Helicobacter pylori Antigen HP0986 (TieA) Interacts with Cultured Gastric Epithelial Cells and Induces IL8 Secretion via NF-κB Mediated Pathway
Savita Devi
Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, University of Hyderabad, Hyderabad, 500046 India
Search for more papers by this authorSuhail A. Ansari
Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, University of Hyderabad, Hyderabad, 500046 India
Search for more papers by this authorJamuna Vadivelu
Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, 50603 Malaysia
Search for more papers by this authorFrancis Mégraud
INSERM - U853, Laboratoire de Bacteriologie - C.H.U. Pellegrin, Place Amelie Raba-Leon, 33076 Bordeaux Cedex, France
Search for more papers by this authorShivendra Tenguria
Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, University of Hyderabad, Hyderabad, 500046 India
Search for more papers by this authorCorresponding Author
Niyaz Ahmed
Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, University of Hyderabad, Hyderabad, 500046 India
Institute of Biological Sciences, University of Malaya, Kuala Lumpur, 50603 Malaysia
Reprint requests to: Niyaz Ahmed, Department of Biotechnology and Bioinformatics, University of Hyderabad, School of Life Sciences, HCU Post, Hyderabad 500046, India. E-mail:[email protected]Search for more papers by this authorSavita Devi
Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, University of Hyderabad, Hyderabad, 500046 India
Search for more papers by this authorSuhail A. Ansari
Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, University of Hyderabad, Hyderabad, 500046 India
Search for more papers by this authorJamuna Vadivelu
Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, 50603 Malaysia
Search for more papers by this authorFrancis Mégraud
INSERM - U853, Laboratoire de Bacteriologie - C.H.U. Pellegrin, Place Amelie Raba-Leon, 33076 Bordeaux Cedex, France
Search for more papers by this authorShivendra Tenguria
Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, University of Hyderabad, Hyderabad, 500046 India
Search for more papers by this authorCorresponding Author
Niyaz Ahmed
Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, University of Hyderabad, Hyderabad, 500046 India
Institute of Biological Sciences, University of Malaya, Kuala Lumpur, 50603 Malaysia
Reprint requests to: Niyaz Ahmed, Department of Biotechnology and Bioinformatics, University of Hyderabad, School of Life Sciences, HCU Post, Hyderabad 500046, India. E-mail:[email protected]Search for more papers by this authorAbstract
Background
The envisaged roles and partly understood functional properties of Helicobacter pylori protein HP0986 are significant in the context of proinflammatory and or proapoptotic activities, the two important facilitators of pathogen survival and persistence. In addition, sequence analysis of this gene predicts a restriction endonuclease function which remained unknown thus far. To evaluate the role of HP0986 in gastric inflammation, we studied its expression profile using a large number of clinical isolates but a limited number of biopsies and patient sera. Also, we studied antigenic role of HP0986 in altering cytokine responses of human gastric epithelial (AGS) cells including its interaction with and localization within the AGS cells.
Materials and Methods
For in vitro expression study of HP0986, 110 H. pylori clinical isolates were cultured from patients with functional dyspepsia. For expression analysis by qRT PCR of HP0986, 10 gastric biopsy specimens were studied. HP0986 was also used to detect antibodies in patient sera. AGS cells were incubated with recombinant HP0986 to determine cytokine response and NF-κB activation. Transient transfection with HP0986 cloned in pEGFPN1 was used to study its subcellular localization or homing in AGS cells.
Results
Out of 110 cultured H. pylori strains, 34 (31%) were positive for HP0986 and this observation was correlated with in vitro expression profiles. HP0986 mRNA was detected in 7 of the 10 biopsy specimens. Further, HP0986 induced IL-8 secretion in gastric epithelial cells in a dose and time-dependent manner via NF-κB pathway. Serum antibodies against HP0986 were positively associated with H. pylori positive patients. Transient transfection of AGS cells revealed both cytoplasmic and nuclear localization of HP0986.
Conclusion
HP0986 was moderately prevalent in clinical isolates and its expression profile in cultures and gastric biopsies points to its being naturally expressed. Collective observations including the induction of IL-8 via TNFR1 and NF-κB, subcellular localization, and seropositivity data point to a significant role of HP0986 in gastroduodenal inflammation. We propose to name the HP0986 gene/protein as ‘TNFR1 interacting endonuclease A (TieA or tieA)’.
References
- 1Crabtree JE. Immune and inflammatory responses to Helicobacter pylori infection. Scand J Gastroenterol Suppl 1996; 215: 3–10.
- 2Yamaoka Y, Kita M, Kodama T, Sawai N, Kashima K, Imanishi J. Induction of various cytokines and development of severe mucosal inflammation by cagA gene positive Helicobacter pylori strains. Gut 1997; 41: 442–51.
- 3Yoshida N, Granger DN, Evans DJ Jr, Evans DG, Graham DY, Anderson DC, Wolf RE, Kvietys PR. Mechanisms involved in Helicobacter pylori-induced inflammation. Gastroenterology 1993; 105: 1431–40.
- 4Megraud F. Impact of Helicobacter pylori virulence on the outcome of gastroduodenal diseases: lessons from the microbiologist. Dig Dis 2001; 19: 99–103.
- 5Houghton J, Fox JG, Wang TC. Gastric cancer: laboratory bench to clinic. J Gastroenterol Hepatol 2002; 17: 495–502.
- 6Khalifa MM, Sharaf RR, Aziz RK. Helicobacter pylori: a poor man's gut pathogen? Gut Pathog 2010; 2: 2.
- 7Ahmed N, Tenguria S, Nandanwar N. Helicobacter pylori–a seasoned pathogen by any other name. Gut Pathog 2009; 1: 24.
- 8Brandt S, Kwok T, Hartig R, Konig W, Backert S. NF-kappaB activation and potentiation of proinflammatory responses by the Helicobacter pylori CagA protein. Proc Natl Acad Sci USA 2005; 102: 9300–5.
- 9Covacci A, Censini S, Bugnoli M, Petracca R, Burroni D, Macchia G, Massone A, Papini E, Xiang Z, Figura N. Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer. Proc Natl Acad Sci USA 1993; 90: 5791–5.
- 10Voland P, Hafsi N, Zeitner M, Laforsch S, Wagner H, Prinz C. Antigenic properties of HpaA and Omp18, two outer membrane proteins of Helicobacter pylori. Infect Immun 2003; 71: 3837–43.
- 11Sobala GM, Crabtree JE, Dixon MF, Schorah CJ, Taylor JD, Rathbone BJ, Heatley RV, Axon AT. Acute Helicobacter pylori infection: clinical features, local and systemic immune response, gastric mucosal histology, and gastric juice ascorbic acid concentrations. Gut 1991; 32: 1415–8.
- 12Yamaoka Y, Kodama T, Gutierrez O, Kim JG, Kashima K, Graham DY. Relationship between Helicobacter pylori iceA, cagA, and vacA status and clinical outcome: studies in four different countries. J Clin Microbiol 1999; 37: 2274–9.
- 13Kersulyte D, Lee W, Subramaniam D, et al. Helicobacter Pylori's plasticity zones are novel transposable elements. PLoS ONE 2009; 4: e6859.
- 14Santos A, Queiroz DM, Menard A, Marais A, Rocha GA, Oliveira CA, Nogueira AM, Uzeda M, Mégraud F. New pathogenicity marker found in the plasticity region of the Helicobacter pylori genome. J Clin Microbiol 2003; 41: 1651–5.
- 15Occhialini A, Marais A, Alm R, Garcia F, Sierra R, Megraud F. Distribution of open reading frames of plasticity region of strain J99 in Helicobacter pylori strains isolated from gastric carcinoma and gastritis patients in Costa Rica. Infect Immun 2000; 68: 6240–9.
- 16Yamaoka Y. Roles of the plasticity regions of Helicobacter pylori in gastroduodenal pathogenesis. J Med Microbiol 2008; 57: 545–53.
- 17Van Doorn LJ, Figueiredo C, Megraud F, et al. Geographic distribution of vacA allelic types of Helicobacter pylori. Gastroenterology 1999; 116: 823–30.
- 18Yamaoka Y, Malaty HM, Osato MS, Graham DY. Conservation of Helicobacter pylori genotypes in different ethnic groups in Houston Texas. J Infect Dis 2000; 181: 2083–6.
- 19Rizwan M, Alvi A, Ahmed N. Novel protein antigen (JHP940) from the genomic plasticity region of Helicobacter pylori induces tumor necrosis factor alpha and interleukin-8 secretion by human macrophages. J Bacteriol 2008; 190: 1146–51.
- 20Kim do J, Park KS, Kim JH, et al. Helicobacter pylori proinflammatory protein up-regulates NF-kappaB as a cell-translocating Ser/Thr kinase. Proc Natl Acad Sci USA 2010; 107: 21418–23.
- 21Alvi A, Ansari SA, Ehtesham NZ, Rizwan M, Devi S, Sechi LA, Qureshi IA, Hasnain SE, Ahmed N. Concurrent proinflammatory and apoptotic activity of a Helicobacter pylori protein (HP986) points to its role in chronic persistence. PLoS ONE 2011; 6: e22530.
- 22Gonzalez-Valencia G, Atherton JC, Munoz O, Dehesa M, la Garza AM, Torres J. Helicobacter pylori vacA and cagA genotypes in Mexican adults and children. J Infect Dis 2000; 182: 1450–4.
- 23Leong DT, Gupta A, Bai HF, Wan G, Yoong LF, Too HP, Chew FT, Hutmacher DW. Absolute quantification of gene expression in biomaterials research using real-time PCR. Biomaterials 2007; 28: 203–10.
- 24Hussain MA, Naveed SA, Sechi LA, Ranjan S, Alvi A, Ahmed I, Ranjan A, Mukhopadhyay S, Ahmed N. Isocitrate dehydrogenase of Helicobacter pylori potentially induces humoral immune response in subjects with peptic ulcer disease and gastritis. PLoS ONE 2008; 3: e1481.
- 25Blaser MJ, Atherton JC. Helicobacter pylori persistence: biology and disease. J Clin Investig 2004; 113: 321–33.
- 26Marshall BJ, Warren JR. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1984; 1: 1311–5.
- 27Montecucco C, Rappuoli R. Living dangerously: how Helicobacter pylori survives in the human stomach. Nat Rev Mol Cell Biol 2001; 2: 457–66.
- 28Suerbaum S, Michetti P. Helicobacter pylori infection. N Engl J Medicine 2002; 347: 1175–86.
- 29Odenbreit S, Puls J, Sedlmaier B, Gerland E, Fischer W, Haas R. Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion. Science 2000; 287: 1497–500.
- 30Chan WY, Hui PK, Leung KM, Thomas TM. Modes of Helicobacter colonization and gastric epithelial damage. Histopathology 1992; 21: 521–8.
- 31Necchi V, Candusso ME, Tava F, Luinetti O, Ventura U, Fiocca R, Ricci V, Solcia E. Intracellular, intercellular, and stromal invasion of gastric mucosa, preneoplastic lesions, and cancer by Helicobacter pylori. Gastroenterology 2007; 132: 1009–23.
- 32Janssen PJ, Audit B, Ouzounis CA. Strain-specific genes of Helicobacter pylori: distribution, function and dynamics. Nucleic Acids Res 2001; 29: 4395–404.
- 33Fischer W, Windhager L, Rohrer S, Zeiller M, Karnholz A, Hoffmann R, Zimmer R, Haas R. Strain-specific genes of Helicobacter pylori: genome evolution driven by a novel type IV secretion system and genomic island transfer. Nucleic Acids Res 2010; 38: 6089–101.
- 34Shiota S, Matsunari O, Watada M, Hanada K, Yamaoka Y. Systematic review and meta-analysis: the relationship between the Helicobacter pylori dupA gene and clinical outcomes. Gut Pathog 2010; 2: 13.
- 35Alm RA, Trust TJ. Analysis of the genetic diversity of Helicobacter pylori: the tale of two genomes. J Mol Med 1999; 77: 834–46.
- 36Romo-Gonzalez C, Salama NR, Burgeno-Ferreira J, Ponce-Castaneda V, Lazcano-Ponce E, Camorlinga-Ponce M, Torres J. Differences in genome content among Helicobacter pylori isolates from patients with gastritis, duodenal ulcer, or gastric cancer reveal novel disease-associated genes. Infect Immun 2009; 77: 2201–11.
- 37de Jonge R, Kuipers EJ, Langeveld SC, Loffeld RJ, Stoof J, van Vliet AH, Kusters JG. The Helicobacter pylori plasticity region locus jhp0947-jhp0949 is associated with duodenal ulcer disease and interleukin-12 production in monocyte cells. FEMS Immunol Med Microbiol 2004; 41: 161–7.
- 38Hussein NR. The association of dupA and Helicobacter pylori-related gastroduodenal diseases. Eur J Clin Microbiol Infect Dis 2010; 29: 817–21.
- 39Yakoob J, Abid S, Abbas Z, Jafri W, Ahmad Z, Ahmed R, Islam M. Distribution of Helicobacter pylori virulence markers in patients with gastroduodenal diseases in Pakistan. BMC Gastroenterol 2009; 9: 87.
- 40Schmidt HM, Andres S, Kaakoush NO, et al. The prevalence of the duodenal ulcer promoting gene (dupA) in Helicobacter pylori isolates varies by ethnic group and is not universally associated with disease development: a case-control study. Gut Pathog 2009; 1: 5.
- 41Sugimoto M, Watada M, Jung SW, Graham DY, Yamaoka Y. Role of Helicobacter pylori plasticity region genes in development of gastroduodenal diseases. J Clin Microbiol 2012; 50: 441–8.
- 42Avasthi TS, Devi SH, Taylor TD, Kumar N, Baddam R, Kondo S, Suzuki Y, Lamouliatte H, Mégraud F, Ahmed N. Genomes of two chronological isolates (Helicobacter pylori 2017 and 2018) of the West African Helicobacter pylori strain 908 obtained from a single patient. J Bacteriol 2011; 193: 3385–6.
- 43Blaser MJ. Ecology of Helicobacter pylori in the human stomach. J Clin Investig 1997; 100: 759–62.
- 44Blaser MJ, Kirschner D. Dynamics of Helicobacter pylori colonization in relation to the host response. Proc Natl Acad Sci USA 1999; 96: 8359–64.
- 45Aviles-Jimenez F, Reyes-Leon A, Nieto-Patlan E, et al. In vivo expression of Helicobacter pylori virulence genes in patients with gastritis, ulcer, and gastric cancer. Infect Immun 2012; 80: 594–601.
- 46Merrell DS, Goodrich ML, Otto G, Tompkins LS, Falkow S. pH-regulated gene expression of the gastric pathogen Helicobacter pylori. Infect Immun 2003; 71: 3529–39.
- 47Cao P, McClain MS, Forsyth MH, Cover TL. Extracellular release of antigenic proteins by Helicobacter pylori. Infect Immun 1998; 66: 2984–6.
- 48Selbach M, Moese S, Meyer TF, Backert S. Functional analysis of the Helicobacter pylori cag pathogenicity island reveals both VirD4-CagA-dependent and VirD4-CagA-independent mechanisms. Infect Immun 2002; 70: 665–71.
- 49Gorrell RJ, Guan J, Xin Y, Tafreshi MA, Hutton ML, McGuckin MA, Ferrero RL, Kwok T. A novel NOD1- and CagA-independent pathway of interleukin-8 induction mediated by the Helicobacter pylori type IV secretion system. Cell Microbiol 2012; 15: 554–70.
- 50Zhao Y, Yokota K, Ayada K, Yamamoto Y, Okada T, Shen L, Oguma K. Helicobacter pylori heat-shock protein 60 induces interleukin-8 via a Toll-like receptor (TLR)2 and mitogen-activated protein (MAP) kinase pathway in human monocytes. J Med Microbiol 2007; 56: 154–64.
- 51Hussein NR, Argent RH, Marx CK, Patel SR, Robinson K, Atherton JC. Helicobacter pylori dupA is polymorphic, and its active form induces proinflammatory cytokine secretion by mononuclear cells. J Infect Dis 2010; 202: 261–9.
- 52Sharma SA, Tummuru MK, Blaser MJ, Kerr LD. Activation of IL-8 gene expression by Helicobacter pylori is regulated by transcription factor nuclear factor-kappa B in gastric epithelial cells. J Immunol 1998; 160: 2401–7.
- 53Segal ED, Falkow S, Tompkins LS. Helicobacter pylori attachment to gastric cells induces cytoskeletal rearrangements and tyrosine phosphorylation of host cell proteins. Proc Natl Acad Sci USA 1996; 93: 1259–64.
- 54Finn RD, Tate J, Mistry J, et al. The Pfam protein families database. Nucleic Acids Res. 2008; 36: D281–8.
- 55Banerjee S, Nandyala AK, Raviprasad P, Ahmed N, Hasnain SE. Iron-dependent RNA-binding activity of Mycobacterium tuberculosis aconitase. J Bacteriol 2007; 189: 4046–52.
- 56Banerjee S, Nandyala A, Podili R, Katoch VM, Murthy KJR, Hasnain SE. Mycobacterium tuberculosis (Mtb) isocitrate dehydrogenases show strong B cell response and distinguish vaccinated controls from TB patients. Proc Natl Acad Sci USA 2004; 101: 12652–7.
- 57Kumar A, Lewin A, Rani PS, Qureshi IA, Devi S, Majid M, Hasnain SE, Ahmed N. Dormancy Associated Translation Inhibitor (DATIN/Rv0079) of Mycobacterium tuberculosis interacts with TLR2 and induces proinflammatory cytokine expression. Cytokine 2013; 64: 258–64.
- 58Kumar A, Majid M, Kunisch R, Rani PS, Qureshi IA, Lewin A, Hasnain SE, Ahmed N. Mycobacterium tuberculosis DosR regulon gene Rv0079 encodes a putative, ‘dormancy associated translation inhibitor (DATIN)’. PLoS ONE 2012; 7: e38709.
- 59Xu Q, Morgan RD, Roberts RJ, Xu SY, van Doorn LJ, Donahue JP, Miller GG, Blaser MJ. Functional analysis of iceA1, a CATG-recognizing restriction endonuclease gene in Helicobacter pylori. Nucleic Acids Res 2002; 30: 3839–47.
