Beta-defensins and analogs in Helicobacter pylori infections: mRNA expression levels, DNA methylation, and antibacterial activity

Autoři: Raffaela Pero aff001;  Tiziana Angrisano aff003;  Mariarita Brancaccio aff004;  Annarita Falanga aff005;  Lucia Lombardi aff006;  Francesco Natale aff003;  Sonia Laneri aff005;  Barbara Lombardo aff001;  Stefania Galdiero aff005;  Olga Scudiero aff001
Působiště autorů: Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università degli Studi di Napoli “Federico II”, Napoli, Italy aff001;  Task Force sugli Studi del Microbioma, Università degli Studi di Napoli “Federico II”, Napoli, Italy aff002;  Dipartimento di Biologia, Università degli Studi di Napoli “Federico II”, Napoli, Italy aff003;  Dipartimento di Biologia ed Evoluzione degli Organismi Marini, Stazione Zoologica Anton Dohrn, Napoli, Italy aff004;  Dipartimento di Farmacia, Università degli Studi di Napoli “Federico II”, Napoli, Italy aff005;  Dipartimento di Agraria, Università degli Studi di Napoli “Federico II”, Napoli, Italy aff006;  CEINGE-Biotecnologie Avanzate Scarl, Napoli, Italy aff007
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: 10.1371/journal.pone.0222295


Antimicrobial peptides can protect the gastric mucosa from bacteria, but Helicobacter pylori (H. pylori) can equally colonize the gastric apparatus. To understand beta-defensin function in H. pylori-associated chronic gastritis, we investigated susceptibility, human beta-defensin mRNA expression, and DNA methylation changes to promoters in the gastric mucosa with or without H. pylori infection. We studied the expression of HBD2 (gene name DEFB4A), HBD3 (DEFB103A), and HBD4 (DEFB104) using real-time PCR in 15 control and 10 H. pylori infection patient gastric specimens. This study demonstrates that H. pylori infection is related to gastric enhancement of inducible HBD2, but inducible HBD3 and HBD4 expression levels remained unchanged. HBD2 gene methylation levels were overall higher in H. pylori-negative samples than in H. pylori-positive samples. We also assessed antimicrobial susceptibility using growth on blood agar. The H. pylori strain Tox+ was susceptible to all defensins tested and their analogs (3N, 3NI). These results show that HBD2 is involved in gastritis development driven by H. pylori, which facilitates the creation of an epigenetic field during H. pylori-associated gastric tumorigenesis.

Klíčová slova:

Biology and life sciences – Cell biology – Chromosome biology – Chromatin – Chromatin modification – DNA methylation – Genetics – Epigenetics – DNA modification – Gene expression – DNA – Biochemistry – Nucleic acids – Microbiology – Medical microbiology – Microbial pathogens – Bacterial pathogens – Helicobacter pylori – Microbial control – Antimicrobials – Organisms – Bacteria – Helicobacter – Medicine and health sciences – Pathology and laboratory medicine – Pathogens – Gastroenterology and hepatology – Gastritis – Gastrointestinal infections – Immunology – Immune response – Inflammation – Diagnostic medicine – Signs and symptoms – Pharmacology – Drugs – Research and analysis methods – Database and informatics methods – Bioinformatics – Sequence analysis – DNA sequence analysis


1. Itzkowitz SH, Yio X. Inflammation and cancer IV. Colorectal cancer in inflammatory bowel disease: the role of inflammation. Am J Physiol Gastrointest Liver Physiol. 2004; 287: G7–17. doi: 10.1152/ajpgi.00079.2004 15194558

2. Coretti L, Natale A, Cuomo M, Florio E, Keller S, Lembo F, et al. The Interplay between Defensins and Microbiota in Crohn’s Disease. Mediators Inflamm. 2017; 2017: 8392523. doi: 10.1155/2017/8392523 28246439

3. Stauffer JK, Scarzello A, Jiang Q, Wiltrout RH. Chronic inflammation, immune escape and oncogenesis in the liver: a unique neighbourhood for novel intersections. Hepatology. 2012; 56: 1567–74. doi: 10.1002/hep.25674 22378061

4. Macarthur M., Hold GL, El-Omar EM. Inflammation and Cancer II. Role of chronic inflammation and cytokine gene polymorphisms in the pathogenesis of gastrointestinal malignancy. Am J Physiol Gastrointest Liver Physiol. 2004; 286: G515–G520. doi: 10.1152/ajpgi.00475.2003 15010360

5. Lu H, Ouyang W, Huang C. Inflammation, a key event in cancer development. Mol Cancer Res. 2006; 4: 221–233. doi: 10.1158/1541-7786.MCR-05-0261 16603636

6. Chiariotti L, Coretti L, Pero R, Lembo F. Epigenetic Alterations Induced by Bacterial Lipopolysaccharides. Adv Exp Med Biol. 2016; 879: 91–105. doi: 10.1007/978-3-319-24738-0_5 26659265

7. Rutter M, Saunders B, Wilkinson K, Rumbles S, Schofield G, Kamm M, et al. Severity of inflammation is a risk factor for colorectal neoplasia in ulcerative colitis. Gastroenterology. 2004; 126: 451–459. doi: 10.1053/j.gastro.2003.11.010 14762782

8. Chen CJ, Yang HI, Su J, Jen CL, You SL, Lu SN, et al. Risk of hepatocellular carcinoma across a biological gradient of serum hepatitis B virus DNA level. JAMA. 2006; 295: 65–73. doi: 10.1001/jama.295.1.65 16391218

9. Yang HI, Yeh SH, Chen PJ, Iloeje UH, Jen CL, Su J, et al. Associations between hepatitis B virus genotype and mutants and the risk of hepatocellular carcinoma. J Natl Cancer Inst. 2008; 100: 1134–1143. doi: 10.1093/jnci/djn243 18695135

10. Correa P, Houghton J. Carcinogenesis of Helicobacter pylori. Gastroenterology. 2007; 133: 659–667. doi: 10.1053/j.gastro.2007.06.026 17681184

11. Pero R, Coretti L, Nigro E, Lembo F, Laneri S, Lombardo B, et al. β-Defensins in the Fight against Helicobacter pylori. Molecules. 2017; 7: 3.

12. Feinberg AP, Ohlsson R, Henikoff S. The epigenetic progenitor origin of human cancer. Nat Rev Genet. 2006; 7: 21–33. doi: 10.1038/nrg1748 16369569

13. Ushijima T. Epigenetic field for cancerization. J Biochem Mol Biol. 2007; 40: 142–150. 17394762

14. Niwa T, Tsukamoto T, Toyoda T, Mori A, Tanaka H, Maekita T, et al. Inflammatory processes triggered by Helicobacter pylori infection cause aberrant DNA methylation in gastric epithelial cells. Cancer Res. 2010; 70: 1430–1440. doi: 10.1158/0008-5472.CAN-09-2755 20124475

15. Maekita T, Nakazawa K, Mihara M, Nakajima T, Yanaoka K, Iguchi M, et al. High levels of aberrant DNA methylation in Helicobacter pylori infected gastric mucosae and its possible association with gastric cancer risk. Clin Cancer Res. 2006; 12: 989–995. doi: 10.1158/1078-0432.CCR-05-2096 16467114

16. Enomoto S, Maekita T, Tsukamoto T, Nakajima T, Nakazawa K, Tatematsu M, et al. Lack of association between CpG island methylator phenotype in human gastric cancers and methylation in their background non-cancerous gastric mucosae. Cancer Sci. 2007; 98: 1853–1861. doi: 10.1111/j.1349-7006.2007.00625.x 17900260

17. Kondo Y, Kanai Y, Sakamoto M, Mizokami M, Ueda R, Hirohashi S. Genetic instability and aberrant DNA methylation in chronic hepatitis and cirrhosis: a comprehensive study of loss of heterozygosity and microsatellite instability at 39 loci and DNA hypermethylation on 8 CpG islands in micro dissected specimens from patients with hepatocellular carcinoma. Hepatology. 2000; 32: 970–979. doi: 10.1053/jhep.2000.19797 11050047

18. Issa JP, Ahuja N, Toyota M, Bronner MP, Brentnall TA. Accelerated age-related CpG island methylation in ulcerative colitis. Cancer Res. 2001; 61: 3573–3577. 11325821

19. Chiariotti L, Angrisano T, Keller S, Florio E, Affinito O, Pallante P, et al. Epigenetic modifications induced by Helicobacter pylori infection through a direct microbe-gastric epithelial cells cross-talk. Med Microbiol Immunol. 2013; 202: 327–337. doi: 10.1007/s00430-013-0301-6 23715627

20. Ohata H, Kitauchi S, Yoshimura N, Mugitani K, Iwane M, Nakamura H, et al. Progression of chronic atrophic gastritis associated with Helicobacter pylori infection increases risk of gastric cancer. Int J Cancer. 2004; 109: 138–143. doi: 10.1002/ijc.11680 14735480

21. Yakirevich E, Resnick MB. Pathology of gastric cancer and its precursor lesions. Gastroenterol Clin North Am. 2013; 42: 261–284. doi: 10.1016/j.gtc.2013.01.004 23639640

22. Gigek CO, Chen ES, Calcagno DQ, Wisnieski F, Burbano R, Smith MA. Epigenetic mechanisms in gastric cancer. Epigenomics. 2012; 4: 279–294. doi: 10.2217/epi.12.22 22690664

23. Zhao CX. Promoter methylation of tumor-related genes in gastric carcinogenesis. Histol Histopathol. 2012; 27: 1271–1282. doi: 10.14670/HH-27.1271 22936446

24. Chan AO, Lam SK, Wong BC, Wong WM, Yuen MF, Yeung YH, et al. Promoter methylation of E-cadherin gene in gastric mucosa associated with Helicobacter pylori infection and in gastric cancer. Gut. 2003; 52: 502–506. doi: 10.1136/gut.52.4.502 12631658

25. Chan AO, Rashid A. CpG island methylation in precursors of gastrointestinal malignancies. Curr Mol Med. 2006; 6: 401–408. 16900663

26. Nigro E, Colavita I, Sarnataro D, Scudiero O, Zambrano G, Granata V, et al. An ancestral host defence peptide within human β-defensin recapitulates the antibacterial and antiviral activity of the full-length molecule. Sci Rep. 2015; 21: 5–18450.

27. Boughan PK, Argent RH, Body-Malapel M, Park JH, Ewings KE, Bowie AG, et al. Nucleotide-binding oligomerization domain-1 and epidermal growth factor receptor: critical regulators of beta-defensins during Helicobacter pylori infection. J Biol Chem. 2006; 281: 11637–11648. doi: 10.1074/jbc.M510275200 16513653

28. Grubman A, Kaparakis M, Viala J, Allison C, Badea L, Karrar A, et al. The innate immune molecule, NOD1, regulates direct killing of Helicobacter pylori by antimicrobial peptides. Cell Microbiol. 2010; 12: 626–639. doi: 10.1111/j.1462-5822.2009.01421.x 20039881

29. Bauer B, Wex T, Kuester D, Meyer T, Malfertheiner P. Differential expression of human beta defensin 2 and 3 in gastric mucosa of Helicobacter pylori-infected individuals. Helicobacter. 2013; 18: 6–12. doi: 10.1111/hel.12000 23067102

30. Muhammad JS, Zaidi SF, Zhou Y, Sakurai H, Sugiyama T. Novel epidermal growth factor receptor pathway mediates release of human β-defensin 3 from Helicobacter pylori-infected gastric epithelial cells. Pathog Dis. 2016; 74.

31. O’Neil DA. Regulation of expression of beta-defensins: endogenous enteric peptide antibiotics. Mol Immunol. 2003; 40: 445–450. doi: 10.1016/s0161-5890(03)00161-5 14568390

32. Taha AS, Faccenda E, Angerson WJ, Balsitis M, Kelly RW. Gastric epithelial anti-microbial peptides-histological correlation and influence of anatomical site and peptic ulcer disease. Dig Liver Dis. 2005; 37: 51–56. doi: 10.1016/j.dld.2004.07.019 15702860

33. Patel SR, Smith K, Letley DP, Cook KW, Memon AA, Ingram RJ, et al. Helicobacter pylori downregulates expression of human β-defensin 1 in the gastric mucosa in a type IV secretion-dependent fashion. Cell Microbiol. 2013; 15: 2080–2092. doi: 10.1111/cmi.12174 23870035

34. Selsted ME, Ouellette AJ. Mammalian defensins in the antimicrobial immune response. Nat Immunol. 2005; 6: 551–557. doi: 10.1038/ni1206 15908936

35. Scudiero O, Galdiero S, Cantisani M, Di Noto R, Vitiello M, Galdiero M, et al. Novel synthetic, salt-resistant analogs of human beta-defensins 1 and 3 endowed with enhanced antimicrobial activity. Antimicrob Agents Chemother. 2010; 54: 2312–2322. doi: 10.1128/AAC.01550-09 20308372

36. Scudiero O, Galdiero S, Nigro E, Del Vecchio L, Di Noto R, Cantisani M. et al. Chimeric beta-defensin analogs, including the novel 3NI analog, display salt-resistant antimicrobial activity and lack toxicity in human epithelial cell lines. Antimicrob Agents Chemother. 2013; 57: 1701–1708. doi: 10.1128/AAC.00934-12 23357761

37. Lombardi L, Shi Y, Falanga A, Galdiero E, De Alteriis E, Franci G et al. Enhancing the potency of antimicrobial peptides through molecular engineering and self-assembly. Biomacromolecules. 2019; 20: 1362–1374. doi: 10.1021/acs.biomac.8b01740 30735368

38. Scudiero O, Nigro E, Cantisani M, Colavita I, Leone M, Mercurio FA, et al. Design and activity of a cyclic mini-β-defensin analog: a novel antimicrobial tool. Int J Nanomedicine. 2015; 10: 6523–6539. doi: 10.2147/IJN.S89610 26508857

39. Bajaj-Elliott M, Fedeli P, Smith GV, Domizio P, Maher L, Ali RS, et al. Modulation of host antimicrobial peptide (beta-defensins 1 and 2) expression during gastritis. Gut. 2002; 51: 356–361. doi: 10.1136/gut.51.3.356 12171956

40. Hamanaka Y, Nakashima M, Wada A, Ito M, Kurazono H, Hojo H, et al. Expression of human beta-defensin 2 (hBD-2) in Helicobacter pylori induced gastritis: antibacterial effect of hBD-2 against Helicobacter pylori. Gut. 2001; 49; 481–487. doi: 10.1136/gut.49.4.481 11559643

41. Wehkamp J, Schmidt K, Herrlinger KR, Baxmann S, Behling S, Wohlschläger C, et al. Defensin pattern in chronic gastritis: HBD-2 is differentially expressed with respect to Helicobacter pylori status. J Clin Pathol. 2003; 56: 352–357. doi: 10.1136/jcp.56.5.352 12719455

42. Kawauchi K, Yagihashi A, Tsuji N, Uehara N, Furuya D, Kobayashi D, et al. Human beta-defensin-3 induction in H. pylori-infected gastric mucosal tissues. World J Gastroenterol. 2006; 12: 5793–5797. doi: 10.3748/wjg.v12.i36.5793 17007044

43. di Martino O, Troiano A, Guarino AM, Pollice A, Vivo M, La Mantia G, et al. ΔNp63α controls YB-1 protein stability: evidence on YB-1 as a new player in keratinocyte differentiation. Genes Cells. 2016; 21: 648–660. doi: 10.1111/gtc.12373 27168020

44. Keller S, Angrisano T, Florio E, Pero R, Decaussin-Petrucci M, Troncone G et al. DNA methylation state of the galectin-3 gene represents a potential new marker of thyroid malignancy. Oncol Lett. 2013; 6: 86–90. doi: 10.3892/ol.2013.1312 23946782

45. Angrisano T, Pero R, Brancaccio M, Coretti L, Florio E, Pezone A, et al. Cyclical DNA Methylation and Histone Changes Are Induced by LPS to Activate COX-2 in Human Intestinal Epithelial Cells. PLoS. One. 2016; 11: e0156671. doi: 10.1371/journal.pone.0156671 27253528

46. Hase K, Murakami M, Iimura M, Cole SP, Horibe Y, Ohtake T, et al. Expression of LL-37 by human gastric epithelial cells as a potential host defense mechanism against Helicobacter pylori. Gastroenterology. 2003; 125: 1613–1625. doi: 10.1053/j.gastro.2003.08.028 14724813

47. Otte JM, Neumann HM, Brand S, Schrader H, Schmidt WE, Schmitz F. Expression of beta-defensin 4 is increased in human gastritis. Eur J Clin Invest. 2009; 39: 126–128. doi: 10.1111/j.1365-2362.2008.02071.x 19200166

48. Tsunoda T, Takagi T. Estimating transcription factor bind ability on DNA. Bioinformatics. 1999; 815: 622–630.

49. Isomoto H, Mukae H, Ishimoto H, Nishi Y, Wen CY, Wada A, et al. High concentrations of human beta-defensin 2 in gastric juice of patients with Helicobacter pylori infection. World J Gastroenterol. 2005; 11: 4782–4787. doi: 10.3748/wjg.v11.i31.4782 16097044

50. Nishi Y, Isomoto H, Mukae H, Ishimoto H, Wen CY, Wada A, et al. Concentrations of alpha- and beta-defensins in gastric juice of patients with various gastroduodenal diseases. World J Gastroenterol. 2005; 11: 99–103. doi: 10.3748/wjg.v11.i1.99 15609405

51. Uehara N, Yagihashi A, Kondoh K, Tsuji N, Fujita T, Hamada H, et al. Human beta-defensin-2 induction in Helicobacter pylori-infected gastric mucosal tissues: antimicrobial effect of overexpression. J Med Microbiol. 2003; 52: 41–45. doi: 10.1099/jmm.0.04985-0 12488564

52. Vordenbäumen S, Pilic D, Otte JM, Schmitz F, Schmidt-Choudhury A. Defensin-mRNA expression in the upper gastrointestinal tract is modulated in children with celiac disease and Helicobacter pylori positive gastritis. J Pediatr Gastroenterol Nutr. 2010; 50: 596–600. doi: 10.1097/MPG.0b013e3181cd26cd 20400909

53. Islam D, Bandholtz L, Nilsson J, Wigzel H, Christensson B, Agerberth B, et al. Downregulation of bactericidal peptides in enteric infections: a novel immune escape mechanism with bacterial DNA as a potential regulator. Nat Med. 2001; 7: 180–185. doi: 10.1038/84627 11175848

54. Hosaka Y, Koslowski M, Nuding S, Wang G, Schlee M, Schäfer C, et al. Antimicrobial host defense in the upper gastrointestinal tract. Eur J Gastroenterol Hepatol. 2008; 20: 1151–1158. doi: 10.1097/MEG.0b013e3283052ddb 18989140

55. Resnick MB, Sabo E, Meitner PA, Kim SS, Cho Y, Kim HK, et al. Global analysis of the human gastric epithelial transcriptome altered by Helicobacter pylori eradication in vivo. Gut. 2006; 55: 1717–1724. doi: 10.1136/gut.2006.095646 16641130

56. Ohara T, Morishita T, Suzuki H, Masaoka T, Nishizawa T, Hibi T. Investigation of the possibility of human-beta defensin 2 (hBD2) as a molecular marker of gastric mucosal inflammation. Hepatogastroenterology. 2005; 52: 1320–1324. 16201065

57. Wada A, Mori N, Oishi K, Hojo H, Nakahara Y, Hamanaka Y, et al. Induction of human beta-defensin-2 mRNA expression by Helicobacter pylori in human gastric cell line MKN45 cells on cag pathogenicity island. Biochem Biophys Res Commun. 1999; 263: 770–774. doi: 10.1006/bbrc.1999.1452 10512755

58. Angrisano T, Sacchetti S, Natale F, Cerrato A, Pero R, Keller S, et al. Chromatin and DNA methylation dynamics during retinoic acid-induced RET gene transcriptional activation in neuroblastoma cells. Nucleic Acids Res. 2011; 39:1993–2006. doi: 10.1093/nar/gkq864 20952403

59. Murayama A, Sakura K, Nakama M, Yasuzawa-Tanaka K, Fujita E, Tateishi Y, et al. specific CpG site demethylation in the human interleukin 2 gene promoter is an epigenetic memory. EMBO J. 2006; 25: 1081–1092. doi: 10.1038/sj.emboj.7601012 16498406

60. Belot MP, Castell AL, Le Fur S, Bougnères P. Dynamic demethylation of the IL2RA promoter during in vitro CD4+ T cell activation in association with IL2RA expression. Epigenetics. 2018; 13: 459–472. doi: 10.1080/15592294.2018.1469893 30096258

61. Sobiak B, Leśniak W. The Effect of Single CpG Demethylation on the Pattern of DNA-Protein Binding. Int J Mol Sci. 2019; 20: 914.

62. Ushijima T. Detection and interpretation of altered methylation patterns in cancer cells. Nat Rev Cancer. 2005; 5: 223–231. doi: 10.1038/nrc1571 15719030

63. Shin CM, Kim N, Jung Y, Park JH, Kang GH, Kim JS, et al. Role of Helicobacter pylori infection in aberrant DNA methylation along multistep gastric carcinogenesis. Cancer Sci. 2010; 101: 1337–1346. doi: 10.1111/j.1349-7006.2010.01535.x 20345486

64. Hayashi Y, Tsujii M, Wan J, Kondo J, Akasaka T, Jin Y, et al. Cag A mediates epigenetic regulation to attenuate let-7 expression in Helicobacter pylori-related carcinogenesis. Gut. 2013; 62: 1536–1546. doi: 10.1136/gutjnl-2011-301625 22936674

Článek vyšel v časopise


2019 Číslo 9