- Email: [email protected]
Helicobacter pylori Factors Associated With Disease Development
GASTROENTEROLOGY 1997;113:521-528
Helicobacter pylori Factors Associated With
Disease Development
HARRY L. T. MOBLEY Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland
Although certain factors appear to predispose the host to infection by Helicobacter pylori, clearly the bacterium possesses a well-defined battery of virulence factors that allow the organism to: (1) colonize the gastric mucosa (urease, flagella, adhesins, acidinhibitory protein, iron acquisition proteins, and heat shock proteins); (2) evade host defense (shedding of surface proteins, catalase, superoxide dismutase, and poorly reactive lipopolysaccharide); and (3) damage host tissue (vacuolating cytotoxin, protease, CagArelated factors, inducers of cytokines, and chemotaxins). Together these factors allow H. pylori to persist in the host, establishing a chronic infection. Although many of these virulence factors are produced by all strains of H. pylori, there are also well-defined pathogenicity islands (contiguous stretches of chromosomal DNA) present in some strains that encode additional proteins including CagA that potentiate virulence. Strains possessing these "virulence cassettes" are isolated more frequently from patients with the more serious clinical manifestations associated with duodenal ulcer than from patients with gastritis alone or nonulcer dyspepsia.
A
true bacterial pathogen is an organism that is capable of causing disease in an immunocompetent host. Such bacteria possess defined mechanisms of pathogenesis. An infection is defined as the successful establishment and persistence of a pathogen. 1 Replication of a bacterium to form many bacteria is sufficient to maintain infection and may represent the ultimate goal of the microbe. Multiplication and persistence alone, however, does not usually lead to disease. Disease may actually be an unfavorable or "unwanted" outcome for the infecting microbe and results from an infection that causes significant overt damage to the host. 1 There is, of course, interplay between the host and bacterium. Although certain factors appear to predispose the host to infection by Helicobacter pylori (see article by Go in this supplement), clearly this bacterium possesses a well-defined battery of virulence factors that allow the organism to colonize the gastric mucosa, evade host defense, and damage host tissue. These factors will be
discussed in the context of a model for microbial pathogenesis. Although numerous virulence factors have been identified, urease has emerged as one of the few proteins with proven involvement in the development of infection and disease. 2- 6 The activity of urease is regulated by its nickel content in the active site of the enzyme,7 Protein systems responsible for transport and binding of nickel have been described recently, and their role will be reviewed. 8- 15
H. pylori shows tremendous strain-to-strain variability.16-21 This heterogeneity is manifested in a number of ways, bur the discovery of pathogenicity islands (contiguous stretches of chromosomal DNA encoding virulence factors), associated with the more virulent strains,22,23 has perhaps allowed us to define H. pylori as a true pathogen.
Model for Pathogenesis In the study of microbial pathogenesis, microbiologists have attempted to divide the steps required for the development of infection and disease into overlapping categories. First, the bacterium must enter the host and colonize. Specific colonization factors are required for this process. Next, the microbe must avoid host defenses so that it can multiply and persist. Finally, an optional step for infection, but a required step for the development of disease, is the eliciting of direct or indirect damage to the host.
Colonization It is generally accepted that H. pylori is acquired in childhood by a fecal-oral or oral-oral spread from another infected human. 24 There is no dramatic evidence showing the existence of an active reservoir that actually plays a role in transmission beyond the human host. Therefore, the bacterium is most likely adapted to its host and synthesizing the proteins and metabolites required for
Abbreviations used in this paper: LPS, lipopolysaccharide; peRRFLP, polymerase chain reaction-restriction fragment length polymorphism. © 1997 by the American Gastroenterological Association 0016-5085/97/$3.00
S22
GASTROENTEROLOGY Vol. 113, No. 6
HARRY L. T. MOBLEY
Ii. H~
Ii.
l
HI
e e e e e e
Figure 1. Colonization by H. pylori. Products produced by H. pylori that may allow the bacterium to establish itself in the gastric mucosa. HSP, heat shock protein. Ni++, nickel ions; H+, protons.
Figure 2. Avoidance of host defense by H. pylori. Protein products and LPS that allow H. pylori to overcome innate defense including acid and immune surveillance. O2 , toxic oxygen radical.
colonization of a new host. Several virulence factors are putatively involved in this process (Figure 1). Urease is one of the most striking features of this organism. This high-molecular-weight (550-kilodalton) enzyme hydrolyzes urea, the nitrogenous waste product of humans, to liberate ammonia and carbon dioxide. 25 ,26 The enzyme is one of the few nickel metalloenzymes in nature and requires the divalent cation in its active site to successfully hydrolyze its substrate. 27 H. pylori synthesizes urease in extraordinarily high amounts, making up as much as 5 % of the total cell protein. 26 ,28,29 Its proposed role in pathogenesis is quite appealing. As the organism enters the stomach, it is undoubtedly faced with abundant gastric acid. Because H. pylori is not an acidophile and does not thrive or even survive in vitro at low pH, it must protect itself from acid. 3o The hydrolysis of urea provides ammonia as a neutralizing agent for hydrochloric acid. The transient development of achlorhydria and, indeed, the development of neutral pH may allow the organism time to burrow through the gastric mucus to the gastric epithelium, which provides a more hospitable environment. Urease-negative mutants developed by both chemical mutagenesis 3 and allelic exchange mutagenesis4,3 1 are unable to colonize the gastric mucosa of the gnotobiotic piglet, an accepted animal model of H. pylori infection. Indeed, the flagella-mediated burrowing motility is also essential for survival. The spiral shape of the bacterium coupled with the tuft of polar flagella make the organism well suited for traversing the mucous layer. The recent discovery of genes associated with chemotaxis now suggests that H. pylori can chemotax toward positive stimuli and away from toxic substances. 32 Isogenic mutants of H. pylori lacking flagella have been unable to colonize the gnotobiotic piglet, whereas the wild-type strain can colonize and persist. 33 ,34 Once the bacterium has traversed the gastric mucus, a
small percentage of organisms attach intimately to the surface of gastric epithelial cells. This event is coupled to actin rearrangement 35 and host protein phosphorylation 36 within the epithelial celP5,37 Because H. pylori is so highly specific for humans and indeed gastric tissue (the duodenum is colonized only when there is spread of the epithelium by gastric metaplasia 38 ; esophageal epithelium is also not colonized), it is appealing to envision highly specific adherence factors. Although no adherence factor has been proven to be required for colonization, a number of such candidates have been described including hemagglutinins,39 an intimin-like protein,35 Lewis blood group antigen-binding adhesin,4o and an adhesin lipoprotein. 41 Other factors that may play a role in colonization include an acid-inhibitory protein, which can inactivate the gastric proton pump,42 again neutralizing hazardous acid. Like most other pathogenic bacteria, the organism must have strategies for acquiring iron, tightly sequestered by the host. Although no siderophores have yet been described, there are undoubtedly iron acquisition systems that take up the iron required for cytochrome production. Finally, the high levels of production of GroEL- and GroES-like heat shock proteins (HspB and HspA, respectively) may explain the ability to transiently tolerate the extreme conditions met by the bacterium during the early stages of colonization. 42 ,43 These chaperone-like proteins may assist in maintaining the integrity of proteins in the bacterium during this period of stress.
Avoidance of Immune Response Once colonization is complete, H. pylori must avoid the immune response that could result in clearance. A number of mechanisms for avoidance have been proposed (Figure 2). Shedding of proteins is one mechanism. Various proteins have been localized to the surface of the bacterium using immunochemical approaches.
H. PYLORI: BACTERIAL FACTORS AND MUCOSAL DISEASE
December Supplement 1997
Vocuoe'Wlo (')toIOlUf'l
Figure 3. Damage to host by H. pylori. Products of H. pylori that directly or indirectly damage the host epithelium. NH 3 , ammonia; CagA, cytotoxin-associated gene A; PieS, promotes induction of cytokines gene S; IL-8, interleukin 8.
Urease, which is strictly a cytoplasmic protein in nonHelicobacter bacterial species, is present in very high amounts on the surface. Phadnis and Dunn 44 suggest that the mechanism is nonspecific and involves "altruistic lysis" whereby a certain proportion of the population undergoes lysis and coats the remaining viable bacteria with normally cytosolic proteins. These shed proteins could function to saturate the binding sites of secretory immunoglobulin specific for these proteins and other immune receptors and prevent a bactericidal response. Catalase and superoxide dismurase are H. pylori enzymes that detoxifY toxic oxygen radicals.45 .4 6 However, their actual roles have not been shown in H. pylori. Interestingly mutations in superoxide dismutase genes appear to be lethal; this is not the case for catalase because a murant has been constructed. The lipopolysaccharide (LPS) of H. pylori may also favor persistence of the bacterium. The isolated polymers are approximately lOOO-fold less toxic than the LPS of other gram-negative bacteria; therefore, the LPS molecule may not stimulate the immune response to the degree necessary to clear the organism,47 and in some cases mimics the Lewis x and Lewis y antigen. 48 .49 In addition, an immunoglobulin A (IgA) protease that specifically cleaves secretory IgA molecules has been proposed for H. pylori,5o but no direct evidence supports their existence or role in pathogenesis. Finally, one way that several bacterial species escape the immune response is by invasion of eukaryotic cells. Although H. pylori bacteria have been observed "within" epithelial cells in electron micrographs of thin sections, it is not believed that invasion contributes to the pathogenesis of H. pylori infection.
Damage to the Host Certain virulence factors are associated with direct or indirect damage to the gastric mucosa (Figure 3). In
S23
addition to the role of urease in colonization, there is evidence that ammonium hydroxide, the product of urea hydrolysis, contributes significantly to histological damage. The hydroxide ion generated by the equilibration of ammonia with water seems to be the toxic species; the ammonium ion itself is not toxic. To show the cytotoxic effect of urease, cell cultures of a human gastric adenocarcinoma cell line were seeded with H. pylori and supplemented with various concentrations of urea. 51 Cell viability was found to be inversely proportional to ammonia concentrations generated by urea hydtolysis. Viability was improved when the urease inhibitor, acetohydroxamic acid, was added to the culture before the exposure to H. pylori. Acetohydroxamic acid slowed the liberation of ammonia and reduced the cytotoxic effect. These data suggested that histological damage may result directly from the localized generation of ammonia due to the hydrolysis of urea. It has also been postulated that ammonia produced by urea hydrolysis has an additional effect. 52 Ammonia may interfere with normal hydrogen ion back-diffusion across gastric mucosa, resulting in cytotoxicity to the underlying epithelium. The vacuolating cytotoxin is derived from a largemolecular-size polypeptide (139 kilodaltons) that is processed at both the N terminus (traditional signal sequence) and at the C terminus (-50 kilodaltons). 53 The remaining subunit of -87 kilodaltons assembles into six copies to form the holotoxin. 54 The toxin protein is not homologous to other known proteins and acts to form vacuoles within its target cells. 55 The vacA gene is present in all H. pylori strains, but the toxin is secreted in detectable amounts in only about half of the isolates. In general, these strains are cagA positive. CagA itself, however, is not responsible for toxin export. This will be discussed further below. LPS of H. pylori is a complex molecule with several domains. It has been reported that purified LPS can function to induce pepsinogen and elicit mucous modification.56 These findings were determined by the use of Ussing chambers and have not been confirmed in vivo. The presence of hemolysins has been suggested by some investigators, but the evidence is not substantial. 57 Zones of clearing are not generally found around the colonies on the agar plates, but hemolysis of erythrocytes in suspension occurs in the presence of H. pylori. This activity, although weak, is calcium dependent, as for other hemolysins. In addition, hemolytic muranes that do not display the phenotype have been isolated. At this poine, the existence of hemolysins is not well supported by the available data. H. pylori also produces phospholipases and alcohol
S24
HARRY L. T. MOBLEY
dehydrogenase, but their role in disease is currently speculative. 58 Urease activity may also be indirectly responsible for damage to the gastric epithelium via its interaction with the immune system. H. pylori whole cells can stimulate an oxidative burst in human neutrophils. 59 Urease can also cause activation of monocytes and polymorphonuclear leukocytes, and recruitment of inflammatory response cells, resulting in indirect damage to the gastric epithelium. Water extracts of H. pylori, known to contain urease in high concentration, can activate monocytes by an iPS-independent pathway.60 In vitro stimulation of human monocytes led to secretion of inflammatory cytokines and reactive oxygen intermediates, all of which may be involved in mediating the inflammatory response in the gastric epithelium. Further investigation has shown that sonicates of H. pylori strains could prime and also cause direct activation of the oxidative burst in human polymorphonuclear leukocytes and monocytes. 61 There is also evidence of urease or urease-containing fractions from H. pylori acting as chemotactic factors for leukocytes, causing further local inflammation. 62 ,63 Such chemotactic activity for human monocytes and neutrophils was present in purified urease samples and could be inhibited by specific antibody to the UreB urease subunit. Further, a 20-amino acid peptide based on the amino terminus of the UreB subunit protein also exhibited similar levels of chemotaxis in a microchamber test system. 63 Immunocytochemical staining showed urease closely associated with the crypt cells in the lamina propria of patients with duodenal ulcers. It is posrulated that urease is absorbed into the mucosa where it attracts leukocytes and causes mucosal inflammation. 64 Urease, by a variety of mechanisms, is at least partly responsible for the initial recruitment of monocytes and neurrophils and further activation and stimulation of the immune system to produce the local inflammatory lesion associated with H. pylori infection.
Contribution to Virulence Examined Using Defined Mutation in Clinical Strains Only a few putative virulence factors have been tested appropriately65 using isogenic mutants and suitable animal models to determine the contribution to virulence of specific genes or proteins encoded by these genes. These include urease, flagella, and vacuolating cytotoxin. Of these, urease-negative and flagellumnegative mutants were unable to colonize the gastric mucosa of gnotobiotic piglets. 3--6,33,34 Other candidate virulence factors have been implicated only indirectly in virulence.
GASTROENTEROLOGY Vol. 113, No.6
H. pyIori
Agure 4. Proteins involved in nickel management as regulators of urease activity. Urease, a metalloenzyme, requires nickel ions in the active site for catalytic activity (hydrolysis of urea). Proteins involved in transport or binding of Ni 2 + can regulate urease activity by controlling the amount of nickel available for insertion into the apoprotein. NixA, high-affinity nickel transport protein; Hpn, histidinerich protein; HspA, heat shock protein A. For details, see text.
Urease Activity Is Regulated by Proteins That Transport and Bind Nickel Ions The most prominent and first described virulence factor of H. pylori is urease. 25 ,66,67 The activity of the enzyme requires that two nickel ions (NiH) be inserted into each of six active sites of each holoenzyme (composed of six copies each of two structural subunits, UreA and UreB) (data extrapolated from Mobley et al.,2 Dunn et al.,28 and Jabri et al. 68 ). It seems that the actual process of nickel ion insertion into the apoenzyme is accomplished by accessory proteins that are encoded by genes within the urease gene clusterJ,69 Insertion of nickel ions, however, requires that the metal ion be available to the accessory protein system. A number of proteins have now emerged as elements that appear to control availability of nickel ions and thus serve to regulate urease catalytic activity by regulating nickel ion availability. NixA,8,9 a P-type adenosine triphosphatase (ATPase),lO,l1 an ABC transporter,13 Hpn,12 and HspA 14,15 all appear to contribute to this process by either transporting or binding nickel ions (Figure 4). NixA is a high-affinity nickel transport protein found in the cytoplasmic membrane of H. pylori. 8 It was discovered by its ability to intensify urease activity of Escherichia coli cotransformed with H. pylori urease genes and a nixA-containing gene bank clone. The 35kilodalton protein does not seem to be part of a larger complex and can act alone to import nickel ions into the cell. NixA-deficient mutants have reduced urease activity (42% reduction in one strain) because of reduced nickel ion availability.9 Significant urease activity remains, however, indicating that nickel ions can also be acquired by other systems. A P-type ATPase has been described by two groups, which appears to play an important role in regulation of urease activity.lO,l1 The 648-amino acid polypeptide also
December Supplement 1997
seems to be an integral cytoplasmic membrane protein. 11 Adenosine triphosphate (ATP) binding motifs and an N-terminal metal ion binding motif have been identified. Mutation of the ATPase gene in H. pylori results in a dramatic reduction in urease activity (>99%). Although there has been no direct demonstration of nickel transport by the ATPase, transport appears to be its function. An ABC (ATP-binding cassette) transporter system has also been described rece~tly in our laboratory.13 This four-component system also uses ATP energy for transport. One component, AbcC, a homologue of a nickel transporting protein in E. coli (nikD), has ATP-binding domains. Another component is clearly a cytoplasmic membrane protein. Mutation of these genes in H. pylori also dramatically decreases urease activity (72% reduction) but not activity of other enzymes such as catalase and oxidase. 13 A heat shock protein, HspA, may also playa role in nickel ion management. 14,15 This protein is highly homologous to other GroES proteins but contains a striking histidine-rich motif at its C terminus. The motif is capable of specifically binding nickel ions and could act as a reservoir for the ion. Mutation of hspA is lethal, and its direct contribution to urease activity cannot be assessed in the parent organism. Another histidine-rich protein, Hpn, can also bind nickel and other ions and may contribute to nickel ion management. 12 Mutation of the hpn gene, however, does not reduce urease activity.
Strain Heterogeneity There is clearly tremendous strain-to-strain variability for H. pylori with respect to chromosomal DNA sequence and gene arrangement. Strains cultured from gastric biopsy specimens can, more often than not, be differentiated from other strains by molecular analySiS. I 6-21 If the same gene is analyzed for many isolates, frequent base pair substitution in the DNA sequence can be noted. 21 If the arrangement of genes on the chromosome is analyzed, frequent rearrangements of their order are also observed. 7o-72 In addition, more virulent isolates such as those associated with duodenal ulceration, for example, may carry additional sequences that may be completely absent in strains associated with gastritis alone.7 3 ,74 However, these descriptions of variability or heterogeneity do not indicate that the H. pylori genome is unstable or in constant flux. On the contrary, when strains have been cultured from biopsy specimens of patients over periods of months or years, their DNA sequence of selected genes has been found to remain
H. PYLORI: BACTERIAL FACTORS AND MUCOSAL DISEASE
S25
unchanged. 21 Identical strains, as determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), have also been isolated from several generations of family members.7 5
Diverse RFLP Patterns The first identification of the tremendous diversity among H. pylori strains was the recognition of diverse RFLP patterns of HindIII-digested chromosomal DNA isolated from different strains electrophoresed on an agarose gel. 16 These data suggested that strains isolated from different patients had numerous base pair substitutions or gene arrangements or both. This has been confirmed by a number of investigators using a variety of techniques including PCR-RFLP,17 PCR-DNA sequence typing,21 arbitrarily primed PCR, or random amplified polymorphic DNA 18,76 methods. In each case, these methods were used to identify different strains or confirm identity of strains.
Mosaicism in Virulence Genes Looking more closely at the diversity within specific genes, it was noted that the gene encoding the vacuolating cytotoxin displayed what was described as mosaicism. Although all strains possess the vacA genes, only about half the strains secrete an active cytotoxin. 55 Although the specific reason for this deficiency is not known, genetic differences have been identified between the vacA allele of toxin-secreting strains and vacA allele of toxin-negative strains.7 7 These differences have been localized to two regions: first, in the signal sequence (N-terminal amino acid sequences required for protein secretion by the general secretion pathway) and, second, in the midregion of the gene where considerable heterogeneity exists. Although these differences do not directly explain variation in toxin phenotypes, they do provide a starting point for such investigations.
Pathogenicity Island Diversity of isolates can also be explained by the presence or absence of specific gene sequences. Recent work has identified a region of DNA in the chromosome of H. pylori that is unique to some strains. These "pathogenicity islands," which appear to include the cagA gene, have been characterized by nucleotide sequencing and encode homologues of proteins involved in various secretory pathways including protein and DNA.7 3 One of the predicted gene products has been provisionally termed "Pic" for "promotes induction of cytokines."78
S26
HARRY L. T. MOBLEY
Why to Look Out for CagA: Pathogenicity Islands and Virulence CagA, another intriguing protein that has received considerable attention, is a 128-kilodalton immunodominant outer membrane protein that was originally thought to represent the cytotoxin itself. Indeed, expression of the protein correlates strongly with cytotoxin production. 22 ,23,53,55 It has since been discovered that the gene encoding this protein, cagA (cytotoxin-associated gene), is distinct and maps at some distance (300 kilobases 72 ) from the vacA cytotoxin gene. Nevertheless, the presence of the cagA gene, and expression of the CagA protein, is present significantly more often in strains isolated from patients with more serious clinical manifestations including duodenal and gastric ulcer than from strains isolated from patients with gastritis alone. 44 .49 For example, in a study of patients with chronic gastritis, the presence of anti-CagA antibodies was measuredJ9 In patients with no ulcers, 63% (32 of 51 patients) were CagA positive. In patients with peptic ulcers, 100% (25 of 25 patients) were CagA positive. In another study, when the presence of the cagA gene in the infecting H. pylori strain was examined, similar results were found. Patients with any gastrointestinal disease (presumably including peptic ulcer) were 75% cagA positive. In patients with duodenal ulcer, 100% of the strains were cagA positive. 22 The cagA gene and CagA protein thus serve as genotypic and phenotypic markers for virulent strains. There are well-defined pathogenicity islands (contiguous stretches of chromosomal DNA) present in some strains of H. pylori that encode CagA and additional proteins that may potentiate virulence. Strains possessing these "virulence cassettes" are isolated more frequently from patients with the more serious clinical manifestations associated with duodenal ulcer than from patients with gastritis alone or nonulcer dyspepsia.
Is There a Gradient of Virulence Among Strains? Because of the tremendous diversity in H. pylori, it not surprising that assignment of virulence is not as clear-cut as identifying the presence or absence of the cagA-containing pathogenicity island. Strains have been identified that run the spectrum from having the "complete" pathogenicity island to those that lack these sequences compietelyJ3 In between, strains may have these pathogenicity island sequences, which are split and located at different points in the genome or may contain only portions of the complete island. Therefore, there may exist a gradient of virulence among isolates. If one IS
GASTROENTEROLOGY Vol. 113, No.6
superimposes this continuum of virulence upon the predisposition of certain human hosts for acquiring infection, then it is not surprising that there are so many degrees of clinical manifestations of H. pylori infections that include gastritis alone, gastric ulcer, duodenal ulcer, and gastric malignancy.
Conclusion H. pylori can be considered a true pathogen. Although all strains seem to be capable of causing gastritis in humans and are thus virulent, a certain subset of strains possesses additional genes encoded by pathogenicity islands, which render them more virulent. The proteins encoded by these extra genes appear to encode homologues of proteins involved in protein secretion and thus may cause ulcer development via secreted products. These secreted products probably interact directly with the epithelium to trigger cascades of events that lead irreversibly to epithelial cell damage. Alternatively, these secreted proteins could interact with immune cells that in turn liberate cytokines causing the observed effect indirectly.
References 1. Finally BB, Falkow S. Common themes in microbial pathogenic· ity. Microbiol Rev 1989;53:210-230. 2. Mobley HLT, Island MD, Hausinger RP. Molecular biology of microbial ureases. Microbiol Rev 1995;59:451-480. 3. Eaton KA, Brooks CL, Morgan DR, Krakowka S. Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylori in gnotobiotic piglets. Infect Immun 1991;59:24702475. 4. Eaton KA, Krakowka S. Effect of gastriC pH on ureasedependent colonization of gnotobiotic piglets by Helicobacter pylori. Infect Immun 1994;62:3604-3607. 5. Tsuda M, Karita M, Morshed MG, Okita K, Nakasaki T. A urease-negative mutant of Helicobacter pylori constructed by allelic exchange mutagenesis lacks the ability to colonize the nude mouse stomach. Infect Immun 1994;62:3586-3589. 6. Takahashi S, Igarashi H, Nakamura K, Masubuchi N, Saitos S, Aoyagi T, Itoh T, Hirata I. Helicobacter pylori urease activitycomparative study between urease positive and urease negative strain. Jpn J Clin Med 1993;51:3149-3153. 7. Hu L-T, Foxall PA, Russell R, Mobley HLT. Purification of recombinant Helicobacter pylori urease apoenzyme encoded by ureA and ureB. Infect Immun 1992;60:2657-2666. 8. Mobley HLT, Garner RE, Bauerfeind P. Helicobacter pylori nickel transport gene nixA: synthesis of catalytically active urease in E. coli independent of growth conditions. Mol MicrobioI1995;16: 97-109. 9. Bauerfeind P, Garner RM, Mobley HLT. Allelic exchange mutagenesis of nixA in Helicobacter pylori results in reduced nickel transport and urease activity. Infect Immun 1996;64:28772880. 10. Ge Z, Hiratsuka K, Taylor DE. Nucleotide sequence and mutational analysis indicate the two Helicobacter pylori genes encode a P-type ATPase and a cation binding protein associated with copper transport. Mol MicrobioI1995;15:97-106. 11. Melchers K, Weitzenegger T, Buhmann A, Steinhilber W, Sachs G, Schafer KP. Cloning and membrane topology of a P type
December Supplement 1997
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
ATPase from Helicobacter pylori. J Bioi Chem 1996;271:446457. Gilbert JV, Ramakrishna J, Sunderman FIN, Wright A, Plaut AG. Protein Hpn: cloning and characterization of a histidine-rich metal-binding polypeptide in Helicobacter pylori and Helicobacter mustelae. Infect Immun 1995;63:2682-2688. Hendricks JK, Mobley HLT. Helicobacter pylori ABC transporter: effect of allelic exchange mutagenesis on urease activity. J Bacteriol (in press). Suerbaum S, Thiberge J-M, Kansau I, Ferrero RL, Labigne A. Helicobacter pylori hspA-hspB heat-shock gene cluster: nucleotide sequence, expression, putative function and immunogenicity. Mol MicrobioI1994;14:959-974. Kansau I, Guillain F, Thiberge J-M, Labigne A. Nickel binding and immunological properties of the C-terminal domain of the Helicobacter pylori GroES homologue (HspA). Mol Microbiol 1996;22:1013-1023. Langenberg W, Rauws EAJ, Widjojokusumo A, Tytgat GN, Zanen HC. Identification of Campylobacter pyloridis isolates by restriction endonuclease DNA analysis. J Clin MicrobioI1986;24:414417. Foxall PA, Hu L-T, Mobley HLT. Use of polymerase chain reaction: amplified Helicobacter pylori urease structural genes for differentiation of isolates. J Clin MicrobioI1992;30:739-741. Akopanz N, Bukanov NO, Westblom TU, Kresovich S, Berg DE. DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Res 1992;20:5137-5142. Evans DG, Evans DJ Jr, Lampert HC, Graham DY. Restriction fragment length polymorphism in the adhesin gene hpaA of Helicobacter pylori. Am J GastroenteroI1995;90:1282-1288. Clayton CL, Kleanthous H, Morgan DD, Puckey L, Tabaqchali S. Rapid fingerprinting of Helicobacter pylori by polymerase chain reaction and restriction fragment length polymorphism analysis. J Clin MicrobioI1993;31:1420-1425. Labigne A. PCR-DNA sequence typing. In: Clayton CL, Mobley HlT, eds. Helicobacter pylori protocols. Totowa, New Jersey: Humana, 1997:107-116. Covacci A, Censini S, Bugnoli M, Petracca R, Burroni D, Macchia G, Massone A, Papini E, XiangZ, Figura N, Rappoali R. Molecular characterization of the 128 kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer. Proc Natl Acad Sci USA 1993;90:5791-5795. Crabtree JE, Figura N, Taylor JD, Bugnoli M, Armellini D, Tompkins DS. Expression of 120 kilodalton protein and cytotoxicity in Helicobacter pylori. J Clin PathoI1992;45:733-734. Neale KR, Logan RPH. The epidemiology and transmission of Helicobacter pylori infection in children. Aliment Pharmacol Ther 1995;9(Suppl 2):77-84. Mobley HLT, Cortesia MJ, Rosenthal LE, Jones BD. Characterization of urease from Campylobacter pylori. J Clin Microbiol 1988;26:831-836. Hu L-T, Mobley HLT. Purification and N-terminal analysis of urease from Helicobacter pylori. Infect Immun 1990;58:992998. Hawtin PR, Delves HT, Newell DG. The demonstration of nickel in the urease of Helicobacter pylori by atomic absorption spectroscopy. FEMS Microbiol Lett 1991;77:51-54. Dunn BE, Campbell GP, Perez-Perez GI, Blaser MJ. Purification and characterization of urease from Helicobacter pylori. J Bioi Chem 1990;265:9464-9469. Evans DJ Jr, Evans DG, Kirkpatrick SS, Graham DS. Characterization of the Helicobacter pylori urease and purification of its subunits. Microb Pathog 1991;10:15-26. Bauerfeind P, Garner R, Dunn BE, Mobley HLT. Synthesis and activity of Helicobacter pylori urease and catalase at low pH. Gut 1997 (in press).
H. PYLORI: BACTERIAL FACTORS AND MUCOSAL DISEASE
S27
31. Ferrero RL, Cussac V, Courcoux P, Labigne A. Construction of isogenic urease-negative mutants of Helicobacter pylori by allelic exchange. J Bacteriol1992;17 4:4212-4217. 32. Jackson CJ, Kelly DJ, Clayton CL. The cloning and characterization of chemotaxis genes in Helicobacter pylori. Abstracts, Vilith International Workshop on Campylobacters, Helicobacters, and Related Organisms. Winchester, England; 1995:63. 33. Eaton KA, Morgan DR, Krakowka S. Motility as a factor in the colonization of gnotobiotic piglets by Helicobacter pylori. J Med MicrobioI1992;37:123-127. 34. Eaton KA, Suerbaum S, Josenhans C, Krakowka S. Colonization of gnotobiotic piglets by Helicobacter pylori deficient in two flagellin genes. Infect Immun 1996;64:2445-2448. 35. Smoot DT, Resau JH, Naab T, Desbordes BC, Gilliam T, BullHenry K, Curry SB, Nidiry J, Sewchand J, Mills-Robertson K, Frontin K, Abebe E, Dillon M, Chippendale GR, Phelps PC, Scott VF, Mobley HLT. Adherence of Helicobacter pylori to cultured human gastric epithelial cells. Infect Immun 1993;61:350355. 36. Segal ED, Falkow S, Tompkins LS. Helicobacter pylori attachment to gastric cells induces cytoskeletal rearrangements and tyrosine phosphorylation of host cell protein. Proc Natl Acad Sci USA 1996;93:1259-1264. 37. Steer HW. Ultrastructure of Campylobacter pylori in vivo. In: Rathbone BJ, Heatly R, eds. Campylobacter pylori and gastroduodenal disease. Oxford, England: Blackwell Scientific, 1989: 189. 38. Walker SJ, Birch PJ, Stewart M, Stoddard CJ, Hart CA, Day DW. Patterns of colonization of Campylobacter pylori in the esophagus, stomach and duodenum. Gut 1989;30:1334-1338. 39. Logan RPH. Adherence of Helicobacter pylori. Aliment Pharmacol Ther 1996;10(Suppll):3-15. 40. Boren T, Falk P, Roth KA, Larson G, Normark S. Attachment of Helicobacter pylori to human gastric epithelium mediated by blood group antigens. SCience 1993;262:1892-1895. 41. Odenbreit S, Till M, Haas R. Identification of a H. pylori-specific protein involved in adherence to gastric epithelial cells. Abstracts, VI 11th International Workshop on Gastroduodenal Pathology and Helicobacter pylori. Edinburgh, Scotland, 1995. 42. Cave DR, Vargas M. Effect of a Campylobacter pylori protein on acid secretion by parietal cells. Lancet 1989;2:187-189. 43. Ferrero RL, Thiberge J-M, Kansau I, Wuscher N, Huerre M, Labigne A. The GroES homologue of Helicobacter pylori confers protective immunity against mucosal infection in mice. Proc Natl Acad Sci USA 1995;92:6499-6503. 44. Phadnis SH, Dunn BE. Surface localization of H. pylori urease and HP54K requires bacterial lysis. Abstracts, Vilith International Workshop on Gastroduodenal Pathology and Helicobacter pylori. Edinburgh, Scotland; 1995. 45. Hazell SL, Mendz GL. The metabolism and enzymes of Helicobacter pylori: function and potential virulence effects. In: Goodwin CS, Worsley BW, eds. Helicobacter pylori: biology and clinical practice. Boca Raton, FL: CRC, 1993:115-141. 46. Odenbreit S, Wieland B, Haas R. Cloning and genetic characterization of Helicobacter pylori catalase and construction of a catalase-deficient mutant strain. J Bacteriol 1996;178:69606967. 47. Muotiala A, Helander 1M, Pyhala L, Kosunen TV, Moran AP. Low biological activity of Helicobacter pylori lipopolysaccharide. Infect Immun 1992;60:1714-1716. 48. Moran AP, Prendergast MM, Appelmelk BJ. Molecular mimicry of host structures by bacterial lipopolysaccharides and its contribution to disease. FEMS Immunol Med MicrobioI1996;16: 105-115. 49. Aspinall GO, Monteiro MA, Pang H, Walsh EJ, Moran AP. Lipopolysaccharide of the Helicobacter pylori type strain NCTC
S28
50.
51.
52. 53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
HARRY L. T. MOBLEY
11637 (ATCC43504): structure of the 0 antigen chain and core oligosaccharide regions. Biochemistry 1996;35:2489-2497. Windle HJ, Kelleher D. Identification and characterization of a metallo-protease activity from H. pylori which is capable of degrading immunoglobulins. Abstracts, Vilith International Workshop on Gastroduodenal Pathology and Helieobaeter pylori. Edinburgh, Scotland; 1995. Suzuki M, Miura S, Soematsu M, Fukumura D, Kurose I, Suzuki H, Kai A, Kudoh Y, Ohashi M, Tanchiya M. Helieobaeter pyloriassociated ammonia production enhances neutrophil-dependent gastric mucosal cell injury. Am J PhysioI1992;263:G719G725. Hazell SL, Lee A. Campylobaeter pyloridis, urease, hydrogen ion back diffusion, and gastric ulcers. Lancet 1986;2:15-17. Tummuru MK, Cover TL, Blaser MJ. Cloning and expression of a high-molecular-mass major antigen of Helieobaeter pylori: evidence of linkage to cytotOXin production. Infect Immun 1993;61: 1799-1809. Lupetti P, Heuser JE, Manetti R, Massari P, Lanzavecchia S, Bellon PL, Dallai R, Rappuoli R, Telford JL. Oligomeric and subunit structure of the Helicobaeter pylori vacuolating cytotoxin. J Cell Bioi 1996;133:801-807 . Cover TL, Dooley CP, Blaser MJ. Characterization of and human serologic response to proteins in Helicobacter pylori broth culture supernatants with vacuolizing cytotOXin activity. Infect Immun 1990;58:603-610. Young GO, Lastovica AJ, Brown S, McGovern JJ, Moran AP. Effect on pepsinogen release of various sub-fractions of H. pylori lipopolysaccharide. Abstracts, VI 11th International Workshop on Campylobaeters, Helieobaeters, and Related Organisms. Winchester, England, 1995:65. Wetherall BL, McDonald PJ, Johnson AM. Partial characterization of a cell free hemolytic factor produced by Helieobaeter pylori. FEMS MicrobiollmmunoI1992;4:123-128. Lichtenberger LM, Hazell SL, Romero JJ, Graham DY. Helieobaeter pylori hydrolysis of artificial phospholipid monolayers: insight into a potential mechanism of mucosal injury (abstr). Gastroenterology 1990;98:A78. Suzuki M, Miura S, Soematsu M, Fukumura D, Kurose I, Suzuki H, Kai A, Kudoh y, Ohashi M, Tanchiya M. Helicobaeter pyloriassociated ammonia production enhances neutrophil-dependent gastric mucosal cell injury. Am J PhysioI1992;263:G719G725. Mai UEH, Perez-Perez GI, Wahl LM, Wahl SM, Blaser MJ, Smith PD. Soluble surface proteins from Helicobacter pylori activate monocytes/macrophages by lipopolysaccharide-independent mechanism. J Clin Invest 1991;87:894-900. Nielsen P, Anderson LP. Activation of human phagocyte oxidative metabolism by Helieobaeter pylori. Gastroenterology 1992; 103:1747-1753. Craig PM, Territo MC, Karnes WE, Walsh JH. Helieobaeter pylori secretes a chemotactic factor for monocytes and neutrophils. Gut 1992;33:1020-1023. Mai UEH, Perez-Perez GI, Allen JB, Wahl SM, Blaser MJ, Smith PD. Surface proteins from Helicobacter pylori exhibit chemotactic activity for human leukocytes and are present in gastric mucosa. J Exp Med 1992;175:517-525. Mai UEH, Perez-Perez GI, Wahl LM, Wahl SM, Blaser MJ, Smith PD. Soluble surface proteins from Helieobaeter pylori activate monocytes/macrophages by lipopolysaccharide-independent mechanism. J Clin Invest 1991;87:894-900.
GASTROENTEROLOGY Vol. 113, No.6
65. Falkow S. Molecular Koch's postulates applied to microbial pathogeniCity. Rev Infect Dis 1988;10(SuppI2):S274-S276. 66. Langenberg ML, Tytgat GNJ, Schipper MEL Campylobacter-like organisms in the stomach of patients and healthy individuals. Lancet 1984;1:1348. 67. Dent JG, McNulty CAM, Uff JS, Gear MWL, Wilkinson SP. Campylobaeter pylori urease: a new serological test. Lancet 1988;1:1002. 68. Jabri E, Carr MB, Hausinger RP, Karplus PA. The crystal structure of urease from Klebsiella aerogenes. Science 1995; 268:998-1004. 69. Cussac V, Ferrero RL, Labigne A. Expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogenlimiting conditions. J Bacteriol1992;17 4:2466-24 73. 70. Taylor DE, Eaton M, Chang N, Salama S. Construction of a Helieobacter pylori genome map and demonstration of diversity at the genome level. J Bacteriol1992;17 4:2332-2337. 71. Jiang Q, Hiratsuka K, Taylor DE. Variability of gene order in different Helieobacter pylori strains contributes to genome diversity. Mol MicrobioI1996;20:833-842. 72. Bukanov NO, Berg DE. Ordered cosmid library and high resolution physical genetic map of Helicobacter pylori strain NCTC 11638. Mol MicrobioI1994;11:509-523. 73. Censini S, Lange C, Xiang Z, Crabtree JE, Ghiara P, Borodovsky M, Rappuoli R, Covacci A. eag, a pathogeniCity island of Helieobaeter pylori, encodes type I-specific and diseaseassociated virulence factors. Proc Natl Acad Sci USA 1996;93: 14648-14653. 74. Akopyants NS, Kersulyte D, Berg DE. CAGII, a new multigene locus associated with virulence in H. pylori. Abstracts, Vilith International Workshop on Gastroduodenal Pathology and Helicobacter pylori. Edinburgh, Scotland, 1995. 75. Labigne A. PCR-DNA sequence typing. In: Clayton CL, Mobley HLT, eds. Helicobaeter pylori protocols. Totowa, NJ: Humana, 1997:107-116. 76. Berg DE, Lelwala-Guruge J, Incecik ET, Srivastava K, Akopyants NS. H. pylori DNA fingerprinting using the arbitrarily primed PCR (AP-PCR) or random amplified polymorphic DNA (RAPD) method. In: Clayton CL, Mobley HLT, eds. Helieobaeter pylori protocols. Totowa, New Jersey: Humana, 1997:117-132. 77. Atherton JC, Cao P, Peek RM Jr, Tummuru MK, Blaser MJ, Cover TL. MosaiCism in vacuolating cytotOXin alleles of Helieobacter pylori. Association of specific vacA types with cytotoxin production and peptiC ulceration. J Bioi Chem 1995;270:1777117777. 78. Tummuru MKR, Sharma SA, Blaser MJ. Helieobaeter pylori pieB, a homologue of the Bordetella pertussis toxin secretion protein, is required for induction of IL-8 in gastric epithelial cells. Mol MicrobioI1995;18:867-876. 79. Crabtree JE, Taylor JD, Wyatt JI, Heatley RV, Shallcross TM, Tompkins DS, Rathbone BJ. Mucosal IgA recognition of Helicobacter pylori 120 kDa protein, peptic ulceration and gastric pathology. Lancet 1991;338:332-335. Received June 30,1997. Accepted July 22,1997. Address requests for reprints to: Harry L. T. Mobley, Ph.D, Department of Microbiology and Immunology, University of Maryland School of Medicine, 10 South Pine Street, Room 9-00, Baltimore, Maryland 21201. Fax: (410) 706-6751. Supported by Public Health Service Grant AI25567 from the National Institutes of Health.
Helicobacter pylori Factors Associated With
Disease Development
HARRY L. T. MOBLEY Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland
Although certain factors appear to predispose the host to infection by Helicobacter pylori, clearly the bacterium possesses a well-defined battery of virulence factors that allow the organism to: (1) colonize the gastric mucosa (urease, flagella, adhesins, acidinhibitory protein, iron acquisition proteins, and heat shock proteins); (2) evade host defense (shedding of surface proteins, catalase, superoxide dismutase, and poorly reactive lipopolysaccharide); and (3) damage host tissue (vacuolating cytotoxin, protease, CagArelated factors, inducers of cytokines, and chemotaxins). Together these factors allow H. pylori to persist in the host, establishing a chronic infection. Although many of these virulence factors are produced by all strains of H. pylori, there are also well-defined pathogenicity islands (contiguous stretches of chromosomal DNA) present in some strains that encode additional proteins including CagA that potentiate virulence. Strains possessing these "virulence cassettes" are isolated more frequently from patients with the more serious clinical manifestations associated with duodenal ulcer than from patients with gastritis alone or nonulcer dyspepsia.
A
true bacterial pathogen is an organism that is capable of causing disease in an immunocompetent host. Such bacteria possess defined mechanisms of pathogenesis. An infection is defined as the successful establishment and persistence of a pathogen. 1 Replication of a bacterium to form many bacteria is sufficient to maintain infection and may represent the ultimate goal of the microbe. Multiplication and persistence alone, however, does not usually lead to disease. Disease may actually be an unfavorable or "unwanted" outcome for the infecting microbe and results from an infection that causes significant overt damage to the host. 1 There is, of course, interplay between the host and bacterium. Although certain factors appear to predispose the host to infection by Helicobacter pylori (see article by Go in this supplement), clearly this bacterium possesses a well-defined battery of virulence factors that allow the organism to colonize the gastric mucosa, evade host defense, and damage host tissue. These factors will be
discussed in the context of a model for microbial pathogenesis. Although numerous virulence factors have been identified, urease has emerged as one of the few proteins with proven involvement in the development of infection and disease. 2- 6 The activity of urease is regulated by its nickel content in the active site of the enzyme,7 Protein systems responsible for transport and binding of nickel have been described recently, and their role will be reviewed. 8- 15
H. pylori shows tremendous strain-to-strain variability.16-21 This heterogeneity is manifested in a number of ways, bur the discovery of pathogenicity islands (contiguous stretches of chromosomal DNA encoding virulence factors), associated with the more virulent strains,22,23 has perhaps allowed us to define H. pylori as a true pathogen.
Model for Pathogenesis In the study of microbial pathogenesis, microbiologists have attempted to divide the steps required for the development of infection and disease into overlapping categories. First, the bacterium must enter the host and colonize. Specific colonization factors are required for this process. Next, the microbe must avoid host defenses so that it can multiply and persist. Finally, an optional step for infection, but a required step for the development of disease, is the eliciting of direct or indirect damage to the host.
Colonization It is generally accepted that H. pylori is acquired in childhood by a fecal-oral or oral-oral spread from another infected human. 24 There is no dramatic evidence showing the existence of an active reservoir that actually plays a role in transmission beyond the human host. Therefore, the bacterium is most likely adapted to its host and synthesizing the proteins and metabolites required for
Abbreviations used in this paper: LPS, lipopolysaccharide; peRRFLP, polymerase chain reaction-restriction fragment length polymorphism. © 1997 by the American Gastroenterological Association 0016-5085/97/$3.00
S22
GASTROENTEROLOGY Vol. 113, No. 6
HARRY L. T. MOBLEY
Ii. H~
Ii.
l
HI
e e e e e e
Figure 1. Colonization by H. pylori. Products produced by H. pylori that may allow the bacterium to establish itself in the gastric mucosa. HSP, heat shock protein. Ni++, nickel ions; H+, protons.
Figure 2. Avoidance of host defense by H. pylori. Protein products and LPS that allow H. pylori to overcome innate defense including acid and immune surveillance. O2 , toxic oxygen radical.
colonization of a new host. Several virulence factors are putatively involved in this process (Figure 1). Urease is one of the most striking features of this organism. This high-molecular-weight (550-kilodalton) enzyme hydrolyzes urea, the nitrogenous waste product of humans, to liberate ammonia and carbon dioxide. 25 ,26 The enzyme is one of the few nickel metalloenzymes in nature and requires the divalent cation in its active site to successfully hydrolyze its substrate. 27 H. pylori synthesizes urease in extraordinarily high amounts, making up as much as 5 % of the total cell protein. 26 ,28,29 Its proposed role in pathogenesis is quite appealing. As the organism enters the stomach, it is undoubtedly faced with abundant gastric acid. Because H. pylori is not an acidophile and does not thrive or even survive in vitro at low pH, it must protect itself from acid. 3o The hydrolysis of urea provides ammonia as a neutralizing agent for hydrochloric acid. The transient development of achlorhydria and, indeed, the development of neutral pH may allow the organism time to burrow through the gastric mucus to the gastric epithelium, which provides a more hospitable environment. Urease-negative mutants developed by both chemical mutagenesis 3 and allelic exchange mutagenesis4,3 1 are unable to colonize the gastric mucosa of the gnotobiotic piglet, an accepted animal model of H. pylori infection. Indeed, the flagella-mediated burrowing motility is also essential for survival. The spiral shape of the bacterium coupled with the tuft of polar flagella make the organism well suited for traversing the mucous layer. The recent discovery of genes associated with chemotaxis now suggests that H. pylori can chemotax toward positive stimuli and away from toxic substances. 32 Isogenic mutants of H. pylori lacking flagella have been unable to colonize the gnotobiotic piglet, whereas the wild-type strain can colonize and persist. 33 ,34 Once the bacterium has traversed the gastric mucus, a
small percentage of organisms attach intimately to the surface of gastric epithelial cells. This event is coupled to actin rearrangement 35 and host protein phosphorylation 36 within the epithelial celP5,37 Because H. pylori is so highly specific for humans and indeed gastric tissue (the duodenum is colonized only when there is spread of the epithelium by gastric metaplasia 38 ; esophageal epithelium is also not colonized), it is appealing to envision highly specific adherence factors. Although no adherence factor has been proven to be required for colonization, a number of such candidates have been described including hemagglutinins,39 an intimin-like protein,35 Lewis blood group antigen-binding adhesin,4o and an adhesin lipoprotein. 41 Other factors that may play a role in colonization include an acid-inhibitory protein, which can inactivate the gastric proton pump,42 again neutralizing hazardous acid. Like most other pathogenic bacteria, the organism must have strategies for acquiring iron, tightly sequestered by the host. Although no siderophores have yet been described, there are undoubtedly iron acquisition systems that take up the iron required for cytochrome production. Finally, the high levels of production of GroEL- and GroES-like heat shock proteins (HspB and HspA, respectively) may explain the ability to transiently tolerate the extreme conditions met by the bacterium during the early stages of colonization. 42 ,43 These chaperone-like proteins may assist in maintaining the integrity of proteins in the bacterium during this period of stress.
Avoidance of Immune Response Once colonization is complete, H. pylori must avoid the immune response that could result in clearance. A number of mechanisms for avoidance have been proposed (Figure 2). Shedding of proteins is one mechanism. Various proteins have been localized to the surface of the bacterium using immunochemical approaches.
H. PYLORI: BACTERIAL FACTORS AND MUCOSAL DISEASE
December Supplement 1997
Vocuoe'Wlo (')toIOlUf'l
Figure 3. Damage to host by H. pylori. Products of H. pylori that directly or indirectly damage the host epithelium. NH 3 , ammonia; CagA, cytotoxin-associated gene A; PieS, promotes induction of cytokines gene S; IL-8, interleukin 8.
Urease, which is strictly a cytoplasmic protein in nonHelicobacter bacterial species, is present in very high amounts on the surface. Phadnis and Dunn 44 suggest that the mechanism is nonspecific and involves "altruistic lysis" whereby a certain proportion of the population undergoes lysis and coats the remaining viable bacteria with normally cytosolic proteins. These shed proteins could function to saturate the binding sites of secretory immunoglobulin specific for these proteins and other immune receptors and prevent a bactericidal response. Catalase and superoxide dismurase are H. pylori enzymes that detoxifY toxic oxygen radicals.45 .4 6 However, their actual roles have not been shown in H. pylori. Interestingly mutations in superoxide dismutase genes appear to be lethal; this is not the case for catalase because a murant has been constructed. The lipopolysaccharide (LPS) of H. pylori may also favor persistence of the bacterium. The isolated polymers are approximately lOOO-fold less toxic than the LPS of other gram-negative bacteria; therefore, the LPS molecule may not stimulate the immune response to the degree necessary to clear the organism,47 and in some cases mimics the Lewis x and Lewis y antigen. 48 .49 In addition, an immunoglobulin A (IgA) protease that specifically cleaves secretory IgA molecules has been proposed for H. pylori,5o but no direct evidence supports their existence or role in pathogenesis. Finally, one way that several bacterial species escape the immune response is by invasion of eukaryotic cells. Although H. pylori bacteria have been observed "within" epithelial cells in electron micrographs of thin sections, it is not believed that invasion contributes to the pathogenesis of H. pylori infection.
Damage to the Host Certain virulence factors are associated with direct or indirect damage to the gastric mucosa (Figure 3). In
S23
addition to the role of urease in colonization, there is evidence that ammonium hydroxide, the product of urea hydrolysis, contributes significantly to histological damage. The hydroxide ion generated by the equilibration of ammonia with water seems to be the toxic species; the ammonium ion itself is not toxic. To show the cytotoxic effect of urease, cell cultures of a human gastric adenocarcinoma cell line were seeded with H. pylori and supplemented with various concentrations of urea. 51 Cell viability was found to be inversely proportional to ammonia concentrations generated by urea hydtolysis. Viability was improved when the urease inhibitor, acetohydroxamic acid, was added to the culture before the exposure to H. pylori. Acetohydroxamic acid slowed the liberation of ammonia and reduced the cytotoxic effect. These data suggested that histological damage may result directly from the localized generation of ammonia due to the hydrolysis of urea. It has also been postulated that ammonia produced by urea hydrolysis has an additional effect. 52 Ammonia may interfere with normal hydrogen ion back-diffusion across gastric mucosa, resulting in cytotoxicity to the underlying epithelium. The vacuolating cytotoxin is derived from a largemolecular-size polypeptide (139 kilodaltons) that is processed at both the N terminus (traditional signal sequence) and at the C terminus (-50 kilodaltons). 53 The remaining subunit of -87 kilodaltons assembles into six copies to form the holotoxin. 54 The toxin protein is not homologous to other known proteins and acts to form vacuoles within its target cells. 55 The vacA gene is present in all H. pylori strains, but the toxin is secreted in detectable amounts in only about half of the isolates. In general, these strains are cagA positive. CagA itself, however, is not responsible for toxin export. This will be discussed further below. LPS of H. pylori is a complex molecule with several domains. It has been reported that purified LPS can function to induce pepsinogen and elicit mucous modification.56 These findings were determined by the use of Ussing chambers and have not been confirmed in vivo. The presence of hemolysins has been suggested by some investigators, but the evidence is not substantial. 57 Zones of clearing are not generally found around the colonies on the agar plates, but hemolysis of erythrocytes in suspension occurs in the presence of H. pylori. This activity, although weak, is calcium dependent, as for other hemolysins. In addition, hemolytic muranes that do not display the phenotype have been isolated. At this poine, the existence of hemolysins is not well supported by the available data. H. pylori also produces phospholipases and alcohol
S24
HARRY L. T. MOBLEY
dehydrogenase, but their role in disease is currently speculative. 58 Urease activity may also be indirectly responsible for damage to the gastric epithelium via its interaction with the immune system. H. pylori whole cells can stimulate an oxidative burst in human neutrophils. 59 Urease can also cause activation of monocytes and polymorphonuclear leukocytes, and recruitment of inflammatory response cells, resulting in indirect damage to the gastric epithelium. Water extracts of H. pylori, known to contain urease in high concentration, can activate monocytes by an iPS-independent pathway.60 In vitro stimulation of human monocytes led to secretion of inflammatory cytokines and reactive oxygen intermediates, all of which may be involved in mediating the inflammatory response in the gastric epithelium. Further investigation has shown that sonicates of H. pylori strains could prime and also cause direct activation of the oxidative burst in human polymorphonuclear leukocytes and monocytes. 61 There is also evidence of urease or urease-containing fractions from H. pylori acting as chemotactic factors for leukocytes, causing further local inflammation. 62 ,63 Such chemotactic activity for human monocytes and neutrophils was present in purified urease samples and could be inhibited by specific antibody to the UreB urease subunit. Further, a 20-amino acid peptide based on the amino terminus of the UreB subunit protein also exhibited similar levels of chemotaxis in a microchamber test system. 63 Immunocytochemical staining showed urease closely associated with the crypt cells in the lamina propria of patients with duodenal ulcers. It is posrulated that urease is absorbed into the mucosa where it attracts leukocytes and causes mucosal inflammation. 64 Urease, by a variety of mechanisms, is at least partly responsible for the initial recruitment of monocytes and neurrophils and further activation and stimulation of the immune system to produce the local inflammatory lesion associated with H. pylori infection.
Contribution to Virulence Examined Using Defined Mutation in Clinical Strains Only a few putative virulence factors have been tested appropriately65 using isogenic mutants and suitable animal models to determine the contribution to virulence of specific genes or proteins encoded by these genes. These include urease, flagella, and vacuolating cytotoxin. Of these, urease-negative and flagellumnegative mutants were unable to colonize the gastric mucosa of gnotobiotic piglets. 3--6,33,34 Other candidate virulence factors have been implicated only indirectly in virulence.
GASTROENTEROLOGY Vol. 113, No.6
H. pyIori
Agure 4. Proteins involved in nickel management as regulators of urease activity. Urease, a metalloenzyme, requires nickel ions in the active site for catalytic activity (hydrolysis of urea). Proteins involved in transport or binding of Ni 2 + can regulate urease activity by controlling the amount of nickel available for insertion into the apoprotein. NixA, high-affinity nickel transport protein; Hpn, histidinerich protein; HspA, heat shock protein A. For details, see text.
Urease Activity Is Regulated by Proteins That Transport and Bind Nickel Ions The most prominent and first described virulence factor of H. pylori is urease. 25 ,66,67 The activity of the enzyme requires that two nickel ions (NiH) be inserted into each of six active sites of each holoenzyme (composed of six copies each of two structural subunits, UreA and UreB) (data extrapolated from Mobley et al.,2 Dunn et al.,28 and Jabri et al. 68 ). It seems that the actual process of nickel ion insertion into the apoenzyme is accomplished by accessory proteins that are encoded by genes within the urease gene clusterJ,69 Insertion of nickel ions, however, requires that the metal ion be available to the accessory protein system. A number of proteins have now emerged as elements that appear to control availability of nickel ions and thus serve to regulate urease catalytic activity by regulating nickel ion availability. NixA,8,9 a P-type adenosine triphosphatase (ATPase),lO,l1 an ABC transporter,13 Hpn,12 and HspA 14,15 all appear to contribute to this process by either transporting or binding nickel ions (Figure 4). NixA is a high-affinity nickel transport protein found in the cytoplasmic membrane of H. pylori. 8 It was discovered by its ability to intensify urease activity of Escherichia coli cotransformed with H. pylori urease genes and a nixA-containing gene bank clone. The 35kilodalton protein does not seem to be part of a larger complex and can act alone to import nickel ions into the cell. NixA-deficient mutants have reduced urease activity (42% reduction in one strain) because of reduced nickel ion availability.9 Significant urease activity remains, however, indicating that nickel ions can also be acquired by other systems. A P-type ATPase has been described by two groups, which appears to play an important role in regulation of urease activity.lO,l1 The 648-amino acid polypeptide also
December Supplement 1997
seems to be an integral cytoplasmic membrane protein. 11 Adenosine triphosphate (ATP) binding motifs and an N-terminal metal ion binding motif have been identified. Mutation of the ATPase gene in H. pylori results in a dramatic reduction in urease activity (>99%). Although there has been no direct demonstration of nickel transport by the ATPase, transport appears to be its function. An ABC (ATP-binding cassette) transporter system has also been described rece~tly in our laboratory.13 This four-component system also uses ATP energy for transport. One component, AbcC, a homologue of a nickel transporting protein in E. coli (nikD), has ATP-binding domains. Another component is clearly a cytoplasmic membrane protein. Mutation of these genes in H. pylori also dramatically decreases urease activity (72% reduction) but not activity of other enzymes such as catalase and oxidase. 13 A heat shock protein, HspA, may also playa role in nickel ion management. 14,15 This protein is highly homologous to other GroES proteins but contains a striking histidine-rich motif at its C terminus. The motif is capable of specifically binding nickel ions and could act as a reservoir for the ion. Mutation of hspA is lethal, and its direct contribution to urease activity cannot be assessed in the parent organism. Another histidine-rich protein, Hpn, can also bind nickel and other ions and may contribute to nickel ion management. 12 Mutation of the hpn gene, however, does not reduce urease activity.
Strain Heterogeneity There is clearly tremendous strain-to-strain variability for H. pylori with respect to chromosomal DNA sequence and gene arrangement. Strains cultured from gastric biopsy specimens can, more often than not, be differentiated from other strains by molecular analySiS. I 6-21 If the same gene is analyzed for many isolates, frequent base pair substitution in the DNA sequence can be noted. 21 If the arrangement of genes on the chromosome is analyzed, frequent rearrangements of their order are also observed. 7o-72 In addition, more virulent isolates such as those associated with duodenal ulceration, for example, may carry additional sequences that may be completely absent in strains associated with gastritis alone.7 3 ,74 However, these descriptions of variability or heterogeneity do not indicate that the H. pylori genome is unstable or in constant flux. On the contrary, when strains have been cultured from biopsy specimens of patients over periods of months or years, their DNA sequence of selected genes has been found to remain
H. PYLORI: BACTERIAL FACTORS AND MUCOSAL DISEASE
S25
unchanged. 21 Identical strains, as determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), have also been isolated from several generations of family members.7 5
Diverse RFLP Patterns The first identification of the tremendous diversity among H. pylori strains was the recognition of diverse RFLP patterns of HindIII-digested chromosomal DNA isolated from different strains electrophoresed on an agarose gel. 16 These data suggested that strains isolated from different patients had numerous base pair substitutions or gene arrangements or both. This has been confirmed by a number of investigators using a variety of techniques including PCR-RFLP,17 PCR-DNA sequence typing,21 arbitrarily primed PCR, or random amplified polymorphic DNA 18,76 methods. In each case, these methods were used to identify different strains or confirm identity of strains.
Mosaicism in Virulence Genes Looking more closely at the diversity within specific genes, it was noted that the gene encoding the vacuolating cytotoxin displayed what was described as mosaicism. Although all strains possess the vacA genes, only about half the strains secrete an active cytotoxin. 55 Although the specific reason for this deficiency is not known, genetic differences have been identified between the vacA allele of toxin-secreting strains and vacA allele of toxin-negative strains.7 7 These differences have been localized to two regions: first, in the signal sequence (N-terminal amino acid sequences required for protein secretion by the general secretion pathway) and, second, in the midregion of the gene where considerable heterogeneity exists. Although these differences do not directly explain variation in toxin phenotypes, they do provide a starting point for such investigations.
Pathogenicity Island Diversity of isolates can also be explained by the presence or absence of specific gene sequences. Recent work has identified a region of DNA in the chromosome of H. pylori that is unique to some strains. These "pathogenicity islands," which appear to include the cagA gene, have been characterized by nucleotide sequencing and encode homologues of proteins involved in various secretory pathways including protein and DNA.7 3 One of the predicted gene products has been provisionally termed "Pic" for "promotes induction of cytokines."78
S26
HARRY L. T. MOBLEY
Why to Look Out for CagA: Pathogenicity Islands and Virulence CagA, another intriguing protein that has received considerable attention, is a 128-kilodalton immunodominant outer membrane protein that was originally thought to represent the cytotoxin itself. Indeed, expression of the protein correlates strongly with cytotoxin production. 22 ,23,53,55 It has since been discovered that the gene encoding this protein, cagA (cytotoxin-associated gene), is distinct and maps at some distance (300 kilobases 72 ) from the vacA cytotoxin gene. Nevertheless, the presence of the cagA gene, and expression of the CagA protein, is present significantly more often in strains isolated from patients with more serious clinical manifestations including duodenal and gastric ulcer than from strains isolated from patients with gastritis alone. 44 .49 For example, in a study of patients with chronic gastritis, the presence of anti-CagA antibodies was measuredJ9 In patients with no ulcers, 63% (32 of 51 patients) were CagA positive. In patients with peptic ulcers, 100% (25 of 25 patients) were CagA positive. In another study, when the presence of the cagA gene in the infecting H. pylori strain was examined, similar results were found. Patients with any gastrointestinal disease (presumably including peptic ulcer) were 75% cagA positive. In patients with duodenal ulcer, 100% of the strains were cagA positive. 22 The cagA gene and CagA protein thus serve as genotypic and phenotypic markers for virulent strains. There are well-defined pathogenicity islands (contiguous stretches of chromosomal DNA) present in some strains of H. pylori that encode CagA and additional proteins that may potentiate virulence. Strains possessing these "virulence cassettes" are isolated more frequently from patients with the more serious clinical manifestations associated with duodenal ulcer than from patients with gastritis alone or nonulcer dyspepsia.
Is There a Gradient of Virulence Among Strains? Because of the tremendous diversity in H. pylori, it not surprising that assignment of virulence is not as clear-cut as identifying the presence or absence of the cagA-containing pathogenicity island. Strains have been identified that run the spectrum from having the "complete" pathogenicity island to those that lack these sequences compietelyJ3 In between, strains may have these pathogenicity island sequences, which are split and located at different points in the genome or may contain only portions of the complete island. Therefore, there may exist a gradient of virulence among isolates. If one IS
GASTROENTEROLOGY Vol. 113, No.6
superimposes this continuum of virulence upon the predisposition of certain human hosts for acquiring infection, then it is not surprising that there are so many degrees of clinical manifestations of H. pylori infections that include gastritis alone, gastric ulcer, duodenal ulcer, and gastric malignancy.
Conclusion H. pylori can be considered a true pathogen. Although all strains seem to be capable of causing gastritis in humans and are thus virulent, a certain subset of strains possesses additional genes encoded by pathogenicity islands, which render them more virulent. The proteins encoded by these extra genes appear to encode homologues of proteins involved in protein secretion and thus may cause ulcer development via secreted products. These secreted products probably interact directly with the epithelium to trigger cascades of events that lead irreversibly to epithelial cell damage. Alternatively, these secreted proteins could interact with immune cells that in turn liberate cytokines causing the observed effect indirectly.
References 1. Finally BB, Falkow S. Common themes in microbial pathogenic· ity. Microbiol Rev 1989;53:210-230. 2. Mobley HLT, Island MD, Hausinger RP. Molecular biology of microbial ureases. Microbiol Rev 1995;59:451-480. 3. Eaton KA, Brooks CL, Morgan DR, Krakowka S. Essential role of urease in pathogenesis of gastritis induced by Helicobacter pylori in gnotobiotic piglets. Infect Immun 1991;59:24702475. 4. Eaton KA, Krakowka S. Effect of gastriC pH on ureasedependent colonization of gnotobiotic piglets by Helicobacter pylori. Infect Immun 1994;62:3604-3607. 5. Tsuda M, Karita M, Morshed MG, Okita K, Nakasaki T. A urease-negative mutant of Helicobacter pylori constructed by allelic exchange mutagenesis lacks the ability to colonize the nude mouse stomach. Infect Immun 1994;62:3586-3589. 6. Takahashi S, Igarashi H, Nakamura K, Masubuchi N, Saitos S, Aoyagi T, Itoh T, Hirata I. Helicobacter pylori urease activitycomparative study between urease positive and urease negative strain. Jpn J Clin Med 1993;51:3149-3153. 7. Hu L-T, Foxall PA, Russell R, Mobley HLT. Purification of recombinant Helicobacter pylori urease apoenzyme encoded by ureA and ureB. Infect Immun 1992;60:2657-2666. 8. Mobley HLT, Garner RE, Bauerfeind P. Helicobacter pylori nickel transport gene nixA: synthesis of catalytically active urease in E. coli independent of growth conditions. Mol MicrobioI1995;16: 97-109. 9. Bauerfeind P, Garner RM, Mobley HLT. Allelic exchange mutagenesis of nixA in Helicobacter pylori results in reduced nickel transport and urease activity. Infect Immun 1996;64:28772880. 10. Ge Z, Hiratsuka K, Taylor DE. Nucleotide sequence and mutational analysis indicate the two Helicobacter pylori genes encode a P-type ATPase and a cation binding protein associated with copper transport. Mol MicrobioI1995;15:97-106. 11. Melchers K, Weitzenegger T, Buhmann A, Steinhilber W, Sachs G, Schafer KP. Cloning and membrane topology of a P type
December Supplement 1997
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
ATPase from Helicobacter pylori. J Bioi Chem 1996;271:446457. Gilbert JV, Ramakrishna J, Sunderman FIN, Wright A, Plaut AG. Protein Hpn: cloning and characterization of a histidine-rich metal-binding polypeptide in Helicobacter pylori and Helicobacter mustelae. Infect Immun 1995;63:2682-2688. Hendricks JK, Mobley HLT. Helicobacter pylori ABC transporter: effect of allelic exchange mutagenesis on urease activity. J Bacteriol (in press). Suerbaum S, Thiberge J-M, Kansau I, Ferrero RL, Labigne A. Helicobacter pylori hspA-hspB heat-shock gene cluster: nucleotide sequence, expression, putative function and immunogenicity. Mol MicrobioI1994;14:959-974. Kansau I, Guillain F, Thiberge J-M, Labigne A. Nickel binding and immunological properties of the C-terminal domain of the Helicobacter pylori GroES homologue (HspA). Mol Microbiol 1996;22:1013-1023. Langenberg W, Rauws EAJ, Widjojokusumo A, Tytgat GN, Zanen HC. Identification of Campylobacter pyloridis isolates by restriction endonuclease DNA analysis. J Clin MicrobioI1986;24:414417. Foxall PA, Hu L-T, Mobley HLT. Use of polymerase chain reaction: amplified Helicobacter pylori urease structural genes for differentiation of isolates. J Clin MicrobioI1992;30:739-741. Akopanz N, Bukanov NO, Westblom TU, Kresovich S, Berg DE. DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Res 1992;20:5137-5142. Evans DG, Evans DJ Jr, Lampert HC, Graham DY. Restriction fragment length polymorphism in the adhesin gene hpaA of Helicobacter pylori. Am J GastroenteroI1995;90:1282-1288. Clayton CL, Kleanthous H, Morgan DD, Puckey L, Tabaqchali S. Rapid fingerprinting of Helicobacter pylori by polymerase chain reaction and restriction fragment length polymorphism analysis. J Clin MicrobioI1993;31:1420-1425. Labigne A. PCR-DNA sequence typing. In: Clayton CL, Mobley HlT, eds. Helicobacter pylori protocols. Totowa, New Jersey: Humana, 1997:107-116. Covacci A, Censini S, Bugnoli M, Petracca R, Burroni D, Macchia G, Massone A, Papini E, XiangZ, Figura N, Rappoali R. Molecular characterization of the 128 kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer. Proc Natl Acad Sci USA 1993;90:5791-5795. Crabtree JE, Figura N, Taylor JD, Bugnoli M, Armellini D, Tompkins DS. Expression of 120 kilodalton protein and cytotoxicity in Helicobacter pylori. J Clin PathoI1992;45:733-734. Neale KR, Logan RPH. The epidemiology and transmission of Helicobacter pylori infection in children. Aliment Pharmacol Ther 1995;9(Suppl 2):77-84. Mobley HLT, Cortesia MJ, Rosenthal LE, Jones BD. Characterization of urease from Campylobacter pylori. J Clin Microbiol 1988;26:831-836. Hu L-T, Mobley HLT. Purification and N-terminal analysis of urease from Helicobacter pylori. Infect Immun 1990;58:992998. Hawtin PR, Delves HT, Newell DG. The demonstration of nickel in the urease of Helicobacter pylori by atomic absorption spectroscopy. FEMS Microbiol Lett 1991;77:51-54. Dunn BE, Campbell GP, Perez-Perez GI, Blaser MJ. Purification and characterization of urease from Helicobacter pylori. J Bioi Chem 1990;265:9464-9469. Evans DJ Jr, Evans DG, Kirkpatrick SS, Graham DS. Characterization of the Helicobacter pylori urease and purification of its subunits. Microb Pathog 1991;10:15-26. Bauerfeind P, Garner R, Dunn BE, Mobley HLT. Synthesis and activity of Helicobacter pylori urease and catalase at low pH. Gut 1997 (in press).
H. PYLORI: BACTERIAL FACTORS AND MUCOSAL DISEASE
S27
31. Ferrero RL, Cussac V, Courcoux P, Labigne A. Construction of isogenic urease-negative mutants of Helicobacter pylori by allelic exchange. J Bacteriol1992;17 4:4212-4217. 32. Jackson CJ, Kelly DJ, Clayton CL. The cloning and characterization of chemotaxis genes in Helicobacter pylori. Abstracts, Vilith International Workshop on Campylobacters, Helicobacters, and Related Organisms. Winchester, England; 1995:63. 33. Eaton KA, Morgan DR, Krakowka S. Motility as a factor in the colonization of gnotobiotic piglets by Helicobacter pylori. J Med MicrobioI1992;37:123-127. 34. Eaton KA, Suerbaum S, Josenhans C, Krakowka S. Colonization of gnotobiotic piglets by Helicobacter pylori deficient in two flagellin genes. Infect Immun 1996;64:2445-2448. 35. Smoot DT, Resau JH, Naab T, Desbordes BC, Gilliam T, BullHenry K, Curry SB, Nidiry J, Sewchand J, Mills-Robertson K, Frontin K, Abebe E, Dillon M, Chippendale GR, Phelps PC, Scott VF, Mobley HLT. Adherence of Helicobacter pylori to cultured human gastric epithelial cells. Infect Immun 1993;61:350355. 36. Segal ED, Falkow S, Tompkins LS. Helicobacter pylori attachment to gastric cells induces cytoskeletal rearrangements and tyrosine phosphorylation of host cell protein. Proc Natl Acad Sci USA 1996;93:1259-1264. 37. Steer HW. Ultrastructure of Campylobacter pylori in vivo. In: Rathbone BJ, Heatly R, eds. Campylobacter pylori and gastroduodenal disease. Oxford, England: Blackwell Scientific, 1989: 189. 38. Walker SJ, Birch PJ, Stewart M, Stoddard CJ, Hart CA, Day DW. Patterns of colonization of Campylobacter pylori in the esophagus, stomach and duodenum. Gut 1989;30:1334-1338. 39. Logan RPH. Adherence of Helicobacter pylori. Aliment Pharmacol Ther 1996;10(Suppll):3-15. 40. Boren T, Falk P, Roth KA, Larson G, Normark S. Attachment of Helicobacter pylori to human gastric epithelium mediated by blood group antigens. SCience 1993;262:1892-1895. 41. Odenbreit S, Till M, Haas R. Identification of a H. pylori-specific protein involved in adherence to gastric epithelial cells. Abstracts, VI 11th International Workshop on Gastroduodenal Pathology and Helicobacter pylori. Edinburgh, Scotland, 1995. 42. Cave DR, Vargas M. Effect of a Campylobacter pylori protein on acid secretion by parietal cells. Lancet 1989;2:187-189. 43. Ferrero RL, Thiberge J-M, Kansau I, Wuscher N, Huerre M, Labigne A. The GroES homologue of Helicobacter pylori confers protective immunity against mucosal infection in mice. Proc Natl Acad Sci USA 1995;92:6499-6503. 44. Phadnis SH, Dunn BE. Surface localization of H. pylori urease and HP54K requires bacterial lysis. Abstracts, Vilith International Workshop on Gastroduodenal Pathology and Helicobacter pylori. Edinburgh, Scotland; 1995. 45. Hazell SL, Mendz GL. The metabolism and enzymes of Helicobacter pylori: function and potential virulence effects. In: Goodwin CS, Worsley BW, eds. Helicobacter pylori: biology and clinical practice. Boca Raton, FL: CRC, 1993:115-141. 46. Odenbreit S, Wieland B, Haas R. Cloning and genetic characterization of Helicobacter pylori catalase and construction of a catalase-deficient mutant strain. J Bacteriol 1996;178:69606967. 47. Muotiala A, Helander 1M, Pyhala L, Kosunen TV, Moran AP. Low biological activity of Helicobacter pylori lipopolysaccharide. Infect Immun 1992;60:1714-1716. 48. Moran AP, Prendergast MM, Appelmelk BJ. Molecular mimicry of host structures by bacterial lipopolysaccharides and its contribution to disease. FEMS Immunol Med MicrobioI1996;16: 105-115. 49. Aspinall GO, Monteiro MA, Pang H, Walsh EJ, Moran AP. Lipopolysaccharide of the Helicobacter pylori type strain NCTC
S28
50.
51.
52. 53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
HARRY L. T. MOBLEY
11637 (ATCC43504): structure of the 0 antigen chain and core oligosaccharide regions. Biochemistry 1996;35:2489-2497. Windle HJ, Kelleher D. Identification and characterization of a metallo-protease activity from H. pylori which is capable of degrading immunoglobulins. Abstracts, Vilith International Workshop on Gastroduodenal Pathology and Helieobaeter pylori. Edinburgh, Scotland; 1995. Suzuki M, Miura S, Soematsu M, Fukumura D, Kurose I, Suzuki H, Kai A, Kudoh Y, Ohashi M, Tanchiya M. Helieobaeter pyloriassociated ammonia production enhances neutrophil-dependent gastric mucosal cell injury. Am J PhysioI1992;263:G719G725. Hazell SL, Lee A. Campylobaeter pyloridis, urease, hydrogen ion back diffusion, and gastric ulcers. Lancet 1986;2:15-17. Tummuru MK, Cover TL, Blaser MJ. Cloning and expression of a high-molecular-mass major antigen of Helieobaeter pylori: evidence of linkage to cytotOXin production. Infect Immun 1993;61: 1799-1809. Lupetti P, Heuser JE, Manetti R, Massari P, Lanzavecchia S, Bellon PL, Dallai R, Rappuoli R, Telford JL. Oligomeric and subunit structure of the Helicobaeter pylori vacuolating cytotoxin. J Cell Bioi 1996;133:801-807 . Cover TL, Dooley CP, Blaser MJ. Characterization of and human serologic response to proteins in Helicobacter pylori broth culture supernatants with vacuolizing cytotOXin activity. Infect Immun 1990;58:603-610. Young GO, Lastovica AJ, Brown S, McGovern JJ, Moran AP. Effect on pepsinogen release of various sub-fractions of H. pylori lipopolysaccharide. Abstracts, VI 11th International Workshop on Campylobaeters, Helieobaeters, and Related Organisms. Winchester, England, 1995:65. Wetherall BL, McDonald PJ, Johnson AM. Partial characterization of a cell free hemolytic factor produced by Helieobaeter pylori. FEMS MicrobiollmmunoI1992;4:123-128. Lichtenberger LM, Hazell SL, Romero JJ, Graham DY. Helieobaeter pylori hydrolysis of artificial phospholipid monolayers: insight into a potential mechanism of mucosal injury (abstr). Gastroenterology 1990;98:A78. Suzuki M, Miura S, Soematsu M, Fukumura D, Kurose I, Suzuki H, Kai A, Kudoh y, Ohashi M, Tanchiya M. Helicobaeter pyloriassociated ammonia production enhances neutrophil-dependent gastric mucosal cell injury. Am J PhysioI1992;263:G719G725. Mai UEH, Perez-Perez GI, Wahl LM, Wahl SM, Blaser MJ, Smith PD. Soluble surface proteins from Helicobacter pylori activate monocytes/macrophages by lipopolysaccharide-independent mechanism. J Clin Invest 1991;87:894-900. Nielsen P, Anderson LP. Activation of human phagocyte oxidative metabolism by Helieobaeter pylori. Gastroenterology 1992; 103:1747-1753. Craig PM, Territo MC, Karnes WE, Walsh JH. Helieobaeter pylori secretes a chemotactic factor for monocytes and neutrophils. Gut 1992;33:1020-1023. Mai UEH, Perez-Perez GI, Allen JB, Wahl SM, Blaser MJ, Smith PD. Surface proteins from Helicobacter pylori exhibit chemotactic activity for human leukocytes and are present in gastric mucosa. J Exp Med 1992;175:517-525. Mai UEH, Perez-Perez GI, Wahl LM, Wahl SM, Blaser MJ, Smith PD. Soluble surface proteins from Helieobaeter pylori activate monocytes/macrophages by lipopolysaccharide-independent mechanism. J Clin Invest 1991;87:894-900.
GASTROENTEROLOGY Vol. 113, No.6
65. Falkow S. Molecular Koch's postulates applied to microbial pathogeniCity. Rev Infect Dis 1988;10(SuppI2):S274-S276. 66. Langenberg ML, Tytgat GNJ, Schipper MEL Campylobacter-like organisms in the stomach of patients and healthy individuals. Lancet 1984;1:1348. 67. Dent JG, McNulty CAM, Uff JS, Gear MWL, Wilkinson SP. Campylobaeter pylori urease: a new serological test. Lancet 1988;1:1002. 68. Jabri E, Carr MB, Hausinger RP, Karplus PA. The crystal structure of urease from Klebsiella aerogenes. Science 1995; 268:998-1004. 69. Cussac V, Ferrero RL, Labigne A. Expression of Helicobacter pylori urease genes in Escherichia coli grown under nitrogenlimiting conditions. J Bacteriol1992;17 4:2466-24 73. 70. Taylor DE, Eaton M, Chang N, Salama S. Construction of a Helieobacter pylori genome map and demonstration of diversity at the genome level. J Bacteriol1992;17 4:2332-2337. 71. Jiang Q, Hiratsuka K, Taylor DE. Variability of gene order in different Helieobacter pylori strains contributes to genome diversity. Mol MicrobioI1996;20:833-842. 72. Bukanov NO, Berg DE. Ordered cosmid library and high resolution physical genetic map of Helicobacter pylori strain NCTC 11638. Mol MicrobioI1994;11:509-523. 73. Censini S, Lange C, Xiang Z, Crabtree JE, Ghiara P, Borodovsky M, Rappuoli R, Covacci A. eag, a pathogeniCity island of Helieobaeter pylori, encodes type I-specific and diseaseassociated virulence factors. Proc Natl Acad Sci USA 1996;93: 14648-14653. 74. Akopyants NS, Kersulyte D, Berg DE. CAGII, a new multigene locus associated with virulence in H. pylori. Abstracts, Vilith International Workshop on Gastroduodenal Pathology and Helicobacter pylori. Edinburgh, Scotland, 1995. 75. Labigne A. PCR-DNA sequence typing. In: Clayton CL, Mobley HLT, eds. Helicobaeter pylori protocols. Totowa, NJ: Humana, 1997:107-116. 76. Berg DE, Lelwala-Guruge J, Incecik ET, Srivastava K, Akopyants NS. H. pylori DNA fingerprinting using the arbitrarily primed PCR (AP-PCR) or random amplified polymorphic DNA (RAPD) method. In: Clayton CL, Mobley HLT, eds. Helieobaeter pylori protocols. Totowa, New Jersey: Humana, 1997:117-132. 77. Atherton JC, Cao P, Peek RM Jr, Tummuru MK, Blaser MJ, Cover TL. MosaiCism in vacuolating cytotOXin alleles of Helieobacter pylori. Association of specific vacA types with cytotoxin production and peptiC ulceration. J Bioi Chem 1995;270:1777117777. 78. Tummuru MKR, Sharma SA, Blaser MJ. Helieobaeter pylori pieB, a homologue of the Bordetella pertussis toxin secretion protein, is required for induction of IL-8 in gastric epithelial cells. Mol MicrobioI1995;18:867-876. 79. Crabtree JE, Taylor JD, Wyatt JI, Heatley RV, Shallcross TM, Tompkins DS, Rathbone BJ. Mucosal IgA recognition of Helicobacter pylori 120 kDa protein, peptic ulceration and gastric pathology. Lancet 1991;338:332-335. Received June 30,1997. Accepted July 22,1997. Address requests for reprints to: Harry L. T. Mobley, Ph.D, Department of Microbiology and Immunology, University of Maryland School of Medicine, 10 South Pine Street, Room 9-00, Baltimore, Maryland 21201. Fax: (410) 706-6751. Supported by Public Health Service Grant AI25567 from the National Institutes of Health.