1 Helicobacter pylori arginase inhibits nitric oxide production by eukaryotic cells: A strategy for bacterial survival PNAS November 20, 2001 vol. 98 no –13849 Reporter : 翁國曜 Oct. 15,2002 結構基因體特論

2 Nitric oxide-dependent killing Some cytokines can also induce phagocytic cells, particularly macrophages, to produce nitric oxide (NO), which is toxic to microorganisms and malignant cells Innate Immunity !!

3 NOS catalyzes the net reaction: L-Arginine + n NADPH + m O2 = Citrulline + Nitric oxide + n NADP+ with the intermediate N-(omega)-Hydroxyarginine (C05933) Three forms of nitric oxide synthase - a neuronal type called nNOS, an epithelial type called eNOS, and an inducible form called iNOS, iNOS is expressed under certain conditions like immune system regulation by cytokines or pathological induction in the presence of endotoxins (bacterial lipopolysaccharide) and cytotoxins (which affect cytokine secretion). Nitric oxide synthase Bacterial endotoxins (e.g. from H.pylori or E.coli infections) induce the iNOS gene, which in turn produces high levels of NO damaging pathogenic DNA and inhibiting respiration

4 In mammalian L-arginine NO synthases O2O2 NO + citrulline arginase Urea + L-ornithine rocF gene Toxic (H. P.) Subject H.P. infection

5 Bacteria H. pylori strains SS1 and Cells murine macrophage (cell line RAW 264.7) peritoneal macrophages,(c57BL/6 WT and iNOS-/- mice) NO,NO 2 - Concentration L-Arginine Concentration Northern Blot Analysis Materials and Methods HP1399 arginase (rocF) Mutant : inserting aphA3 cassette into rocF gene NO2 was assessed by the Griess reaction, NO by NO analyzer By HPLC

6 Results (coculture H.P. and cell line macrophages) WTrocF - MOI multiplicity of infection Unpaired p test *, p<0.05 **, p<0.01 ***, p<0.001 L-arginine concern. 0.4 mM --Very similar results in H. P The competitive inhibition between bacterial arginase and macrophage iNOS L-arginine NO synthases O2O2 NO + citrulline arginase Urea + L-ornithine Observing product

7 L-arginine NO synthases O2O2 NO + citrulline arginase Urea + L-ornithine Observing reactant Results (coculture H.P. and cell line macrophages) iNOS mRNA level of macrophage -- H.P. arginase effectively decreases L-arginine in the medium, leading to the decrease of macrophage NO production by means of loss of substrate availability for iNOS. -- Bacterial arginase regulates macrophage NO production independently of effects on iNOS expression.

8 L-arginine iNO synthases O2O2 NO + citrulline arginase Urea + L-ornithine IFN-  -activated macrophages -- Bacterial arginase acts to inhibit NO release by preformed iNOS. Results (coculture H.P. and cell line macrophages) live dead -- Viable bacteria efficiently consume L-arginine available in the extracellular environment, resulting in effective inhibition of NO production. WT rocF -

9 Results (coculture H.P. and macrophages) cell line macrophages Mice peritoneal macrophages -- Bacterial arginase contributes to the viability of H. pylori. WT rocF fold decrease in survival with the WT No killing with iNOS -/- killing of H. pylori was NO-dependent rocF - H.P. in WT and iNOS-/- macrophages

10 Discussion Pros: 1. The extracellular H. pylori can be killed by NO released from activated macrophages. 2. H. pylori arginase competes with host cell iNOS for the common substrate L-arginine. 3. These observations are independent of any effect on iNOS transcriptional regulation. 4. a unique pathway of immune escape by extracellular-infecting bacteria. Cons: The SS1 rocF mutant strain used in the current study has attenuated ability to colonize mice => the role of iNOS-derived NO in H.pylori infection in vivo? Aftering infecting iNOS and WT C57BL6 mice with WT SS1, No significant difference in colonization levels or histologic injury scores, despite consistent expression of iNOS in the WT mouse stomach !!

11 Discussion Hypothesis (based on the in vitro data) The lack of difference between iNOS-deficient and WT mice in H. pylori infection could be related to the ability of H. pylori arginase to modulate mucosal NO synthesis by iNOS in WT mice. => Further experiments have been initiated to compare the rocF::aphA3 to WT SS1 strains in iNOS/ vs. WT mice Possible reasons: 1. H. pylori arginase could directly modulate the iNOS-derived NO levels because of this close interaction between bacteria and the iNOS-expressing cells. 2. There is also nonenzymatically formed NO in the gastric juice caused by acidification of salivary nitrate and nitrite.

12 Discussion 1. L-Arginine is not synthesized by H. pylori; H.P. must obtain this amino acid from extracellular sources. L-arginine consumption by WT H. pylori strains explains the inhibition of host NO production. 2. Altered L-arginine uptake by the macrophages or metabolism of L-arginine by other biochemical pathways. In the case, the lack of significant NO production by macrophage iNOS, at the residual level of 0.1 mM L-arginine in the medium. 3. Viable bacteria efficiently consume L-arginine available in the extracellular environment, providing a means to decrease macrophage NO production. (c.f. heat-killed, water extracts and French press lysates cases) 4. H. pylori arginase has several unusual characteristics for enhancement of bacterial survival, -- Not require de novo gene transcription to prevents NO production -- H. pylori arginase is constitutively expressed and present in all strains, suggesting that arginase has evolved in H. pylori as an important enzyme.

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