Volume 120, Issue 2, Pages 470-479 (February 2001) Survival of Helicobacter pylori from complement lysis by binding of GPI-anchored protectin (CD59)  Riina Rautemaa, Hilpi Rautelin, Pauli Puolakkainen, Arto Kokkola, Päivi Karkkainen, Seppo Meri  Gastroenterology  Volume 120, Issue 2, Pages 470-479 (February 2001) DOI: 10.1053/gast.2001.21197 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Complement activation in gastric mucosa of H. pylori–infected and noninfected patients as demonstrated by immunofluorescence microscopy. (A and B) C3 and C and D) C5b-9 deposits are located below the basement membrane or on the surface of the epithelium in the gastric mucosa of H. pylori–infected patients. H. pylori bacteria on the surface epithelium (arrows) are coated with C3 (B) and C5b-9 (D). In the gastric mucosa of noninfected patients, staining for (E) C3 and (F) C5b-9 is less intense. Controls: FITC-conjugated antibodies against (G) rabbit and (H) mouse IgG. (Original magnifications: A, 200×; B,C, E–H, 400×; D, 1000×.) Gastroenterology 2001 120, 470-479DOI: (10.1053/gast.2001.21197) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Expression of membrane regulators of complement in H. pylori–infected and noninfected gastric mucosa. (A and B) Protectin (CD59) and (D) DAF become stained on cell membranes. The vascular endothelia stain most prominently for CD59 (arrows in A and B). (B) In the infected mucosa, staining for CD59 decreases in the gastric pits toward the apical surface and increases in the more superficial gastric epithelium. (D) Staining for DAF is weak but more intense in the infected mucosa. (C) Control: irrelevant antibody AF16.1 as primary antibody. (Original magnifications: A–D, 400×.) Gastroenterology 2001 120, 470-479DOI: (10.1053/gast.2001.21197) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 Reciprocal staining of H. pylori for CD59 and C5b-9 in vivo. Sections from H. pylori–infected patients were immunostained for (A) C5b-9 and (B) CD59 or (D) H. pylori and (E) CD59 by double staining. Enlargements of the selected areas are shown in insets in corresponding left or right corners. Coiled rod–like H. pylori positive for CD59 can be seen deep in the gastric pits (red, right in B). In areas where the H. pylori bacteria stain positive for CD59, they stain negative for C5b-9 (green, right in A and C). C5b-9 deposits can be seen on more superficial bacteria (left in A and C). They have a more coccoid shape and stain negative for CD59 (left in B). (F) Double-exposure image: positive staining with an antibody against H. pylori (green, D) colocalizes with the CD59 staining (red, E) of the pits. Controls: (G) irrelevant antibody AF16.1 as primary antibody and (H) rhodamine neutravidin conjugate alone. (Original magnifications: A–H, 1000×; insets, 3300×.) Gastroenterology 2001 120, 470-479DOI: (10.1053/gast.2001.21197) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 Binding of glycolipid-tailed CD59 to H. pylori bacteria in vitro. H. pylori strain NCTC 11637 and 2 clinical isolates 9:0 and 67:20 were incubated with increasing concentrations of 125I-labeled lipid-tailed (m) or soluble (s) CD59 in the presence or absence of 2.5 mmol/L CaCl2. CD59 binding depends on the glycolipid-moiety because no binding of soluble urinary protectin (sCD59) to H. pylori can be detected. The CagA+ strains 11637 and 9:0 bind significantly more mCD59 (P < 0.01) than the CagA− 67:20 isolate. Ca2+ enhances the binding only slightly. Background has been subtracted from the curves. Gastroenterology 2001 120, 470-479DOI: (10.1053/gast.2001.21197) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 Visualization of CD59 on H. pylori in vivo and in vitro. (A) H. pylori bacteria in the gastric pits stain positive for CD59. (B) H. pylori bacteria incubated in vitro with mCD59 stain positive for CD59 similarly as in vivo. Strain 11637 bacteria were first incubated with mCD59 (2 μg/109 bacteria), washed, and spread on microscope slides and immunostained for CD59. (Original magnifications, 1000×.) Gastroenterology 2001 120, 470-479DOI: (10.1053/gast.2001.21197) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 Protection of H. pylori against complement lysis by mCD59. H. pylori strain NCTC 11637 and 2 clinical isolates 9:0 and 67:20 were first incubated with mCD59 (●, 0.01 μg/2 × 105 bacteria in the presence of 2.5 mmol/L Ca2+) and then exposed to indicated concentrations of NHS or NHSi (30 minutes at 37°C). ○, Without mCD59. The CFUs of serial 10-fold dilutions of bacterial suspensions were counted. The survival of serum-treated bacteria is expressed as percentage of the surviving bacteria exposed to NHSi. The binding of mCD59 increases the resistance of all 3 H. pylori strains against NHS. The increase is statistically significant for the CagA+ strains 11637 and 9:0 when serum concentrations up to 1% (P < 0.01) or 3% (P < 0.05) are used, respectively. The difference is not significant for the CagA− 67:20 isolate. Gastroenterology 2001 120, 470-479DOI: (10.1053/gast.2001.21197) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 (A) H. pylori–bound CD59 inhibits C5b-9 neoepitope formation on CagA+ bacteria in vitro. The 3 strains of H. pylori were incubated with mCD59 (●, 2 μg/109 bacteria in the presence of 2.5 mmol/L Ca2+) treated with NHS or NHSi as for Figure 6. ○, Without mCD59. The formation of C5b-9 neoepitope or the binding of CD59 on the bacteria was examined with a whole-cell ELISA method using specific antibodies. Results from control incubations performed by omitting the primary antibody have been subtracted as background (OD < 0.3). Absorbances in wells with NHSi-incubated bacteria were subtracted as background. Results are shown as mean ± SD (n = 6). Bacteria-bound CD59 inhibited C5b-9 neoepitope formation on CagA+ strains 11637 and 9:0 significantly (P < 0.05) when 5% serum was used. (B) The inhibition of C5b-9 formation is inversely proportional to CD59 binding to the CagA+ strains 11637 and 9:0. Correlation between CD59 binding and C5b-9 assembly on H. pylori was analyzed by simple linear regression. The correlation is significant for the CagA+ strains (P < 0.01). Gastroenterology 2001 120, 470-479DOI: (10.1053/gast.2001.21197) Copyright © 2001 American Gastroenterological Association Terms and Conditions