Helicobacter pylori: 20 years later
By: Kushen Ramessur
University of Westminster
London W1 W6UW.
Helicobacter pylori (H.pylori) are a `slow' bacterial pathogen and the name comes from Latin meaning `spiral rod of the lower part of the stomach' (Goldwin et al., 1989). It was first isolated in 1983 in Australia by Warren and Marshall and was found to be present in patients suffering from type B gastritis (Allen, 2000). In 1989, the human pathogen formerly known as Campylobacter pylori was transferred to a new genus, Helicobacter pylori (Goldwin et al., 1989). Since its discovery the diagnosis and treatment of upper gastro duodenal disease has dramatically changed. The peptic ulcers are now considered as infectious disease and the role of H.pylori in gastric cancers and other diseases of the upper gastrointestinal tract is being recognised or is being actively evaluated (Suerbaum & Michetti, 2002).
H.pylori is a small microaerophilic, non-sporing, gram-negative bacteria. It is like curved rods, 3.5 ¼m long and 0.5- 1 ¼m wide, with multiple unipolar-sheathed flagella. It has a spiral shape in young cultures and can assume a coccoid form in older cultures (Baron, 1991). The H.pylori genome has 1.65 million base pairs (bp) and codes for about 1500 proteins (Tomb et al., 1997). In 1992, Taylor et al. showed that there is a genetic diversity among H.pylori strains. It has also been demonstrated that the severity of the disease is associated with a subset of the strain. The more severe pathology is caused by H.pylori strains, type I, which contains a 40 kilobase pairs of alien DNA called Cag pathogenicity island (PAI) (Suerbaum & Michetti, 2002). The Cag PAI includes a gene Cag A that encodes for an approximately 120 KDa immunodominant surface antigen. These strains produce a potent toxin, vacuolating cytotoxin A (Vac A) which is believed to cause tissue erosion and induce the production of a pro-inflammatory cytokine, Interleukin 8 (IL-8). The latter is responsible for the observed inflammation in the gastric mucosa (Segal et al., 1997). H.pylori, type II, lack the expression of the cag pathogenicity island and they do not produce the functional Vac A toxin. They cause mild gastritis in the model mouse model (Marhetti et al., 1995). The genome of H.pylori changes continuously during chronic colonisation by importing small pieces of DNA from other H.pylori strains (Falush et al., 2002).
Epidemiology and transmission:
Infection of H.pylori is worldwide but its prevalence in a particular region depends on the socio-economic conditions prevailing there (Malaty & Graham, 1994). In the developing countries H.pylori colonizes 70-90 % of the population before the age of 10 as compared to about 50 % in the developed world. But only 10-20 % of the infected individuals will develop H.pylori (Telford et al., 1997). It is most probably transmitted by the faecal-oral route but may be transmitted in vomits and saliva (Parsonnet et al., 1999). The severe pathology appears only years later (Murray et al., 2002). There is currently no evidence of zoonotic transmission even if H.pylori is found in non-human primates and occasionally other animals. Development of the symptoms as a result of an infection with H.pylori occurs after a long period of infection. During this time the host immune system mounts a vigorous immunological response. However, the response may also contribute to the severity of the disease. The most common pathology associated with H.pylori infection is chronic active gastritis and peptic ulceration. However, long-term chronic infection can give rise to gastric adenocarcinoma and gastric mucosa- associated lymphoma type (MALT) B-cell lymphoma ( Murray et al., 2002).
Pathogenesis and Immunity:
The mechanism by which H.pylori causes gastric damage and inflammation is summarised below.
1. After being ingested by the host, H.pylori produce an abundant amount of urease in the gastric lumen and the latter activity is regulated by a unique PH-gated urea channel Ure I (Week et al., 2000). The urease hydrolyses urea into carbon dioxide and ammonia. This allows the pathogen to survive in the acidic environment of the gastric lumen (Suerbaum & Michetti, 2002). The lipopolysaccharide of H.pylori (V-shaped on fig. 3) contains the human blood group antigens Lex and Ley and hence, the bacteria can exert antigen mimicry to evade the host immune response. This can lead to the production of antibodies against the gastric epithelium. The flagella make H.pylori very mobile and allow it to swim in the gastric lumen and enter the mucous layer. H.pylori then bind to the epithelial cell by multiple bacterial surface components like Bab A (Ilver et al., 1998).
2. Upon contact, the type I bacteria, containing PAI, induce the epithelium to synthesize and release IL-8. The later attracts polymorphonuclear leukocytes (PMN's) by chemotaxis. There is also release of virulence factors (refer below) that can cause epithelial damage and apoptosis of epithelial cells. Vac A, in type I strains only, is the major factor that can give rise to peptic ulcer (Allen, 2000).
3. H.pylori antigens also, cross the epithelial layer to activate macrophages to release several pro-inflammatory cytokines like IL-8, IL-6, IL-1, and possible IL-12 (Suerbaum & Michetti, 2002).
4. The IL-12 cytokine and H.pylori antigens polarise the CD4+ T helper cells (Th) into a prominent Th1 phenotype. There is release of pro-inflammatory cytokines like tissue necrosing factor ± (TNF-±) and interferon ³ (IFN-³) at the site of the infection. This contribute to maintain the gastritis. IFN-³ induces expression of class II MHC and accessory molecules B7-1 and B7-2 by epithelial cells making them competent for antigen presentation (Suerbaum & Michetti, 2002). TNF-± induces a decrease in the number of astral D cells leading to decreased somatostatin production and indirectly enhancing acid production (Suerbaum & Michetti, 2002).
The following points should also worth to be noted. Multiple virulence factors contribute to gastric inflammation, alteration of gastric acid production and tissue destruction in the infected host. They also facilitate initial colonisation. Murray et al. (2002) give a good summary of these virulence factors below.
Urease: Neutralises gastric juice; stimulates monocytes and neutrophils chemotaxis; stimulates production of inflammatory cytokines.
Heat shock protein (HspB): Enhances expression of urease
Acid-inhibitory protein: Induces hyperchlorhydria during acute infection by blocking acid secretion from parietal cells.
Flagella: Allow penetration into gastric mucous layer and protection from acid environment.
Adhesins: Mediate binding to host cells; examples of adhesions are haem agglutinins, sialic acid-binding adhesin, and Lewis blood group adhesin.
Mucinase: Disrupts gastric mucus
Phospholipases: Disrupt gastric mucus
Superoxide dismutase: Prevents phagocytic killing by neutralising oxygen metabolites
Catalase: Prevents phagocytic killing by neutralising peroxides
Vacuolating cytotoxin: Induces vacuolation in epithelial cells, stimulates neutrophil migration into mucosa.
Poorly defined factors: H.pylori
1. Stimulates interleukin-8 secretion by gastric epithelial cells, which recruits and activates neutrophils
2. Stimulates gastric mucosal cells to produce platelet-activating factor (PAF), which stimulates gastric acid secretion.
3. Induces nitric oxide synthase in gastric epithelial cells, which mediates tissue injury
4. Induces death of gastric epithelial cells
A characteristic feature of H.pylori-induced inflammation is also, the massive attraction of phagocytes (particularly neutrophils) to the site of infection. This can be achieved as H.pylori has a nap A gene, which codes for the production of H.pylori neutrophil-activating protein (Hp-NAP). The latter was found to promote the adhesion of neutrophils to endothelial cells by up regulating adhesion receptors of the ²2-integrin family (Satin et al., 2000). Satin et al. (2000) showed that Hp-NAP stimulates nicotiamide adenine dinucleotide phosphate in the reduced form (NADPH) oxidase assembly and production of reactive oxygen species (ROS). Also, as neutrophils consistently outnumber macrophages in H.pylori infected stomach it induces a state of `chronic acute inflammation' (Satin et al., 2000). The MHC - class II on the human gastric epithelial cells act as receptors for H.pylori (Fan et al., 2000) and the latter induces apoptosis of the epithelial cells (Fan et al., 2000).
H.pylori has been a highly successful human pathogen and it can remain in the gastric mucosa for year's inspite of the host immune response. The following mechanisms have been postulated to explain how H.pylori evades phagocytosis (Andersen et al., 1993).
1. Some unidentified proteins on the surface of some strains of H.pylori help them to regulate their uptake into the phagocytes.
2. Strong binding between H.pylori and phagocytes correlates with a rise in the level of urease on the surface of H.pylori thus retarding phagocytosis and strong respiratory burst in the phagocytes (Telford et al., 1997).
3. Urease prevents opsonisation with complement C3 and antibodies. It also, retards phagocytic process via FcR3 (Fc receptors) and CR3 (Rokita et al., 1998).
4. Opsonins reduce the amount of ROS produced by activating strains of H.pylori (Ratelin et al., 1994).
5. Delayed phagocytosis is linked to intracellular survival, since type I H.pylori persist inside macrophages within a novel vacuole called megasome (Allen et al., 2000).
H.pylori also uses the “ molecular mimicry” phenomenon refers, Fig3, to evade the host immune response as H.pylori displays chemical components like Lewis antigens on its surfaces. This mimicry disguise the H.pylori from the immune system and the immune response is minimised (Lynch, 2002).
Clinical outcomes of Helicobacter pylori:
H.pylori cause continuous gastric inflammation in virtually all infected persons. The clinical course is highly variable and is influenced by both microbial and host factors. Patients with antral-predominant gastritis, the most common cause of gastritis, are predisposed to duodenal ulcer. Patients with lower acid output are more likely to have gastritis in the body of the stomach. Whereas patients with corpus predominant gastritis and multifocal atrophy are more likely to have gastric ulcers, gastric atrophy, intestinal metaplasia, and ultimately gastric carcinoma (Suerbaum & Michetti, 2002). The lifetime risk of peptic ulcer in persons infected with H.pylori ranges from 3% in the U.S.A. to 25 % in Japan (Schlemper et al., 1996).
Gastric cancer is the second most frequent cause of cancer related death. There is strong evidence the H.pylori increases the risk of gastric cancer. H.pylori is also, classified as a type I carcinogen since 1994 by the W.H.O. (Lynch, 2002). 72-98 % of patient with gastric MALT lymphoma are infected with H.pylori (Parsonnet et al., 1994). Eradication of H.pylori induces regression of the lymphoma in 70 to 80 % of cases and patients can stay in remission for years (Lin et al., 2001). Resistance of lymphoma eradication therapy is strongly associated with certain genetic abnormalities in the host like, translocation t (11; 18) (q21; q21) and is often associated with progression to high-grade tumours (Lin et al., 2001). H.pylori may also be implicated in the pathogenesis of many extra gastric diseases like atherosclerosis and skin disease but this association is still controversial (Howden, 1998). However, it has also been found that the presence of H.pylori offer protection against certain gastro oesophageal reflux disease, adenocarcinoma of lower oesophagus and gastric cardia (Murray et al., 2002).
H.pylori can be diagnosed by either non-invasive methods or by endoscopic biopsy of the mucosa. The non-invasive methods are
1. Urea breath test
2. Serologic tests
3. Stool antigen assay
4. Microbiological culture
5. Microscopy of histology specimens from biopsy
6. Antibody testing using ELISA method
The Urea breath test is highly specific and sensitive. The patient usually swallows a small amount of labelled urea. The urease present on the pathogen surface converts urea into bicarbonate, which is expired as carbon dioxide. The labelled carbon dioxide is estimated by the change in colour of the chromogen used to identify it (Goodwin et al., 1997).
The microbiological culture is expensive but is useful to know the antibiotic resistance pattern of the bacteria and is useful in treatment, especially after proven treatment failure (Goodwin et al., 1997).
Serum antibody testing of IgG antibody by the enzyme-linked immunosorbent assay (ELISA) is 95 % sensitive and specific with current reagents used. The principle of the test is as follows:
Partially purified, inactivated H.pylori antigens are pre-coated onto an ELISA plate and patient serum contains antibodies against H.pylori, if they are infected. The antibody will bind to the antigen on the plate. After washing anti-human immunoglobulin coupled with an enzyme is added. The later will bind to the antibodies present. After washing a substrate is added. The enzyme will cleave the substrate and a coloured product is formed. The presence of the coloured product is indicative of a positive reaction (Hanningen, 2000).
The aim of treatment is to have a complete elimination of the organism. For the treatment to be effective it must have a curing rate of at least 80% without major side effects and minimal bacterial resistance (Seurbaum & michetti, 2002). As this cannot be achieved with antibiotics alone the food and drug administration (FDA) in the USA approved the triple-therapies (Suerbaum & Michetti, 2002). The administration of the antibiotic was coupled with bismuth and a proton-pump inhibitor like, omeprazole or metronidazole. The chief antimicrobial agents used are clarithromycin and tetracycline (Goodwin et al., 1997). Studies in France and Italy have shown that this may have a success rate of 78 to 83% ( Suerbaum & Michetti, 2002).
If there is failure of the above therapy a second-line therapy is used. It is recommended to repeat a second course of the above treatment using this time amoxycillin and pantoprozole for 10 days. This has achieved 86% cure success, even in patients with resistant strains (Perri et al., 2001).
Prophylactic and therapeutic vaccination has been successful in animals' models but a vaccine for humans is still not possible because the immunology of the stomach is still poorly understood (Marchetti et al., 1999) and the genome of H.pylori changes continuously during chronic colonisation by importing small pieces of DNA from other H.pylori strains (Falush et al., 2002).
The way diagnosis of the upper gastroduodenal disease and its treatment is approached has changed dramatically since 1983. Peptic ulcer is now approached as an infectious disease, in which the elimination of the causative agent cures the condition. The role of H.pylori infection in gastric cancers is increasingly recognised. Enormous progress has been made in the antimicrobial therapy but there is still no ideal treatment. Also, new association of H.pylori with diminished growth in childhood and coronary heart disease is still been looked into (Goodwin et al. 1997). Vaccine trials have been up to now successful only in animals' models. So, since it was first cultured some 20 years ago H.pylori still remains as one of the most common bacterial infections in the human.
Allen L.A.H. (2000). Modulating phagocyte activation: the pros and cons of H.pylori virulence factors. Journal of experimental medicine; 91 (9): 1451-1454
Allen L.A.H., Schlesinger L.S., Kang B. (2000). Virulent strains of H.pylori demonstrate delayed phagocytosis and stimulate homotypic phagosome fusion in macrophages. Journal of experimental medecine. 191: 115-127
Andersen C.P., Blom J., Nielsen H. (1993). Survival and ultra structural changes of H.pylori after phagocytosis by human polymorphonuclear phagocytes and monocytes. APMIS; 101: 61-72
Covacci A., Telford J.L., Del Giudice G, Parsonnet J., Rappuoli R. (1999). Helicobacter pylori virulence and genetic geography. Science; 274: 1382-1333
Falush D., Kraft C., Taylor N.S. et al. Recombination and mutation during long-term gastric colonisation by Helicobacter pylori: estimates of clockrates, recombination size, and minimal age. Proc. Natl. Acad. Asc. U.S.A.; 98: 15056-15061
Goodwin C.S., Armstrong J.A., Chilvers T., Peters M., Collins M.D., Sly L., Mc Connell W., Harper W.E.S. (1999). Transfer of Campylobacter pylori gen. Nov. as Helicobacter pylori comb.nov. and Helicobacter mustalae comb. Nov. respectively. Int. J. Syst. Bactriol. 39: 397-405
Goodwin C.S., Mendall M.M., Northford T.C. (1997). H.pylori infection. Lancet; 349: 265-269.
Handt L.K., Fox J.G., Dewhirst F.C. et al. (1994). Helicobacter pylori isolated from the domestic cat: public health implications. Infect. Immun.; 62: 2367-2374
Howden C.W., Hunt R.M. (1998). Guidelines in the management of Helicobacter pylori infection. Am. J. gastroenterol; 93: 2330-2338
Hanningen B. (2000): Biomedical Science Explained-Immunology (Ed. Pallister C.J.) Oxford Univ. Press. New York.
Ilver D., Arnqnist A., Ogren J. et al. (1998). Helicobacter pylori adhesion binding fucocylated histo-blood group antigens revealed by retagging. Science; 279: 373-377
Liu H., Ruskon-fourmestaux A., Luvergne-slove A. et al. (2001). Resistance of t(11:18) positive gastric mucosa-associated lymphoid tissue lymphoma to helicobacter pylori eradication therapy; 357: 37-40
Lynch N.A. (2002). Helicobacter Pylori and ulcers: a paradigm revised. Http://www.faseb.org/oper/pylori/pylori.html
Malaty H.M., Graham D.Y. (1994): Importance of childhood socio-economic status in the current prevalence of Helicobacter pylori infection. Gut; 35: 742-745
Marchetti M., Arico B., Burroni D., Figura N., Rappuoli R., Ghiara P. (1995). development of a mouse model of Helicobacter Pylori infection that mimics human disease. Science; 267: 1655-1658
Michetti P., Kreiss C., Kotloff K.L. et al. (1999). Oral immunisation with urease and Escherichia coli heat labile enterotoxin in safe and immunogenic in Helicobacter Pylori infected adults. Gastroenterology; 116: 804-812
Murray P., Rosenthal K.S., Kobayashi G.S., Pfaller M.A. (2002). Medical Microbiology, 4th Ed., Mosby Inc, Missoori, U.S.A.
Parsonnet J., Friedman G.D., Vandersteen D.P. et al. Helicobacter Pylori infection and the risk of gastric carcinoma. New England Journal of Medecine; 325: 1127-1131
Parsonnet J., Hansen S., Rodriguez L. et al. Helicobacter Pylori infection and gastric lymphoma. New England Journal of Medecine; 330: 1267-1271
Perri F., Festa V., Clemente R., et al. (2001). Randomised study of two “rescue” therapies for Helicobacter Pylori-infected patients after failure of standard triple therapies. Am. Journal Gastroenterology; 96: 58-62
Rautelin H., Blomberg G., Jarnerot G., DanielsonD. (1994). Nonopsonic activation of neutrophils and cytotoxin production by Helicobacter Pylori: ulcerogenic markers. Scand. Journal Gastroenterology; 29: 128-132
Rokita E., Malkristathis A., Presterl E., Rotter M.L., Hirschl A.M. (1998). Helicobacter Pylori urease significantly reduces opsonisation by human-complement. Journal of infectious disease; 178: 1521-1525
Satin B., Del Giudice G., Della Bianca V., Dusi S., laudina C., Tonello F., kellcher D., Rappuolli R., Montecullo C., Rossi F. (2000). The neutrophil-activating protein (Hp-Nap) of Helicobacter Pylori is a protective antigen and a major virulence factor. Journal Exp. Med.; 191: 1467-1476
Schlemper R.J., Van Der Werf S.D., Biemond I. (1996). Seroepidemiology of gastritis in Japan and Dutch male employers with and without ulcer disease. Eur. Journal Gastroenterol. Hepatol.; 8:33-39
Segal E.D., Lange C., Tompkins L.S., Falkows S. (1997). Induction of host signal transduction pathways by Helicobacter Pylori. Proc. Natl. Acad. Sci. USA; 94: 7595-7599
Suerbaum S. and Michetti P. (2002). Helicobacter infection. New England Journal of Medecine; 347 (15): 1175-1186
Taylor D.E., Eaton M., Chang N., Salama S.M. (1992). Construction of a Helicobacter Pylori genome map and demonstration of diversity at the genome level. Journal of Bacteriology; 174: 6800-6806
Telford J.L., Covacci A., Rappuoli R., Ghiara (1997). Immunobiology of Helicobacter Pylori infection. Current opinion in Immunology; 9: 498-503
Tomb J.F., White O., Kerlavage A.R. et al. (1999). The complete genome sequence of gastric pathogen Helicobacter Pylori . Nature; 388: 539-547
Week D.L., Eskandari S., Scott D.R., Sachis G. (2000). An H+ gated urea channel: the link between Helicobacter Pylori urease and gastric colonisation. Science; 287: 482-485.