Peptidoglycan Deacetylation in Helicobacter pylori Contributes to ...

INFECTION AND IMMUNITY, Nov. 2010, p. 4660–4666 0019-9567/10/$12.00 doi:10.1128/IAI.00307-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Vol. 78, No. 11

Peptidoglycan Deacetylation in Helicobacter pylori Contributes to Bacterial Survival by Mitigating Host Immune Responses䌤 Ge Wang, Susan E. Maier, Leja F. Lo, George Maier, Shruti Dosi, and Robert J. Maier* Department of Microbiology, University of Georgia, Athens, Georgia 30602 Received 26 March 2010/Returned for modification 6 May 2010/Accepted 17 August 2010

An oxidative stress-induced enzyme, peptidoglycan deacetylase (PgdA), in the human gastric pathogen Helicobacter pylori was previously identified and characterized. In this study, we constructed H. pylori pgdA mutants in two mouse-adapted strains, X47 and B128, to investigate the role of PgdA in vivo (to determine the mutants’ abilities to colonize mice and to induce an immune response). H. pylori pgdA mutant cells showed increased sensitivity to lysozyme compared to the sensitivities of the parent strains. We demonstrated that the expression of PgdA was significantly induced (3.5-fold) when H. pylori cells were in contact with macrophages, similar to the effect observed with oxidative stress as the environmental inducer. Using a mouse infection model, we first examined the mouse colonization ability of an H. pylori pgdA mutant in X47, a strain deficient in the major pathway (cag pathogenicity island [PAI] encoded) for delivery of peptidoglycan into host cells. No animal colonization difference between the wild type and the mutant was observed 3 weeks after inoculation. However, the pgdA mutant showed a significantly attenuated ability to colonize mouse stomachs (9-fold-lower bacterial load) at 9 weeks postinoculation. With the cag PAI-positive strain B128, a significant colonization difference between the wild type and the pgdA mutant was observed at 3 weeks postinoculation (1.32 ⴛ 104 versus 1.85 ⴛ 103 CFU/gram of stomach). To monitor the immune responses elicited by H. pylori in the mouse infection model, we determined the concentrations of cytokines present in mouse sera. In the mice infected with the pgdA mutant strain, we observed a highly significant increase in the level of MIP-2. In addition, significant increases in interleukin-10 and tumor necrosis factor alpha in the pgdA mutant-infected mice compared to the levels in the wild-type H. pylori-infected mice were also observed. These results indicated that H. pylori peptidoglycan deacetylation is an important mechanism for mitigating host immune detection; this likely contributes to pathogen persistence. island (PAI), resulting in the induction of interleukin-8 (IL-8) production through the Nod1 pathway (31). It remains unclear whether H. pylori evades or alters a robust host immune response by modifying its PG structure. Recently, we identified and characterized an H. pylori protein (HP310) whose expression was significantly induced under oxidative stress conditions (36). HP310 turned out to be an enzyme catalyzing PG modification: it has PG N-deacetylase activity. Now we rename it PgdA, as it is functionally homologous to the PgdAs from Listeria (5) and Streptococcus (11, 32) species, although it has limited sequence homology to its Gram-positive counterparts. The true homologues of H. pylori PgdA are present in several Gram-negative pathogenic bacteria, but none of them has been studied. Thus, H. pylori PgdA is a representative of a new subfamily of bacterial PG deacetylases. PG N-deacetylation in Listeria (5) and Streptococcus (11) species was shown to be a virulence factor, playing an important role in evasion of the host innate immune response so that the pathogen survives in vivo. In this study, we investigated the role of H. pylori PgdA in bacterial survival/persistence and in circumventing part of the host immune response by using a mouse infection model.

A critical step in the innate immune response is the identification of an invading organism as foreign. This step involves interaction between host receptors and microbial structural motifs (19). In recent years, bacterial peptidoglycan (PG), one of the main protective barriers in the bacterial cell wall, has been shown to contain structural motifs that can be recognized by host receptors such as Nod1 and Nod2 (18, 29). For example, mammalian Nod1 specifically senses PG degradation products of Gram-negative bacteria, resulting in activation of the transcription factor NF-␬B pathway (14, 15, 18). Helicobacter pylori, a pathogenic bacterium infecting over 50% of humans, is the etiologic agent for gastritis, peptic ulcer, and gastric cancer (9). During the process of colonizing the host, H. pylori induces a strong inflammatory response that generates large amounts of reactive oxygen species (ROS). However, H. pylori survives these oxidative stress conditions via a battery of diverse activities (detoxification and repair), and it persistently colonizes the gastric mucosa (33). In addition to the detoxification and repair activities, H. pylori likely uses other mechanisms to evade host immune response, contributing to life-long infection. As a noninvasive pathogen, H. pylori can inject its PG into the host epithelial cells via a bacterial type IV secretion system encoded by the cag pathogenicity

MATERIALS AND METHODS

* Corresponding author. Mailing address: Department of Microbiology, 815 Biological Sciences Building, University of Georgia, Athens, GA 30602. Phone: (706) 542-2323. Fax: (706) 542-2674. E-mail: rmaier @uga.edu. 䌤 Published ahead of print on 30 August 2010.

H. pylori strains and growth conditions. H. pylori strains X47 and B128 represent the wild-type (WT) strains used for the studies described herein. Cultures were grown microaerobically at 37°C in CO2 incubators under a controlled oxygen concentration as indicated below. Brucella agar (BA; Difco) was used as the base ingredient in plates that were supplemented with 10% defibrinated

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VOL. 78, 2010 sheep’s blood (Gibson Laboratories, Inc.). Kanamycin and chloramphenicol antibiotics were used at a concentration of 40 ␮g/ml as indicated below. pgdA mutant construction in mouse-colonizing strains. A plasmid containing the H. pylori pgdA gene (HP310) sequence disrupted with a kanamycin resistance cassette (pGEM-pgdA:Kan from reference 36) was used to transform H. pylori wild-type strains X47 and B128 by natural transformation. The mutants were selected on blood agar plates supplemented with kanamycin. Genomic DNA was prepared from the mutant clones, and the disruption of the pgdA gene on the genome was confirmed by a 1.4-kb-larger increment of the PCR amplicon due to the insertion of the antibiotic cassette within the gene. Protein gel electrophoresis and Western blotting. Bacteria were harvested from the plates and resuspended in phosphate-buffered saline (PBS) containing 20 mM sodium phosphate and 150 mM NaCl, pH 8.0. After one wash with the buffer, cells were resuspended in the same buffer and were broken by two passages through a French pressure cell at 138,000 kPa (SLM Instruments, Inc.). The cell lysates were obtained by centrifugation (8,000 rpm for 10 min), and the supernatant was transferred to a clean tube. The protein concentration of the cell extract was determined with a Bradford protein assay (Bio-Rad). Seven micrograms of cell extract was mixed with the SDS buffer and incubated at 90°C for 5 min. Proteins were then separated on a 12.5% SDS–PAGE gel by electrophoresis for 1.5 h at 100 V. The gels were either stained with Coomassie blue to visualize total proteins or subjected to Western blotting to identify the PgdA protein. For the Western blot assay, the proteins on the gel were electrotransferred onto a nitrocellulose membrane. The membrane was then incubated with anti-PgdA antiserum (36) (1:500 dilution), followed by incubation with secondary goat anti-rabbit IgG conjugated with alkaline phosphatase (1:1,000 dilution; Bio-Rad). PgdA induction in H. pylori cells during contact with macrophages. HL-60 human polymorphonuclear leukocyte (PMN)-like cells were grown at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) with 10% heatinactivated fetal bovine serum in a tissue culture flask. The cells were passaged every 3 to 5 days as required. Prior to the assays, the HL-60 cells were incubated with 1 ␮M retinoic acid (Sigma, St. Louis, MO) in a 24-well plate for 4 days to stimulate differentiation and maturation of the PMNs. Separately, H. pylori cells were grown on blood agar plates to late log phase (1.5 days for the WT and 2 days for the pgdA mutant) and harvested by suspension in PBS at a concentration of ⬃108 CFU/ml. A 20-␮l amount of this bacterial suspension was added into 1 ml of the macrophage culture (one well in a 24-well plate) containing differentiated HL-60 cells adhering on the bottom of the plate (at a ratio of ⬃20 CFU bacteria per macrophage). As a control, the bacterial suspension was added to a well containing 1 ml DMEM medium with no macrophages. Phagocytosis was synchronized by centrifuging the 24-well plate at 1,000 ⫻ g for 5 min, and the plate was incubated at 37°C in an atmosphere composed of 5% CO2, 4% O2, and the balance N2 (partial pressures). After 4 h of incubation, the 24-well plate was centrifuged again at 1,000 ⫻ g for 5 min, the medium was removed, and H. pylori cells together with macrophages were suspended in PBS. The samples were further analyzed by protein SDS-gel electrophoresis and Western blotting as described above. The anti-PgdA antiserum (1:500 dilution) and anti-UreA antiserum (for an internal control, 1:1,000 dilution) were used simultaneously in this experiment. Cell survival assay for assessing lysozyme sensitivity. H. pylori strains were grown on BA plates to late log phase, and the cells were suspended in PBS at a concentration of ⬃109 cells/ml. Upon the addition of lysozyme (final concentration, 50 mg/ml), the cell suspensions were incubated at 37°C in a 2% O2 atmosphere with occasional shaking. Samples were then removed at various time points (0, 2, 4, and 6 h), serially diluted, and spread onto BA plates. Colony counts were recorded after 4 days of incubation in a microaerobic atmosphere (2% O2) at 37°C. Mouse colonization. The mouse colonization assays have been well described in reports from our laboratory (34, 35, 37). The conventional procedure is briefly described as follows. H. pylori cells were harvested after 48 h of growth on BA plates (37°C, 5% oxygen) and suspended in PBS to an optical density at 600 nm (OD600) of 1.7. Headspace in the tube was sparged with argon gas to minimize oxygen exposure, and the tube was tightly sealed. Food and water were withheld from the mice for 1.5 to 2 h prior to inoculation. The bacterial suspensions were administered to C57BL/6J mice (3 ⫻ 108 H. pylori cells/mouse) twice, with each of the oral deliveries made 2 days apart. Three weeks after the first inoculation, the mice were sacrificed and the stomachs were removed, weighed, and homogenized in argon-sparged PBS (28) to avoid O2 exposure. Stomach homogenate dilutions (dilutions were made in argon-sparged buffer in sealed tubes) were plated on BA plates supplemented with bacitracin (100 ␮g/ml), vancomycin (10 ␮g/ml), and amphotericin B (10 ␮g/ml), and the plates were rapidly transported into an incubator containing sustained 5% (partial pressure) O2. After incuba-

ROLE OF H. PYLORI PgdA IN VIVO

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FIG. 1. Western blot analysis for PgdA in H. pylori strains. H. pylori wild-type or pgdA mutant strains were grown to late log phase in an atmosphere containing 4% O2. Seven micrograms of crude cell extract was resolved on an SDS–12.5% polyacrylamide gel, followed by transfer onto a nitrocellulose membrane, and the PgdA protein was detected using anti-PgdA antibody. Lanes: 1, X47 WT cells; 2, X47 pgdA::Kan mutant cells; 3, B128 pgdA::Kan mutant cells. 4, B128 WT cells; P, purified 6⫻His-tagged PgdA protein.

tion for 5 to 7 days, the fresh H. pylori colonies were enumerated and the data expressed as CFU per gram of stomach. In this study, two mouse-adapted H. pylori strains, X47 and B128, as well as the corresponding pgdA mutants, were examined for mouse colonization ability. In addition to the conventional time frame (examination at 3 weeks after the first inoculation), survival counts were done at different time spans (4 days, 3 weeks, 6 weeks, or 9 weeks after the first inoculation) for different H. pylori strains. Determination of cytokine concentrations in mouse serum. As described in the mouse colonization experiments, the mice were inoculated with H. pylori (WT or pgdA mutant) two times (day 1 and day 3) with a dose of 3 ⫻ 108 viable cells administered per animal each time. Three weeks after the first inoculation, blood samples (from 8 mice for each group) were collected upon guillotining each anesthetized (overdose) animal and collecting 4 to 6 drops of blood from the animal. The concentrations of 9 cytokines in the mouse sera were determined simultaneously by using Bio-Plex cytokine assay kits (Bio-Rad). This is a multiplex bead-based assay with a sandwich immunoassay format (beads-antibodycytokine-biotinylated detection antibody). The results were read and analyzed with the Bio-Plex suspension array system (Bio-Rad).

RESULTS Construction of H. pylori pgdA mutants with mouse-colonizing strains. The primary goal of this study was to investigate the effect of PgdA disruption on the ability of H. pylori to colonize host (mouse) stomachs and to induce an immune response. Previously, the pgdA mutants were constructed in two H. pylori strains, 43504 and 26695 (36), which are not strains adapted for mouse colonization assays. Mouse-adapted H. pylori strains SS1 and X47 have been used in our laboratory for mouse colonization assays (34, 35, 37). However, strain X47 does not contain the cag PAI in its genome (1). Strain SS1 carries an incomplete cag PAI that seems to be nonfunctional (7), and it is unable to induce NF-␬B activation or IL-8 production in gastric epithelial cells (24). Because the major pathway for H. pylori PG to induce host immune response requires functional cag PAI (31), in this study, we used another strain, B128, which contains a functional cag PAI and is a moderate mouse colonizer (16). The pgdA::Kan mutants were constructed in strains X47 and B128. PCR analysis (not shown) indicated the correct insertion of a kanamycin resistance cassette within the pgdA gene on the genome. Crude extracts from wild-type and pgdA mutant cells were resolved on the SDS-PAGE gel, followed by Western blot analysis using anti-PgdA antibody. The PgdA protein band is present in both wild-type strains but is missing in the mutants (Fig. 1), confirming the correct construction of the pgdA mutants in the two mouse-colonizing strains. Previously, we showed that H. pylori pgdA mutant cells are more sensitive to lysozyme than the wild type (36). Although the high concentration of lysozyme used for H. pylori sensitivity

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FIG. 2. Lysozyme sensitivity. Cell survival curves of H. pylori strains after treatment with 50 mg/ml lysozyme for the time indicated on the x axis. The data are the means and standard deviations of the results of three experiments.

tests may not be physiologically relevant, this is nevertheless a significant phenotype that distinguishes the pgdA mutant from the wild type. Therefore, the lysozyme sensitivity of the pgdA mutants in the two mouse-colonizing strains was determined by examining the cell survival curve after treatment with 50 mg/ml lysozyme (Fig. 2). Two hours after the treatment, a slight difference was noticed between the wild type and the mutant, but that difference was statistically significant only at the 90% level (P ⬍ 0.1). However, the number of viable pgdA mutant cells decreased significantly more rapidly than the WT after that point. Six hours after the treatment, about 106 viable cells of the wild-type strains survived, while the mutant cells were completely killed (for B128 pgdA::Kan) or only a few (⬍100) viable cells survived (X47 pgdA::Kan). The differences between the data for the mutants and for the corresponding wild type for the 4-h and 6-h time points analyzed by Student’s t test are highly significant (P ⬍ 0.001). The results clearly indicated that PgdA plays a significant role in protecting H. pylori PG from lysozyme digestion. The expression of PgdA in H. pylori is induced upon contact with macrophages. H. pylori PgdA protein was significantly overexpressed when cells were exposed to oxidative stress conditions, although the enzyme itself is not an oxidative stresscombating factor (36). In this study, the overexpression of PgdA by oxidative stress in the X47 strain was also examined by Western blotting, and a result similar to the one we reported previously was observed: the expression of PgdA in the cells grown at 12% O2 is about 3-fold higher than the level in cells grown at 2% O2 (data not shown). It is known that H. pylori induces macrophages to produce large amounts of reactive oxygen species. Since H. pylori encounters oxidative stress in the host, we hypothesized that oxidative stress serves as a signal for inducing PgdA expression in the host. To test this hypothesis, we cocultured H. pylori with macrophages (HL-60 cell line) in DMEM medium under conditions of 4% O2 atmosphere at 37°C for 4 h. At the beginning of this 4-h incubation period, the samples were subjected to centrifugation so that H. pylori cells were bound to and/or internalized into macrophages. As a control, H. pylori cells were incubated under the

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FIG. 3. PgdA is induced in H. pylori upon contact with macrophages. H. pylori (WT or pgdA mutant) cells were incubated with macrophages (HL-60) in DMEM medium at 4% O2 and 37°C for 4 h. H. pylori cells that were bound to and/or internalized in macrophages were collected, and the proteins were analyzed by SDS-PAGE followed by detection with anti-PgdA antibody and anti-UreA antibody (as an internal loading control). Lanes: 1, H. pylori X47 cultured alone; H. pylori X47 upon contact with macrophages; 3, H. pylori X47 pgdA::Kan mutant cultured alone; 4, H. pylori X47 pgdA::Kan mutant upon contact with macrophages.

same conditions (in DMEM medium) but with no macrophages. H. pylori cells were then collected (together with macrophages), and the proteins were analyzed by SDS-PAGE followed by detection with anti-PgdA antibody and anti-UreA antibody (as an internal loading control) (Fig. 3). The expression of PgdA was significantly induced when the WT H. pylori cells were in contact with macrophages (Fig. 3, lane 2 versus lane 1). Based on densitometry measurement of the protein bands, the induction is 3.5-fold upon contact with macrophages, a level of induction similar to the effect observed previously with oxidative stress as the environmental inducer. As a negative control, PgdA protein was not expressed in the pgdA::Kan mutant cells (Fig. 3, lanes 3 and 4). We also tested a murine cell line (RAW 264.7), with qualitatively similar results to the results for the human cell line (data not shown). H. pylori pgdA mutants have attenuated ability to colonize mouse stomachs. To investigate the physiological role of PgdA in vivo with a mouse model, we first compared the mouse colonization ability of the X47 pgdA::Kan mutant to that of the wild type (Table 1). The two strains were individually inoculated into 10 C57BL/6J mice, and the colonization of stomachs was examined 3 weeks after inoculation. There was no differ-

TABLE 1. Mouse colonization abilities of H. pylori X47 WT and pgdA mutant strainsa H. pylori strain

No. of mice colonizedc

Bacterial load ⫾ SD (CFU/mg of stomach)d

3

X47 WT X47 pgdA::Kan

10/10 10/10

691 ⫾ 103 598 ⫾ 184

6


10/10 10/10

738 ⫾ 109 402 ⫾ 224

9


10/10 7/10

536 ⫾ 212 59 ⫾ 97

Time after inoculation (wk)b

a Groups of 10 mice were inoculated with H. pylori two times, 2 days apart, with a dose of 1.5 ⫻ 108 viable cells administered per animal each time. b Colonization of H. pylori in mouse stomachs was examined 3, 6, or 9 weeks after the first inoculation. c Number of mouse stomachs from which H. pylori was recovered/total number of stomachs assayed. d Number of H. pylori cells colonized in mouse stomach averaged from the 10 mice.

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FIG. 4. Mouse colonization results for H. pylori B128 and its isogenic pgdA mutant. The mice were inoculated with H. pylori two times, 2 days apart, with a dose of 3 ⫻ 108 viable cells administered per animal each time. Colonization of H. pylori in mouse stomachs was examined 3 weeks after the first inoculation. Data are presented as a scatter plot (at log scale) of CFU per gram of stomach as determined by plate counts. Each point represents the CFU count from one mouse stomach, and the solid lines represent the geometric means of the colonization numbers for each group (WT or pgdA mutant). The baseline [log10(CFU/g) ⫽ 2.7] is the detection limit of the assay, which represents a count below 500 CFU/g of stomach.

ence at all between the wild type and the mutant. However, considering that X47 is deficient in the major pathway (cag PAI) for delivery of PG into host cells, the immune response may be a later one. Therefore, we extended the stomach harvest time to 6 and 9 weeks after inoculation to examine the longer-term colonization effect. At 6 weeks, there were lower bacterial loads in the mice infected with the pgdA mutant than in mice infected with the WT H. pylori. A highly significant colonization difference between the strains was observed 9 weeks after inoculation. H. pylori was recovered from all 10 mice that had been inoculated with the wild-type strain, with a mean number of 5.36 ⫻105 CFU/g stomach. In contrast, 7 of 10 mice that were inoculated with the pgdA mutant strain were found to harbor H. pylori, and the mean bacterial load for the mutant was 5.9 ⫻104 CFU/g stomach. According to Wilcoxon signed-rank test analysis, the range of colonization values of the mutant strain is significantly smaller than that of the wild type at the 99% confidence level (P ⬍ 0.01). These results indicated that PgdA plays a significant role in H. pylori survival/colonization in the host. Next, we examined the mouse colonization effect of PgdA

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using the cag PAI-positive strain B128. The wild-type B128 or the pgdA mutant strain was inoculated into 11 mice, and the colonization of H. pylori cells in the mouse stomachs was examined 3 weeks after inoculation (Fig. 4). H. pylori cells were recovered from all 11 mice that had been inoculated with the wild-type B128, with a mean number of 1.32 ⫻104 CFU/g stomach, which is ⬃40-fold lower than that for the other wildtype strain, X47. In contrast, 8 of 11 mice that were inoculated with the pgdA mutant strain were found to harbor H. pylori, and the mean bacterial load was 1.85 ⫻103 CFU/g stomach. According to Wilcoxon rank test analysis, the range of colonization values of the B128 pgdA::Kan mutant strain was significantly smaller than that of the wild-type B128 at the 99% confidence level (P ⬍ 0.01). These results confirmed the role of PgdA in H. pylori survival/colonization in the host and further indicated that the effect takes place earlier with the cag PAI-positive strain (which can deliver bacterial PG into host cells more efficiently). H. pylori pgdA mutants induce a stronger immune response in the host. A key element in the host response to H. pylori infection is the production of proinflammatory cytokines in the gastric mucosa (2). To monitor the immune responses elicited by H. pylori pgdA mutant cells in comparison to those elicited by wild-type cells in the mouse infection model, we proceeded to determine the concentrations of cytokines present in mouse sera. Using Bio-Plex cytokine assay kits from Bio-Rad, we were able to determine the concentrations of multiple cytokines in mouse sera simultaneously. We chose a mouse serum 8-plex kit which determines the concentrations of IL-1␤, IL-2, IL-4, IL-5, IL-10, granulocyte-macrophage colonystimulating factor (GM-CSF), gamma interferon (IFN-␥), and tumor necrosis factor alpha (TNF-␣). In addition, we included macrophage inflammatory protein 2 (MIP-2), a murine chemokine reported to have biological functions analogous to those of human IL-8 (23, 26). Since we observed a significant effect of the B128 pgdA mutation on the mouse colonization ability at 3 weeks, we performed the cytokine assays to compare the effects in the B128 pgdA mutant-infected mice to the effects in the animals infected with the wild type at 3 weeks. The mice were inoculated with H. pylori cells (WT or pgdA mutant, as well as a mock inoculation with PBS) two times, 2 days apart, with a dose of 3 ⫻ 108 viable cells administered per animal each time. Three weeks after the first inoculation, blood samples (from 8 mice for each group) were collected and serum samples were prepared. The concentrations of the 9 cytokines in mouse sera were determined (Table 2). The concentrations of IL-2 and

TABLE 2. Levels of various cytokines in the sera of mice infected with H. pylori B128 WT or pgdA mutant strainsa Concn (ng/ml) of cytokine in mouse sera (mean ⫾ SD)

H. pylori strain or control

IL-1␤

IL-5

IL-10b

GM-CSF

IFN-␥

TNF-␣b

MIP-2b

Mock (PBS) B128 WT B128 pgdA::Kan

182 ⫾ 81 203 ⫾ 134 197 ⫾ 189

104 ⫾ 43 114 ⫾ 112 157 ⫾ 123

9⫾4 13 ⫾ 7 23 ⫾ 11

49 ⫾ 28 57 ⫾ 40 42 ⫾ 27

53 ⫾ 28 62 ⫾ 56 59 ⫾ 41

122 ⫾ 37 145 ⫾ 19 194 ⫾ 64

157 ⫾ 58 188 ⫾ 94 397 ⫾ 175

a Mice were inoculated with H. pylori (WT or pgdA mutant) two times, 2 days apart, with a dose of 3 ⫻ 108 viable cells administered per animal each time. Three weeks after the first inoculation, blood samples (from 8 mice for each group) were collected and serum samples were prepared. The concentrations of 9 cytokines in mouse sera were determined with a Bio-Plex cytokine assay (Bio-Rad). The data for IL-2 and IL-4 were omitted from the table because they were below the detection level. b The concentrations of indicated cytokines in mice infected with the H. pylori pgdA mutant were significantly higher than in the wild-type H. pylori-infected mice, based on Student’s t test analysis.

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IL-4 were below the detection level for all samples; the data for the other 7 cytokines are presented. According to Student’s t test analysis, there were no significant differences among the 3 groups of mice for the concentrations of IL-1␤, IL-5, GM-CSF, and IFN-␥. However, the concentrations of IL-10, TNF-␣, and MIP-2 in the mice infected with the H. pylori pgdA mutant were significantly higher than the concentrations in mice infected with the H. pylori WT strain (P values of 0.05, 0.05, and 0.01, respectively). We also measured the cytokine concentrations at an early time postinoculation. Eight mice were inoculated with H. pylori (WT or pgdA mutant, as well as a mock inoculation with PBS) two times (day 1 and day 3) with a dose of 3 ⫻ 108 viable cells administered per animal each time. Cytokine assays were performed at day 5. Compared to the background levels (in mockinfected mice), there were no significant changes in the serum concentration of any cytokine in mice inoculated with the wildtype H. pylori or the pgdA mutant (data not shown). DISCUSSION H. pylori colonization in human gastric mucosa is usually associated with a chronic inflammatory and immune response that probably accounts for the disease outcomes. However, this immune response does not clear H. pylori infection. This raises the question of how H. pylori evades robust host immune detection and is thus adapted to long-term persistence in the host. In this study, using a mouse infection model, we found that PG deacetylation is an important factor in H. pylori’s mitigation of host immune responses and contributes to the bacterium’s survival/persistence in the host. In our previous study, we discovered a novel PG deacetylase (PgdA) in H. pylori (36) which has a limited sequence homology but a similar function to the known PgdAs from Grampositive bacteria. We showed that the inactivation of pgdA resulted in the absence or significantly reduced amounts of deacetylated muropeptides in H. pylori PG, and we demonstrated a PG deacetylase activity with the purified PgdA protein in vitro. To investigate the role of PgdA in vivo (for the bacterium’s ability to colonize mice and to induce identified immune responses), we constructed H. pylori pgdA mutants in two mouse-adapted strains, X47 and B128. The correct construction of these pgdA mutants was confirmed by Western blot analysis using anti-PgdA antibody, showing that the PgdA protein is expressed in wild-type cells but not in the mutants (Fig. 1). A significant phenotypic differentiation from the wild type in vitro, namely, lysozyme sensitivity, was also demonstrated for the pgdA mutants in the two mouse-colonizing strains (Fig. 2). PG N-deacetylation was previously shown to be the direct cause of lysozyme resistance (36); the new results for lysozyme sensitivity indicate that loss of PgdA results in loss of PG N-deacetylation in the two mouse-colonizing strains. A novel characteristic of H. pylori PgdA is its induction by oxidative stress in vitro. It is well known that H. pylori infection generates oxidative stress in the host. For example, H. pylori stimulates the generation of NO in macrophages through the enzyme-inducible NO synthase (iNOS) (39), and H. pylori induces extracellular release of oxygen radicals from professional phagocytes (25). Macrophages are essential as innate responders to H. pylori-derived products and to signals from epithelial

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cells in direct contact with the bacterium on the surface of the mucosa (38). H. pylori cells have been shown to be bound to erythrocytes within the microvessels of the lamina propria (3). Furthermore, it was demonstrated that H. pylori cells are in direct contact with immune cells of the lamina propria in the majority of cases of gastritis and gastric cancer (21). We sought to test the hypothesis that the contact of H. pylori cells with host immune cells induces the overexpression of PgdA in the same way as it is induced by oxidative stress in vitro. The results from the experiments wherein H. pylori was attached to macrophages (Fig. 3) clearly showed that the expression of PgdA was significantly induced when the H. pylori cells were in contact with macrophages. Thus, oxidative stress encountered by H. pylori in vivo likely serves as a signal to induce the expression of PgdA. Previously, we showed that H. pylori PG purified from wild-type cells cultured in vitro (low-oxygen condition) is partially deacetylated (36). The results presented herein suggest that H. pylori PgdA expression would be induced in vivo, and thus, the extent of bacterial PG deacetylation is likely higher than that observed in vitro. Due to technical limitations, however, we were unable to directly measure the composition of PG within H. pylori cells attached either to macrophages or infected mouse stomachs. Interestingly, recent work on Streptococcus suis PgdA showed that the gene is upregulated upon interaction with porcine neutrophils (11). Thus, the induction of PgdA in vivo (by oxidative stress) may be common in many pathogenic bacteria, resulting in the effects on immune responses and bacterial survival in vivo discussed below. The major goal of this study was to investigate the role of PG deacetylation of H. pylori in escaping a portion of the host immune system and contributing to bacterial survival in the host. Using a mouse model, we first examined the mouse colonization ability of the H. pylori pgdA mutant in the strain X47 background. The results (Table 1) showed that there was no colonization difference between the wild type and the mutant at 3 weeks postinoculation. However, 9 weeks after inoculation, the pgdA mutant has a significantly attenuated ability to colonize mouse stomachs. X47 is deficient in the major pathway (cag PAI) for delivery of PG into host cells. In the absence of a functional cag PAI, PG delivery may still occur but with much lower efficiency (31). Most recently, a novel mechanism for PG delivery was found: gram-negative bacteria, including H. pylori, can deliver PG to cytosolic Nod1 in host cells via outer membrane vesicles (17). This may explain our results of a later response and the colonization effect observed with the X47 pgdA mutant. With the cag PAI-positive strain B128, a significant difference between colonization by the wild type and by the pgdA mutant was observed at 3 weeks postinoculation (Fig. 4). These results indicate that PgdA plays a significant role in H. pylori survival/colonization in the host. Mouse models have been extensively used to study the inflammatory responses induced by H. pylori infection. It is well established for both humans and C57BL/6 mice that H. pylori induces a robust Th1 proinflammatory response that is associated with gastric inflammation, atrophy, epithelial hyperplasia, and dysplasia (12). To investigate the role of H. pylori PgdA in evading host immune responses, we used mouse serum as the starting material for the cytokine assays. IL-8 is known to be a key mediator of the inflammatory responses in H. pylori-infected individuals, but mice lack IL-8. The murine chemokines

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KC (CXCL1) and MIP-2 (CXCL2) were reported to have biological functions analogous to those of human IL-8 (23, 26), and they were produced by a murine gastric epithelial cell line in response to stimulation with H. pylori strains (10). Using the Bio-Plex cytokine assay system, we determined the concentrations of nine cytokines in mouse serum, including IL-1␤, IL-2, IL-4, IL-5, IL-10, GM-CSF, IFN-␥, TNF-␣, and MIP-2. The results (Table 2) revealed that the concentrations of IL-10, TNF-␣, and MIP-2 in the mice infected with the H. pylori pgdA mutant are significantly higher than those in mice infected with the H. pylori wild-type strain. It is well documented that several cytokines are expressed in human gastric epithelial cells in response to H. pylori infection. For example, the gastric mucosa of H. pylori-infected patients has increased levels of proinflammatory cytokines such as IL-8 and TNF-␣ (8, 13, 22, 40). IL-8 and TNF-␣ have a central role in the modification of the cellular microenvironment (4, 30, 40). IL-8, in particular, plays an important role in activating and recruiting neutrophils in response to infection by H. pylori. The epithelium is the greatest source of IL-8 in the gastric mucosa, and the coculture of H. pylori cag PAI-positive strains with epithelial cells stimulates the secretion of IL-8 (6). In our cytokine assay results, the level of MIP-2 (IL-8 analog) in mouse serum increased slightly in response to infection with wild-type H. pylori (188 ⫾ 94 [mean ⫾ standard deviation] versus 157 ⫾ 58 ng/ml). In contrast, a substantial increase in the level of MIP-2 was observed in the mice infected with the pgdA mutant strain (397 ⫾ 175 ng/ml). In addition, increases (at a statistically significant level of 95%) in IL-10 and TNF-␣ in the pgdA mutant-infected mice compared to the levels in the wild-type H. pylori-infected mice were also observed. Tumor necrosis factor ␣ (TNF-␣) is an extremely pleiotropic factor with potent proinflammatory effects that play a major role in initiating and amplifying the immune-inflammatory responses to H. pylori infection (27). TNF-␣ induces fas-mediated apoptosis and disruption of the epithelial barrier to facilitate the translocation of bacterial antigens (30). It was reported that H. pylori primarily stimulated an IL-10 response in blood-derived monocytes (20). Our results showing increases in IL-10 and TNF-␣ in mouse serum after infection with a pgdA mutant strain of H. pylori further support the idea that PgdA lessens some aspects of the host immune response. This is in agreement with the data for mouse colonization showing that the pgdA mutant has less ability to survive in the stomach. In summary, H. pylori PG deacetylation is an important mechanism for mitigating some host immune responses, thereby contributing to persistent and perhaps life-long infection in the host. ACKNOWLEDGMENTS This work was supported by NIH grants no. R01AI077569 to R.J.M. and R21AI076569 to G.W. and by the University of Georgia Foundation. We thank Martin Blaser for providing H. pylori strain B128 and Julie Nelson for help with the Bio-Plex cytokine assay. REFERENCES 1. Akada, J. K., K. Ogura, D. Dailidiene, G. Dailide, J. M. Cheverud, and D. E. Berg. 2003. Helicobacter pylori tissue tropism: mouse-colonizing strains can target different gastric niches. Microbiology 149:1901–1909. 2. Algood, H. M., and T. L. Cover. 2006. Helicobacter pylori persistence: an overview of interactions between H. pylori and host immune defenses. Clin. Microbiol. Rev. 19:597–613.

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