Effect of Helicobacter pylori cdrA on interleukin-8 secretions and nuclear factor kappa B activation

Abstract

Aim: To investigate genetic diversity of Helicobacter pylori (H. pylori) cell division-related gene A (cdrA) and its effect on the host response.

Methods: Inactivation of H. pylori cdrA, which is involved in cell division and morphological elongation, has a role in chronic persistent infections. Genetic property of H. pylori cdrA was evaluated using polymerase chain reaction and sequencing in 128 (77 American and 51 Japanese) clinical isolates obtained from 48 and 51 patients, respectively. Enzyme-linked immunosorbent assay was performed to measure interleukin-8 (IL-8) secretion with gastric biopsy specimens obtained from American patients colonized with cdrA-positive or -negative strains and AGS cells co-cultured with wild-type HPK5 (cdrA-positive) or its derivative HPKT510 (cdrA-disruptant). Furthermore, the cytotoxin-associated gene A (cagA) status (translocation and phosphorylation) and kinetics of transcription factors [nuclear factor-kappa B (NF-κB) and inhibition kappa B] were investigated in AGS cells co-cultured with HPK5, HPKT510 and its derivative HPK5CA (cagA-disruptant) by western blotting analysis with immunoprecipitation.

Results: Genetic diversity of the H. pylori cdrA gene demonstrated that the cdrA status segregated into two categories including four allele types, cdrA-positive (allele types;Iand II) and cdrA-negative (allele types; III and IV) categories, respectively. Almost all Japanese isolates were cdrA-positive (I: 7.8% and II: 90.2%), whereas 16.9% of American isolates were cdrA-positive (II) and 83.1% were cdrA-negative (III: 37.7% and IV: 45.5%), indicating extended diversity of cdrA in individual American isolates. Comparison of each isolate from different regions (antrum and corpus) in the stomach of 29 Americans revealed that cdrA status was identical in both isolates from different regions in 17 cases. However, 12 cases had a different cdrA allele and 6 of them exhibited a different cdrA category between two regions in the stomach. Furthermore, in 5 of the 6 cases possessing a different cdrA category, cdrA-negative isolate existed in the corpus, suggesting that cdrA-negative strain is more adaptable to colonization in the corpus. IL-8 secretions from AGS revealed that IL-8 levels induced by a cdrA-disrupted HPKT510 was significantly lower (P < 0.01) compared to wild-type HPK5: corresponding to 50%-60% of those of wild-type HPK5. These data coincided with in vivo data that an average value of IL-8 in biopsy specimens from cdrA-positive and cdrA-negative groups was 215.6 and 135.9 pg/mL, respectively. Western blotting analysis documented that HPKT510 had no effect on CagA translocation and phosphorylation, however, nuclear accumulation of NF-κB was lower by HPKT510 compared to HPK5.

Conclusion: Colonization by a cdrA-negative or cdrA-dysfunctional strain resulted in decreased IL-8 production and repression of NF-κB, and hence, attenuate the host immunity leading to persistent infection.

Keywords: Genetic diversity; Helicobacter pylori cell division-related gene A; Host immune response; Interleukin-8 secretion; Nuclear factor kappa B; Persistent infection.

Figure 1

Location of primers used for cell division-related gene A in the region downstream of the urease gene cluster of Helicobacter pylori HPK5. Arrows above and below the map depict the direction of transcription and primers, respectively. The open reading frames (HP0063 to HP0067) are shown based on strain 26695.

Figure 2

Interleukin-8 production from AGS cells induced by either wild-type or cell division-related gene A-disrupted mutant strains. ^aP < 0.01.

Figure 3

Cytotoxin-associated gene A status in AGS cells co-cultured with or without Helicobacter pylori strains. The immunoprecipitated proteins with anti-Helicobacter pylori (H. pylori) cytotoxin-associated gene A (CagA) antibody (CagA-Ab) were subjected to Western blotting with CagA-Ab (upper) and PY-99 antibody (PY99-Ab) (bottom), respectively. The assay was carried out with strains at MOI of 150. HPK5: Wild-type; HPKT510: Cell division-related gene A-disrupted mutant; HPK5CA: cagA-disrupted mutant.

Figure 4

Detection of nuclear factor kappa B (p65) (upper), inhibition kappa B (middle) and extracellular signal-regulated kinase 2 (bottom) in nuclear and total extracts, respectively at 3, 6 and 12 h after being co-cultured with Helicobacter pylori. Unphosphorylated extracellular signal–regulated kinase 2 (ERK2) was detected as the control in this study[23]. Molecular weights of nuclear factor kappa B (NF-κB), inhibition kappa B (IκB) and ERK2 are 65, 35-37 and 42 kDa, respectively. The assay was carried out with strains at a MOI of 150. AGS: AGS cells co-cultured without H. pylori; HPK5: AGS cells co-cultured with wild-type HPK5; HPKT510: AGS cells co-cultured with cdrA-disrupted mutant HPKT510.