama H., Takeuchi H., Mizote T., Okita K., Nakazawa T., Urease plays an important role in the chemotactic motility of Helicobacter pylori in a viscous environment, Infect. Immun. 66 (1998) 4832–4837. [30] Yoshiyama H., Mizote T., Nakamura H., Okita K., Nakazawa T., Chemotaxis of Helicobacter pylori: A ureaseindependent response, J. Gastroenterol. 33 (1998) (Suppl X) 1–5. [31] Schneider W.R., Doetsch R.N., Effect of viscosity on bacterial motility, J. Bacteriol. 117 (1974) 696–701. [32] Hazel S.L., Lee A., Brady L., Hennessy W., Campylobacter pyloridis and gastritis: Association with intercellular spaces and adaptation to an environment of mucus as important factors in colonization of the gastric epithelium, J. Infect. Dis. 153 (1986) 658–663. [33] Jung H.C., Kim J.M., Song I.S., Kim C.Y., Increased motility of Helicobacter pylori by methylcellulose could upregulate the expression of proinflammatory cytokines in human gastric epithelial cells, Scand. J. Clin. Lab. Invest. 57 (1997) 263–270. [34] Kirschner D.E., Blaser M.J., The dynamics of Helicobacter pylori infection of the human stomach, J. Theor. Biol. 176 (1995) 281–290. [35] Shimizu T., Akamatsu T., Sugiyama A., Ota H., Katsuyama T., Helicobacter pylori and the surface mucous gel layer of the human stomach, Helicobacter 1 (1996) 207–218. [36] Byrd J.C., Yan P., Sternberg L., Yunker C.K., Scheiman J.M., Bresalier R.S., Aberrant expression of grand-type gastric mucin in the surface epithelium of Helicobacter pylori-infected patients, Gastroenterology 113 (1997) 455–464.
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[37] Nagata K., Satoh H., Iwahi T., Shimoyama T., Tamura T., Potent inhibitory action of gastric proton pump inhibitor lansoprazole against urease activity of Helicobacter pylori: Unique action selective for H. pylori: cells, Antimicrob. Agents Chemother. 37 (1993) 769–774. [38] Phadnis S.H., Parlow M.H., Levy M., Ilver D., Caulkins C.M., Connors J.B., Dunn B.E., Surface localization of Helicobacter pylori urease and a heat shock protein homolog requires bacterial autolysis, Infect. Immun. 64 (1996) 905–912. [39] Smith D.G.E., Russell W.C., Ingledew W.J., Thirkell D., Hydrolysis of urea by Ureaplasma urealyticum generates a transmembrane potential with resultant ATP synthesis, J. Bacteriol. 175 (1993) 3253–3258. [40] Mendz G.L., Hazell S.L., The urea cycle of Helicobacter pylori, Microbiology 142 (1996) 2959–2967. [41] Megraud F., Epidemiology and mechanism of antibiotic resistance in Helicobacter pylori, Gastroenterology 115 (1998) 1278–1282. [42] Dore M.P., Osato M.S., Realdi G., Mura I., Graham D.Y., Sepulveda A.R., Amoxycillin tolerance in Helicobacter pylori, J. Antimicrob. Chemother. 43 (1999) 47–54. [43] Lind T., Megraud F., Unge P., Bayerdorffer E., O’Morain C., Spiller R., Veldhuyzen van Zaiten S., Bardhan K.D., Hellblom M., Wrangstadh M., Zeijlon L., Cederberg C., The MACH2 study: Role of omeprazole in eradication of Helicobacter pylori with 1-week triple therapies, Gastroenterology 116 (1999) 248–253. [44] Nagata K., Takagi E., Tsuda M., Nakazawa T., Satoh H., Nakao M., Okamura H., Tamura T., Inhibitory action of lansoplazole and its analogs against Helicobacter pylori: Inhibition of growth is not related to inhibition of urease, Antimicrob. Agents Chemother. 39 (1995) 567–570. [45] Nakamura H., Yoshiyama H., Okita K., Nakazawa T., Inhibitory effect of proton pump inhibitors against chemotaxis of Helicobacter pylori, (abstracts), The 4th Japan Korea International Symposium on Microbiology, 42.pp. 1998,
Microbes and Infection 2000, 55-60
Review
Unique mechanism of Helicobacter pylori for colonizing the gastric mucus Hironori Yoshiyama, Teruko Nakazawa* Department of Microbiology, Yamaguchi University School of Medicine, Minamikogushi 1 1–1, Ube, Yamaguchi 755–8505, Japan
ABSTRACT – Helicobacter pylori is a human gastric pathogen causing chronic infection. Urease and motility using flagella are essential factors for its colonization. Urease of H. pylori exists both on the surface and in the cytoplasm, and is involved in neutralizing gastric acid and in chemotactic motility. H. pylori senses the concentration gradients of urea in the gastric mucus layer, then moves toward the epithelial surface by chemotactic movement. The energy source for the flagella movement is the proton motive force. The hydrolysis of urea by the cytoplasmic urease possibly generates additional energy for the flagellar rotation in the mucus gel layer. © 2000 Éditions scientifiques et médicales Elsevier SAS Helicobacter pylori / colonization / chemotaxis / motility / urease / mucous layer
1. Helicobacter pylori as a causative agent of upper gastrointestinal diseases Helicobacter pylori is a Gram-negative microaerophilic human-specific bacterium which was first isolated by Warren and Marshal in 1983 from a patient with chronic active gastritis [1]. The fact that this gastric pathogen infects more than half of the human population has had a strong impact on health care, since it had been believed no bacteria can colonize the stomach because of its high acidity. It is now recognized that H. pylori is a primary causative agent of chronic gastritis and peptic ulcer diseases [1, 2]. Recently, a strong association of H. pylori with gastric lymphoma [3] and gastric cancer [4-6] was reported. For the development of more effective therapeutic strategies, it is important to understand the mechanism by which H. pylori colonizes and persists in this highly specialized niche, namely the viscous and acidic mucus layer of the human stomach.
2. Attachment of H. pylori in the stomach of infected patients Both microbial and host factors for attachment of H. pylori are thought responsible for the variety of pathogenic outcomes of infection. The genetic diversity not only of humans, but of H. pylori may provide various combinations of adhesins and receptors [7, 8]. It is predicted by genome sequencing that H. pylori possesses around 1 600 * Correspondence and reprints Microbes and Infection 2000, 55-60
coding genes, in which a supergene family of 32 genes encoding putative outer membrane proteins (OMPs) is included [9]. Among these OMPs, the bacterial adhesin, BabA2 has been identified to bind human blood group antigen Lewis b [10]. Two related genes, babB, having a central region divergent from babA2 and another copy of babA lacking an initiation codon, have also been identified. There is extensive amino acid sequence homology between these three genes, and recombination between duplicate segments would allow adhesin synthesis to be readily switched on or off [10]. Two other OMP members, Alp A and B, are necessary for H. pylori to attach to human gastric tissue: the binding pattern being different from that for the BabA2-mediated adherence to Lewis b [11]. Different receptors may be involved for AlpA, B and for BabA2 [11]. Some strains of H. pylori have Lewis X and Y antigens as components of the lipopolysaccharide O-antigen and thus, mimic the host [12]. Phase variant expression of Lewis X and Y antigens as well as OMPs by slippage or frameshift are possible mechanisms by which this bacterium can escape elimination by the host immune system [13, 14]. H. pylori catalase, HpaA, heat shock protein 60, and a 19.6-kDa conserved protein are also proposed adhesins [15, 16]. For cellular receptors, phosphatidylethanolamine, laminin, and sialic acid-containing molecules are regarded as potential receptors other than Lewis b [17].
3. Urease and chemotaxis of H. pylori H. pylori has a right-handed spiral shaped body with a bundle of sheathed flagella (figure 1). Several factors may 55
Review
Figure 1. Electron micrograph of H. pylori. Spiral bacterial bodies with a bundle of sheathed flagella. Bar = 1 µm.
be involved in the colonization by H. pylori of the gastric mucus [18]. Among them, urease activity and chemotactic motility using flagella are established as essential factors for colonization [19–21]. Urease hydrolyzes urea to produce ammonia and carbon dioxide. The biosynthesis of urease is controlled by two gene clusters, one for structural genes consisting of ureAB, and the other for accessory genes consisting of ureIEFGH for nickel incorporation to form the active center for the enzyme [22], although the role of ureI is not known [23]. The main role of urease is thought to be the neutralization of the acidic microenvironment by producing ammonia. In fact, a stable urease-negative (ureB–) mutant constructed by genetic engineering could not colonize the stomach of nude mice [19]. Furthermore, co-inoculation of a urease-negative (ureG–) strain with a urease-positive strain resulted in preferential colonization of the urease positive bacteria in the stomach of gnotobiotic piglets [20], suggesting that neutralization of the microenvironment is not the sole role of urease for colonization. Motile flagellated bacteria swim toward chemical attractants and away from repellents. Chemotaxis is a response to microenvironmental alteration and is regulated by a concerted regulatory system. When attractants such as amino acids and sugars are added, bacteria sense the concentration gradient of attractants and swim toward them. H. pylori has the ability to sense and move toward urea (ureataxis), bicarbonate ion, and sodium ion at the concentration of 1 to 10 mM [24]. Urea is synthesized in liver, circulated by the blood stream at an average concentration of 5 mM [25], and secreted into the gastric juice through capillary networks beneath the gastric epithelial surface. Thus, a concentration gradient of urea is formed in the gastric mucus layer, which should be sensed by H. 56
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pylori. The concentration gradient of urea must be maintained throughout the infection, since bacterial urease is continuously hydrolyzing urea to produce ammonia and protect the bacteria from attack by gastric acid. Bicarbonate is also secreted to the gastric mucosa by chloride bicarbonate exchangers localized in parietal cells and Na+-H+ exchangers distributed in mucous neck, chief, and surface mucous cells, respectively [26]. Since these concentrations of urea and bicarbonate are in the effective range to attract H. pylori in vitro, the chemotactic motility may operate in vivo. The bacterial flagella rotate by a motor that converts electrochemical energy into mechanical energy. The energy source for the motor is the ion motive force, not the force produced by ATP hydrolysis [27]. Bacterial motors including those of Escherichia coli use proton motive force, except the polar flagellar motor of Vibrio parahaemoliticus, which uses sodium ion motive force [28]. The flagella motor of H. pylori also uses proton motive force, as evidenced by the inhibition of motility by a proton carrier, m-chlorophenylhydrazone (CCCP), but not by a sodium pump inhibitor, amiloride [29, 30].
4. Motility of H. pylori in a viscous environment The velocity at which a bacterium moves in a viscous solution increases with the viscosity to a point, and thereafter decreases [31]. In a viscous solution of 10 centipoise (cp) or more, H. pylori moves faster than E. coli [32]. The extent to which the production of proinflammatory cytokines in human gastric epithelial cells was induced by H. pylori was increased by overlaying epithelial cells with methylcellulose [33], suggesting that high viscosity enhances the bacterial contact with epithelia by increasing the bacterial motility. Most populations of H. pylori reside within the gastric mucus layer, but a few (2%) associate with the gastric epithelia, and the former population is present to replenish the latter [34]. The surface mucous gel layer of the stomach is composed of alternate multilaminae of two types of mucinous layers; the surface mucous cell type layer consisting of MUC5 and the gland mucous cell type layer consisting of MUC6 [35, 36]. According to Shimizu et al. [35], H. pylori is exclusively detected in the surface mucous layer, which becomes thinner in the infected stomach. Since the gel layers turn over rapidly and move to the highly acidic lumen and to the duodenum, bacteria in the mucous layer must move towards the epithelial cell surface against the flow of mucus, crossing the gland mucous gel layer which may contain gastric acid and trypsin (figure 2). For such movement, chemotaxis toward urea and sodium bicarbonate may play an important role.
5. Urease activity is required for the motility in the viscous environment The ureB-disrupted mutant does not show any motility on soft agar, though the wild-type H. pylori strain shows Microbes and Infection 2000, 55-60
Helicobacter pylori colonization of the gastric mucus
Review
Figure 2. Localization of H. pylori in the multilamelar gastric mucus. H. pylori colonizing the stomach mostly (98%) resides in the gastric mucus layer, but a small (2%) population is attached to the gastric epithelia [34]. The gastric mucus is composed of multilaminae of two types of mucinous layers, the surface and gland cell-type mucins. H. pylori is exclusively detected in the surface mucous gel layer [35]. Since the mucus turns over rapidly due to peristalsis and peptic digestion, H. pylori must cross the viscous gel layer towards the epithelial cell surface. Chemotactic movement toward urea and sodium bicarbonate, which can be secreted from epithelia to form a concentration gradient in the mucus, is important.
good motility [29]. In addition, the chemotactic motility of urease-negative bacteria is far less than that of the ureasepositive bacteria, though the chemotaxis itself is independent of the urease activity [24]. It appears that urease is needed for the chemotactic motility in a high-viscous environment. To determine the role of urease in chemotactic motility, we assayed the chemotactic activity of urease-positive and -negative strains toward urea and sodium bicarbonate in the absence or presence of 3% polyvinyl pyrrolidone (PVP), which gives a viscosity of 5.6 cp. To our surprise, the chemotactic motility was markedly stimulated by PVP in urease-positive bacteria, but not in the isogenic ureasenegative mutant. [29]. Two urease inhibitors, flurofamide and acetohydroxamic acid [37], were useful for examining the role of urease activity in a viscous environment. Acetohydroxamic acid is a small molecule (molecular weight, 75.07) which can easily enter the cytoplasm, whereas flurofamide is a large hydrophobic molecule (molecular weight, 217.14) which has difficulty in passing through the membrane [38]. Treatment of the bacterial cells with acetohydroxamic acid prior to chemotaxis assay completely abrogated the chemotactic activity toward sodium bicarbonate concomitant with the loss of the urease activity. In contrast, the inhibitory effect of flurofamide on the chemotactic and urease activities was only partial [29]. It should be noted that the chemotaxis assay was carried out without urea, suggesting that urea is supplied internally from L-arginine by the action of arginase (RocF). Although the presence of complete urea cycle enzymes has been suggested [40], two of the enzymes involved cannot be identified as a result of sequencing [9]. The importance of cytoplasmic urease has been reported in Ureaplasma urealyticum, a small, wall-less Microbes and Infection 2000, 55-60
prokaryote associated with urinary infection [39]. U. urealyticum has a potent cytoplasmic urease and internally hydrolyzes urea from the environment to produce ammonium chemical potential and simultaneously, to increase the proton motive force with resultant de novo ATP synthesis [39]. If urea hydrolysis in H. pylori also generates ammonium chemical potential, urea hydrolysis might provide additional energy for the flagellar motor to rotate in a high-viscosity environment. Based on the above results and considerations, we propose two roles for urease in H. pylori (figure 3). H. pylori is characterized as having potent urease activity, which is essential for colonization in the stomach [19, 20]. It was found that urease was located exclusively within the cytoplasm in fresh log-phase cultures, whereas a significant amount of it was associated with the outer membrane in older preparations [38]. Both extracellular and intracellular urease hydrolyze urea to generate ammonia for neutralizing gastric acidity, which gives bacteria a neutral microenvironment for their survival. The cytoplasmic urease may have an additional function in the motility, possibly through generating membrane potential of proton (figure 4).
6. Adaptation of H. pylori to an ecological niche H. pylori exhibits a right-handed helical morphology with a bundle of unipolar flagella. The flagellar filaments have a sheath, a membrane structure contiguous to the outer membrane that protects the filaments against mechanical and enzymatic digestion. This morphology enables the bacterium to move across the viscous gastric 57
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Figure 3. Two roles of urease in H. pylori. Urease of H. pylori is localizing on the cell surface and in the cytoplasm [38]. Urease localizing on the outer membrane hydrolyzes urea only to neutralize the gastric acid. Cytoplasmic urease hydrolyzes intracellular urea, which is derived extracellularly or intracellularly from arginine by arginase (RocF). Ammonia produced in the cytoplasm is utilized to synthesize glutamine and also converted to ammonium ion coupled with the consumption of proton. Thus, cytoplasmic urease may serve to produce a proton gradient in addition to redox potential (respiration) and provide more proton motive force to the flagellar motor. Ammonium ion, when overproduced, might be exported by a membrane protein, UreI, as has been suggested [23]. It is also possible that UreI functions as a transporter to incorporate urea. AH2 and A; reduced and oxidized metabolite, RocE; basic amino acid permease, RocF; arginase. mucus, so that it can penetrate the gastric mucous layer like a corkscrew and swim fast in the viscous mucus. Because the flagellar motor is powered by a proton motive force, H. pylori might be able to swim fast at low pH. Thus,
orally uptaken H. pylori can promptly evade the acidic periphery of the mucous layer and move toward the epithelial surface and further into the gastric pits by chemoattraction of substances, such as urea and bicarbonate, which diffuse out from the gastric epithelial surface. While urease acts to neutralize the gastric acid to which the bacterium is frequently exposed, it may also act to facilitate the rotation of the flagellar motor. The spiralshaped, strong urease, ureataxis, as well as the complex motility control, should enable this bacterium to adapt to the ecological niche provided by the gastric mucus.
7. Perspectives
Figure 4. A model for the colonization by H. pylori of the gastric mucosa. Details are described in the text. 58
Recent publications highlighting the involvement of H. pylori in gastric carcinogenesis have aroused public interest in the eradication of H. pylori [5, 6]. Unfortunately however, reports of antibiotic-resistant clinical isolates are increasing [41, 42]. Therefore, specific drugs targeting those factors important for bacterial colonization, such as urease and chemotaxis, may be useful to minimize generation of drug-resistant bacteria. Lansoprazole and omeprazole, which are proton pump inhibitors (PPIs) for the H+/K+ ATPase of parietal cells and inhibit the secretion of gastric acid, have been used to eradicate H. pylori in combination with antibiotics [43]. The PPIs are known to have antimicrobial activity and also antiurease activity against H. pylori [44]. The two activities seem to be indeMicrobes and Infection 2000, 55-60
Helicobacter pylori colonization of the gastric mucus
pendent, because both urease-positive and ureasenegative strains of H. pylori have similar minimum inhibitory concentrations of the PPIs. In our recent studies, the PPIs were found to inhibit the chemotactic motility of H. pylori in a high-viscous environment at concentrations similar to those inhibiting urease activity, but much lower for inhibiting growth [45]. It is tempting to speculate that one of the roles of PPIs in the combination therapy for eradication of H. pylori is to inhibit the chemotactic motility of the bacteria in the mucus layer of the stomach. Further studies to elucidate the role of urease in bacterial colonization should facilitate the development of new therapeutic strategies to control H. pylori infection.
Acknowledgments The authors are grateful to Dr H. Konishi for providing the electronmicrograph and to the Ministry of Education, Science, Culture and Sports of Japan for supporting the research with grants (BA08307004, BB08457089).
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