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Research Article
Dual Protective Functions of Helicobacter pylori Peptidoglycan Modifications Impact Early and Late Survival in the Host
Ge Wang* Leja F. Lo, and Robert J. Maier
Department of Microbiology, University of Georgia, USA

ABSTRACT
Structural motifs inherent in bacterial peptidoglycan (PG) are important recognition domains for some efficient host responses to pathogenic bacterial infection, so PG modifications by the infecting bacterium can neutralize host responses. Helicobacter pylori contain two PG modification enzymes, an N-deacetylase (PgdA) and an O-acetyltransferase (PatAB), but some naturally occurring strains lack the latter.  Here the lysozyme resistance and the survival in both macrophages and in lysozyme deficient mice were studied for various H. pylori strains. Resistance to lysozyme killing of H. pylori was conferred by PgdA in naturally-occurring strains that lacked PatA (e.g. B128); the lysozyme sensitivity for B128 pgdA mutant was at the same level as that of a double mutant (pgdA patA) version within parent strain X47. Both PgdA- and PatA-mediated PG modifications are important to H. pylori survival within macrophages. Mouse studies connected the lysozyme and macrophage results to the in vivo condition, in which lysozyme effects are expected early and cytokine production later, pertaining to specific host recognition events. An increased host lysozyme killing effect associated with the double mutant strain (pgdA patA) was indicated from a reduced colonization phenotype (compared to wild type) after a week post-inoculation of lysozyme-positive mice; at this time point some important anti- H. pylori mouse cytokines were shown to be minimal. At 3 weeks post-inoculation, the levels of 3 cytokines (IL-10, TNF-α, and MIP-2) were significantly higher in sera of mice inoculated with the pgdApatA strain, and the pgdApatA strain showed a greatly attenuated ability of mouse colonization, in contrast to the wild type strain, indicating a significant role of PG modifications in persistent infection through mitigating host immune response.  However, the double mutant survived better in lysozyme deficient (LysM-/-) mice, supporting the notion that PgdA- and PatA- mediated PG modifications in H. pylori protect bacteria from killing by host lysozyme.
KEYWORDS
Helicobacter pylori; Peptidoglycan modification; Lysozyme resistance; Macrophage killing; Mouse colonization

INTRODUCTION
Bacterial peptidoglycan (PG), consisting of glycan strands of alternating beta-1,4-linked N-acetylglucosamine (GlucNAc) and N-acetylmuramic acid (MurNAc) which are cross-linked by short peptide chains, is one of the protective barriers of the bacterial cell wall [1]. PG is the target of cleavage by lysozyme, one of the important components of the innate immunity of eukaryotic hosts with invading bacteria [2].  To establish infection, pathogenic bacteria have developed strategies to counteract the hydrolytic activity of lysozyme by modifying their PG structure [3]. In addition, bacterial PG has been shown to contain structural motifs that can be recognized by host receptors such as Nod1 and Nod2 [4,5].  Mammalian Nod1 specifically senses PG degradation products of Gram-negative bacteria, resulting in activation of the transcription factor NF-kB pathway [5-7].  Thus, modification of PG is an important strategy for pathogenic bacteria to evade host immune responses. For example, PG N-deacetylation in Listeria [8] and Streptococcus [9] was shown to be a virulence factor, playing an important role in evasion from the host innate immune response so that the pathogen survives in vivo. Helicobacter pylori, a Gram-negative bacterium, is an important human gastric pathogen that chronically infects 50% of the world's population [10]. During the process of colonizing the host, H. pylori induces a strong inflammatory response. However, H. pylori survives the host immune response to persistently colonize the gastric mucosa, usually for the entire life-span of the host [11].  The mechanisms by which H. pylori evades host immune responses are poorly understood.  Previously we identified and characterized an H. pylori protein (HP310) whose expression was significantly induced under oxidative stress conditions [12]. HP310 turned out to be an enzyme (PgdA) catalyzing N-deacetylation of PG. With a limited sequence homology to PgdAs of Gram-positive bacteria, H. pylori PgdA is a representative of a new subfamily of bacterial PG deacetylases.  A primary phenotype of H. pylori pgdA mutants in vitro is a decrease in lysozyme tolerance compared to its parent strain, if high levels of lysozyme are used in the assay [12]. Subsequently, we showed that the contact of H. pylori to host immune cells (macrophages) also induces over-expression of PgdA, and that H. pylori PG deacetylation is an important mechanism for mitigating host immune responses, contributing to pathogen persistence in the host [13].  Recently, we investigated the possibility that another PG modifier (a component of PG O-acetyltransferase, PatA) also confers lysozyme resistance [14]. Many naturally occurring H. pylori strains do not have a patA gene; in those strains the PgdA may be the only determinant for lysozyme resistance. In this study we investigated lysozyme resistance by comparing the strain that has patA gene (X47) to the strain that has no patA gene (B128). Gram-negative bacteria (like H. pylori) are not generally susceptible to lysozyme because their outer membrane prevents access of the secreted enzyme to the PG layer.  However the outer membrane barrier can be overcome in some cells via host accessory proteins such as lactoferrin, that permeabilize the bacterial outer membrane [15]. Recently we demonstrated that H. pylori cells become sensitive to lysozyme at physiologically relevant lytic enzyme concentrations when a membrane permeabilizer lactoferrin is present [14]. However, the conclusive evidence for the in vivo roles of PG modifications in combating host lysozyme killing is still lacking.  Although we showed that PgdA- and PatA-mediated PG modifications contribute to H. pylori's survival in the host [14], the relative contributions of their two functions (i.e. resisting host lysozyme killing and mitigating host immune response) is not clear. In this study, we performed a macrophage killing assay and used a lysozyme deficient (LysM-/-) mice in the mouse colonization assay, in order to provide conclusive evidence for H. pylori PG modifications conferring lysozyme resistance in vivo. By conducting mouse colonization assays (normal wild type mice) for different time spans and determining the subsequent cytokine levels in the serum of infected mice, we demonstrated that PG modifications have in vivo roles both in combating host lysozyme killing and on escaping host immune recognition.  Both roles protect the bacterium while in the host.
Materials and Methods
H. pylori strains and culture conditions
H. pylori was cultured on Brucella agar (Difco) plates supplemented with 10% defibrinated sheep blood or 5% fetal bovine serum (called BA plates). Chloramphenicol (50 μg/ml) or kanamycin (40 μg/ml) was added to the medium for culturing mutants [13,14]. Cultures of H. pylori were grown microaerobically at 37oC in an incubator under continuously controlled levels of oxygen (4% partial pressure O2, 5% CO2, and the balance was N2). H. pylori strains used in this study, X47 (WT), X47 pgdA patA double mutant, B128 (WT), and B128 pgdA mutant, were described previously [13,14].
Cell survival assay for assessing lysozyme sensitivity in the presence of lactoferrin
H. pylori strains were grown on BA plates to late log phase, and the cells were suspended in PBS to a concentration of ~109 cells/ml. Upon addition of lysozyme (0.3 mg/ml) and lactoferrin (3mg/ml), the cell suspensions were incubated at 37°C under 2% O2  condition with occasional shaking. Samples were then removed at various time points (0,2,4, and 6 hours), serially diluted, and spread onto BA plates. Colony counts are recorded after 4 days of incubation in a microaerobic atmosphere (2% O2) at 37°C.
Macrophage killing assay
The survival of H. pylori cells within macrophages was investigated following the methods published [16-18] with minor modifications.  Briefly, the macrophage RAW264.7 cells were seeded in 24-well plates in the culture medium (0.5 ml) and incubated at 37°C, 5% CO2 for 4 days (cell density is about 105 cells per well).  The medium was replaced by fresh medium to remove the non-adherent cells.  H. pylori cells were added at a ratio of 20 CFU bacteria per macrophage (i.e. 4 μl of H. pylori cell suspension in PBS at a concentration of 5x108 cells/ml was added to one well containing ~105 macrophage cells). Phagocytosis was synchronized by centrifugation at 600 x g for 5 min and then allowed to proceed for 1 h. Extracellular bacteria were removed by washing and incubation in the medium supplemented with gentamicin (100 μg/ml) for 1 h at 37°C, 5% CO2. After three washes to remove the antibiotics the cells were further incubated in fresh medium for 2 h. After removing the medium, the macrophage cells were lysed with ice-cold PBS with 0.1% saponin for 5 min. Appropriate dilutions of the supernatant were plated on BA plates and incubated at 37°C, 5% CO2, 2% O2 for 4 days to count the number of surviving bacteria. The number of surviving bacteria (CFU/ml) is compared with the number of viable bacteria initially added.
Mouse colonization
Mouse colonization assays have been well described in our lab [13,19]. In this study, we used both the conventional WT mice C57BL/6J (purchased from Jackson Laboratories) and lysozyme deficient mice (LysM−/−). The LysM−/ mice were generated (by Prof. T. Graf) by insertion of the Enhanced Green Fluorescent Protein into exon 1/intron 1 of the lysozyme locus followed by backcrossing through ten generations into the FVB/NJ mice [20]. The procedure for mouse colonization assay 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 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 1.5-2 hours prior to inoculation.  The bacterial suspensions were administered through oral deliveries to the mice. One, three, or six weeks after the inoculation, the mice were sacrificed and the stomachs were removed, weighed, and homogenized in argon-sparged PBS to avoid O2 exposure. Stomach homogenate dilutions (dilutions were made in argon-sparged buffer in sealed tubes) were plated onto BA plates supplemented with bacitracin (100 μg/ml), vancomycin (10 μg/ml) and amphotericin B (10 μg/ml), and the plates were rapidly transported into an incubator containing sustained 5% partial pressure O2. After incubation for 5 to 7 days the fresh H. pylori colonies were enumerated and the data expressed as CFU recovered per gram of stomach. In a competition assay, each mouse was inoculated with a mixture of 1.5 x 108 H. pylori wild type cells and 1.5 x 108 pgdA patA mutant cells. The results of  H. pylori colonization were examined after 3 weeks as described above.  Stomach homogenate dilutions were plated on plates either without or with chloramphenicol (50 μg/ml). The number of H. pylori cells on plates without chloramphenicol represents the total number of cells colonizing the mouse stomach, and the number of cells on plates with chloramphenicol represents the number of pgdA patA mutant cells.
Determination of cytokine concentrations in mouse serum
As described in the mouse colonization experiments, the mice were inoculated with H. pylori (WT or pgdA patA mutant) with a dose of 3 x 108 viable cells administered per animal. One or three weeks after the inoculation, the blood samples were collected upon guillotining each anesthetized (overdose) animal and collecting 4-6 drops of blood from the animal. The concentrations of multiple cytokines in mouse serum were determined simultaneously using Bio-Plex Cytokine Assay Kit (Bio-Rad). This is a multiplex bead-based assay with format of sandwich immunoassay (beads- antibody-cytokine-biotinylated detection antibody), and the results were read and analyzed with the Bio-Plex suspension array system (Bio-Rad). We chose a mouse serum 8-plex kit which determines IL-1β, IL-2, IL-4, IL-5, IL-10, GM-CSF, IFN-γ, and TNF-α. In addition, we included the macrophage inflammatory protein 2 (MIP-2), a murine chemokine reported to have biological functions analogous to those of human IL-8 [21,22].
Results and Discussion
PgdA alone is required for lysozyme resistance in those H. pylori strains that do not have a patA gene
Recently we have identified and characterized a patAB locus that functions in O- acetylation of H. pylori PG, and showed that PgdA and PatA contribute synergistically to lysozyme resistance [14]. However, patA gene is present only in a subset of H. pylori strains (e.g. X47 and 26695), but not in many other strains (e.g. B128 and J99).  In an in vitro assay we demonstrated that H. pylori cells become sensitive to physiological levels of lysozyme in the presence of lactoferrin (0.3 mg/ml lysozyme and 3 mg/ml lactoferrin).  The X47 pgdA patA double mutant cells were extremely sensitive to the lysozyme/lactoferrin treatment, while the pgdA or patA single mutant cells were slightly more sensitive than the wild type cells [14]. In this study, we examined the sensitivity of B128 pgdA mutant in comparison to its parent strain using the same assay (Figure 1). H. pylori cell suspensions (~109 cells/ml) were treated with lysozyme/lactoferrin for 2, 4 or 6 hours, and the numbers of surviving cells were determined. After 2 hours of treatment, there was no difference for the viability between the WT and the mutant strains. However, at later time points, the viability of the B128 pgdA mutant cells decreased much faster than the WT. At the 4 hour and 6 hour time points, the viable cell number of the mutant was more than 2 logs and 3 logs, respectively, lower than that of the WT (P<0.001, student t-test).  Compared to the previous results [14], the lysozyme sensitivity of B128 pgdA mutant is at the same level as the X47 pgdA patA double mutant, indicating that PgdA alone is required for full resistance to lysozyme in B128.  A similar result was observed for the J99 pgdA mutant (data not shown).
Figure 1 Lysozyme sensitivity in the presence of lactoferrin: H. pylori cell suspensions (~108 cells/ml) were treated with 0.3 mg/ml lysozyme plus 3 mg/ml lactoferrin for the time indicated on x-axis, and the numbers of surviving cells were determined. The data are the means of three experiments with standard deviation as indicated. Symbols for strains: diamond, B128 WT; circle, B128 pgdA::Kan.

Figure 1

Figure 1 Lysozyme sensitivity in the presence of lactoferrin: H. pylori cell suspensions (~108 cells/ml) were treated with 0.3 mg/ml lysozyme plus 3 mg/ml lactoferrin for the time indicated on x-axis, and the numbers of surviving cells were determined. The data are the means of three experiments with standard deviation as indicated. Symbols for strains: diamond, B128 WT; circle, B128 pgdA::Kan.

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All H. pylori strains contain PG N-deacetylase PgdA, and the PG deacetylation level is likely related to the PgdA expression level. The expression of PgdA in H. pylori is induced under oxidative stress condition or upon contact of H. pylori to host immune cells [12,13], and this induction is regulated by aconitase at the post-transcriptional level (C. Austin and R.J. Maier, to be published).  The PgdA expression level and the PG deacetylation level remain to be determined to address a possible correlation of PG deacetylation to the enzyme level among strains.
H. pylori PG modification mutants are more sensitive to macrophage killing
H. pylori infection induces a strong inflammatory response by the host, with the recruitment of lymphocytes, macrophages, and polymorphonuclear cells; however the bacterium is able to resist this immune response and establish a persistent gastric infection. In particular, although H. pylori can be efficiently ingested by the different types of phagocytic cells, it is able to survive for prolonged periods within these cells [18,23].  In addition to free radicals derived from the phagocytic respiratory burst, eukaryotic immune cells produce a battery of antibacterial peptides including lysozyme and membrane permeabilizers. Here we investigated whether PgdA- and PatA-mediated PG modifications contribute to survival of H. pylori within macrophages.
A macrophage killing assay was performed using a murine macrophage cell line RAW264.7 for H. pylori WT or PG modification defective cells (Figure 2). Similar numbers of the H. pylori WT or PG modification defective mutant cells were inoculated to the macrophage cell culture.  After killing of extracellular bacteria by gentamycin and further incubation for 2 hr, the numbers of surviving H. pylori cells were recovered and determined. As shown in (Figure 2A), a mean number of 3.0 x 106 CFU/ml X47 WT cells survived. In contrast, the same treatment resulted in recovery of a mean number of 2.0 x 105 CFU/ml of the pgdA patA mutant cells (15 fold less than the WT). Based on statistical analysis (Student t-test), the cell survival differences between the WT and the mutant strains are significant (P<0.01). Similar results were obtained when comparing B128 WT and B128 pgdA mutant cells (Figure 2B). These results indicate a role for PgdA- and PatA-mediated PG modifications in survival of H. pylori within macrophages. It is also in agreement with the results above that PgdA alone is required for full resistance to lysozyme in B128. The roles of bacterial PG modifications in survival within macrophage have been observed in several Gram-positive bacteria [8,9,24,25]. Our results are the first example for Gram-negative bacteria.  As the only in vitro phenotype  found for  H. pylori pgdA patA mutant cells is lysozyme resistance (and not oxidative stress resistance), the macrophage killing results herein support the notion that PgdA- and PatA-mediated PG modifications in Gram-negative bacteria play a significant role in protecting bacteria from killing by host lysozyme.
Figure 2 Survival of H. pylori cells in RAW264.7 macrophage cells determined with the gentamycin killing assay: Similar numbers of the H. pylori WT or PG modification defective mutant cells were inoculated to the macrophage cell culture. After extracellular killing by gentamycin and further incubation for 2 hr, the numbers of surviving cells of the WT and the mutant strain were determined.
(A). H. pylori strain X47 (gray bars) and X47 pgdA patA mutant cells (black bars).
(B). H. pylori strain B128 (gray bars) and B128 pgdA mutant cells (black bars). Data are means from three independent determinations with standard deviations.

Figure 2

Figure 2 Survival of H. pylori cells in RAW264.7 macrophage cells determined with the gentamycin killing assay: Similar numbers of the H. pylori WT or PG modification defective mutant cells were inoculated to the macrophage cell culture. After extracellular killing by gentamycin and further incubation for 2 hr, the numbers of surviving cells of the WT and the mutant strain were determined.
(A). H. pylori strain X47 (gray bars) and X47 pgdA patA mutant cells (black bars).
(B). H. pylori strain B128 (gray bars) and B128 pgdA mutant cells (black bars). Data are means from three independent determinations with standard deviations.

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Colonization of H. pylori in lysozyme positive mice and measurement of cytokines
To investigate the in vivo roles of PgdA- and PatA-mediated H. pylori PG modifications in lysozyme resistance and evasion of immune response, we performed time course experiments of mouse (C57BL/6J) colonization assay for X47 pgdA patA mutant compared to the WT strain at 1, 3 and 6 weeks post-inoculation (Table 1).  One week after inoculation, a low colonization number for the WT strain (68 CFU/mg stomach) was observed, but the pgdA patA mutant showed much lower level of colonization  (3 CFU/mg stomach, a level just  above the detection limit).  This early effect is probably due to an efficient host lysozyme killing on the pgdA patA mutant, as lysozyme is constantly present in mucus [26]. Thus, PgdA- and PatA-mediated PG modifications in H. pylori play a significant role in establishing early infection.  At 3 weeks, the mean bacterial load for the double mutant was 11 fold lower than that for the WT strain. Six weeks after inoculation, a consistently high number of colonization for the WT strain was observed, but few pgdA patA mutant cells were recovered. This indicated that PgdA- and PatA- mediated PG modifications in H. pylori play a significant role in persistent infection. A key element in the host response to H. pylori infection is the production of proinflammatory cytokines in the gastric mucosa [27]. To monitor the immune responses elicited by H. pylori pgdA patA mutant cells compared to that of WT cells in the mouse infection model, we proceeded to determine the concentrations of cytokines present in mouse serum. The blood samples (from 10 mice for each group) were collected from the one week and 3 weeks mouse colonization experiments and the serum samples were prepared. Using Bio-Plex Cytokine Assay Kits from Bio-Rad, we were able to determine the concentrations of multiple cytokines in mouse serum simultaneously (Table 2). The concentrations of IL-2 and IL-4 were below the detection level for all the samples, and there were no significant difference between strain groups for the concentrations of IL-1β, IL-5, GM-CSF, and IFN-γ; thus the data for these 6 cytokines are omitted. For one week data, there was no significant difference between the 3 groups (mock, WT, mutant) of mice for the concentrations of any cytokine determined. This indicted that the host immune response has not occurred one week after inoculation.  However, for the 3 week data, the concentrations of IL-10, TNF-α, and MIP-2 in the mice infected with the H. pylori pgdA patA mutant are significantly higher than those infected with the H. pylori WT strain (P<0.05, 0.05, and 0.01, respectively, student's t-test). These 3 cytokines were reported to be important components of human immune response elicited by H. pylori infection [28-31]. Our results support the idea that H. pylori PG modification minimizes some aspects of the host immune response. This is in agreement with the data for the mouse colonization that the pgdA patA mutant has lower ability to survive in the stomach after 3 weeks.
Colonization of H. pylori in lysozyme deficient mice
The role of bacterial PG modifications in lysozyme resistance has thus far been studied in only a few invasive, Gram-positive bacteria [3]. Our previous results [14] showed that the H. pylori X47 pgdA patA double mutant cells were extremely sensitive to physiological levels of lysozyme plus lactoferrin. The results in this study (Figure 2) demonstrated that the double mutant cells are more sensitive to macrophage killing than the WT cells. To obtain conclusive evidence that PgdA- and PatA-mediated PG modifications in H. pylori play a significant role in protecting bacteria from killing by host lysozyme, we performed the mouse colonization assay using lysozyme deficient mice. LysM-/- mice were generated in a different mouse strain (FVB/NJ) from that we conventionally used (C57BL/6J).  No colonization of H. pylori in LysM-/- mice could be detected at one week post-inoculation, even for the wild type H. pylori (not shown). Therefore the assay was done at 3 weeks post-inoculation.  Groups of 4 LysM-/- mice were inoculated with H. pylori (X47 WT or pgdA patA double mutant individually and inoculated as a mixture of the two strains) with a dose of 3 x 108 viable cells administered per animal. Recovery numbers of H. pylori in mouse stomachs was examined 3 weeks after the inoculation (Table 3).  All 4 mice were found to be colonized by the WT H. pylori, with the mean bacterial load of 441 CFU/mg stomach. The pgdA patA double mutant strain colonized 3 of 4 mice, and the mean bacterial load (142 CFU/mg stomach) was about 1/3 of that for the WT strain.  Furthermore, a competition assay was conducted in which a mixture of cells of WT and the pgdA patA mutant (in a 1:1 ratio) was inoculated into LysM-/- mice. Three weeks after inoculation, each mouse stomach homogenate was examined for the total number of H. pylori cells and the number of the mutant cells; the latter carried a chloramphenicol resistance marker. As shown in (Table 3), all of the 4 mice were colonized by the WT H. pylori, with the mean bacterial load of 572 CFU/mg stomach. Similar to the results in separate inoculation, the competitive colonization ability of the pgdApatA mutant was about 1/3 of that for the WT strain.
Table 1 Mouse colonization abilities of H. pylori X47 WT and pgdA patA mutant strains.
Groups of 10 C57BL/6J mice were inoculated with H. pylori with a dose of 3 x 108 viable cells administered per animal.
aColonization of H. pylori in mouse stomachs was examined 1, 3, or 6 weeks after the inoculation. The 3 week data were published previously.
bNumber of mouse stomachs from which H. pylori was recovered / total number of stomachs assayed.
cNumber of H. pylori cells colonized in mouse stomach averaged from the 10 mice with standard deviation. The detection limit of the assay is 0.5 CFU/mg stomach.

Table 1

Time after

inoculation a

Strains

Number of

colonized mice b

Bacterial load

(CFU/mg stomach)

c

1 week

X47 WT

X47 pgdA patA

7 / 10

2 / 10

68 + 13

3 + 2

3 weeks

X47 WT

X47 pgdA patA

10 / 10

6 / 10

691 + 103

60 + 52

6 weeks

X47 WT
X47  pgdA patA

10 / 10

3 / 10

738 + 109

2 + 2

Table 1 Mouse colonization abilities of H. pylori X47 WT and pgdA patA mutant strains.
Groups of 10 C57BL/6J mice were inoculated with H. pylori with a dose of 3 x 108 viable cells administered per animal.
aColonization of H. pylori in mouse stomachs was examined 1, 3, or 6 weeks after the inoculation. The 3 week data were published previously.
bNumber of mouse stomachs from which H. pylori was recovered / total number of stomachs assayed.
cNumber of H. pylori cells colonized in mouse stomach averaged from the 10 mice with standard deviation. The detection limit of the assay is 0.5 CFU/mg stomach.

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Table 2 Levels of various cytokines in the sera of mice infected with H. pylori X47 WT or pgdA patA mutant strains.
Concentration of cytokines (ng/ml) in mouse serum
The mice were inoculated with H. pylori (WT or pgdA patA mutant) with a dose of 3 x 108 viable cells (or PBS buffer as controls) administered per animal. One or three weeks after the inoculation, blood samples (from 10 mice for each group) were collected and the serum samples were prepared. The concentrations of 9 cytokines in mouse serum were determined with Bio- Plex Cytokine Assay (Bio-Rad). The data shown are the mean from 10 mice serum samples with standard deviation. The data for other 6 cytokines were omitted from the table because they were either below the detection level or did not show a significant difference between the WT and mutant strains.

Table 2

Time after inoculation

Strains

IL-10

TNF-α

MIP-2

1 week

Mock (PBS) X47 WT

X47 pgdA patA

11 + 5

13 + 6

15 + 8

105 + 19

118 + 17

109 + 21

146 + 57

154 + 68

162 + 70

3 weeks

Mock (PBS) X47 WT

X47 pgdA patA

10 + 5

15 + 7

28 + 12

112 + 16

132 + 14

183 + 35

141 + 52

169 + 73

388 + 134

Table 2 Levels of various cytokines in the sera of mice infected with H. pylori X47 WT or pgdA patA mutant strains.
Concentration of cytokines (ng/ml) in mouse serum
The mice were inoculated with H. pylori (WT or pgdA patA mutant) with a dose of 3 x 108 viable cells (or PBS buffer as controls) administered per animal. One or three weeks after the inoculation, blood samples (from 10 mice for each group) were collected and the serum samples were prepared. The concentrations of 9 cytokines in mouse serum were determined with Bio- Plex Cytokine Assay (Bio-Rad). The data shown are the mean from 10 mice serum samples with standard deviation. The data for other 6 cytokines were omitted from the table because they were either below the detection level or did not show a significant difference between the WT and mutant strains.

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Table 3 Mouse colonization abilities of H. pylori X47 WT and pgdA patA mutant strains in LysM-/- micea aGroups of 4 LysM-/- mice were inoculated with H. pylori individual strains or the mixture of both strains with a dose of 3 x 108 viable cells administered per animal. Colonization of H. pylori in mouse stomachs was examined 3 weeks after the inoculation. b Number of mouse stomachs from which H. pylori was recovered / total number of stomachs assayed. c Number of H. pylori cells colonized in mouse stomach averaged from the 4 mice with standard deviation. The detection limit of the assay is 0.5 CFU/mg stomach.

Table 3

 

Strains

Number of
colonized mice b

Bacterial load + SD

(CFU/mg stomach) c

Separate inoculation

WT

4 / 4

441 + 219

 

pgdA patA

3 / 4

142 + 76

Mixed inoculation

WT

4 /4

572 + 139

 

pgdA patA

3 / 4

186 + 65

Table 3 Mouse colonization abilities of H. pylori X47 WT and pgdA patA mutant strains in LysM-/- micea aGroups of 4 LysM-/- mice were inoculated with H. pylori individual strains or the mixture of both strains with a dose of 3 x 108 viable cells administered per animal. Colonization of H. pylori in mouse stomachs was examined 3 weeks after the inoculation. b Number of mouse stomachs from which H. pylori was recovered / total number of stomachs assayed. c Number of H. pylori cells colonized in mouse stomach averaged from the 4 mice with standard deviation. The detection limit of the assay is 0.5 CFU/mg stomach.

×
Compared to the results obtained in LysM+/+ mice (Table 1, at 3 weeks post-inoculation), the pgdA patA mutant survived better in LysM-/- mice, supporting the notion that PgdA- and PatA-mediated PG modifications in H. pylori protect bacteria from direct killing by host lysozyme. For the first time, we showed that PgdA- and PatA-mediated PG modifications protect Gram-negative bacteria from direct killing by host lysozyme.  Davis et al. [32] showed that PG modifications by both N-deacetylase (PgdA) and O-acetylase (Adr) in Streptococcus pneumoniae conferred the full resistance to lysozyme in vivo, facilitating bacterial colonization in LysM+/+ mice. However, the combination of PG modifications reduces overall fitness of S. pneumoniae, thus the pgdA adr double mutant displayed a competitive advantage over wild-type pneumococci in LysM−/− mice [32]. In contrast, the colonization ability of H. pylori pgdA patA mutant was still lower than that of the WT in LysM-/- mice. This is likely attributed to a stronger immune response induced by the mutant (Table 2).
Conclusion
Peptidoglycan (PG) break-down products serve as important recognition domains that impact the dynamics of host-pathogen interactions and subsequent host responses.  The two PG modification enzymes, PgdA and PatA from H. pylori serve to alter the PG structure, so the pathogen can partially evade host-mediated anti-bacterial mechanisms.  Here via mutant strain analysis, the in vitro lysozyme resistance was connected to bacterial survival in both macrophages and in lysozyme deficient mice.   Resistance to lysozyme-mediated killing of Helicobacter was conferred by PgdA in naturally-occurring strains that lacked PatA; still, both modification enzymes aid lysozyme tolerance in strains that contain both of the PG modification enzymes. Mutations altering PG have an impact on both early and persistent colonization, while the cytokine data supported the different early and late functions of the PG modifications on host responses.   Our results indicate that H. pylori PG modifications have dual protective functions, both early and late in infection; these are protection against lysozyme-mediated killing and in avoiding host immune recognition.  The use of lysozyme deficient (LysM-/-) mice, supported the proposal that PG modifications in H. pylori (a Gram-negative bacterium) protect the bacteria from killing by lysozyme while they reside in the host.
Acknowledgements
This work was supported by the University of Georgia Foundation and by the National Institutes of Health (R01AI077569). We thank Sue Maier for help with mouse experiments.

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Cite this article: Wang G, Lo LF, Maier RJ (2013) Dual Protective Functions of Helicobacter pylori Peptidoglycan Modifications Impact Early and Late Survival in the Host. JSM Microbiology 1(1): 1001.
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