The Autophagic Induction in Helicobacter pylori -Infected Macrophage

Abstract

Helicobacter pylori has developed several mechanisms to evade the intracellular killing after phagocytosis. In this study, we reported that some Taiwanese clinical isolated H. pylori can multiply in human monocytic cells, such as THP-1 or U937 cells, but not in murine macrophage Raw264.7 cells. After internalization, there was a 5- to 10-fold increment of re-cultivable H. pylori from the infected THP-1 cells at 12 hrs post infection. The dividing H. pylori was found in a double-layer vesicle, which is characteristic of autophagosome. The formation of autophagosomes is associated with the multiplication of H. pylori in THP-1 cells. Its modulation with rapamycin or 3-MA affects the level of H. pylori replication. Furthermore, the VacA or CagA mutants of H. pylori have lower levels of multiplication in macrophages. We conclude that H. pylori infection induces autophagosome formation, and these autophagic vesicles were adapted for the multiplication of H. pylori in the host.

Introduction

Helicobacter pylori is a Gram-negative, flagellated, microaerophilic bacterium that selectively colonizes the human stomach. Its infection occurs worldwide, and its prevalence is correlated with socioeconomic condition (1). The prevalence among middle-aged adults is over 80% in many developing countries, as compared with 20% to 50% in industrialized countries. In Taiwan, the prevalence is around 54% (2). Overt diseases, however, occur in only 10–20% of infected individuals. The most common pathology associated with H. pylori infection is chronic active gastritis and peptic ulceration. A long-term chronic infection will increase the risk to gastric adenocarcinoma and mucosa-associated lymphoid-tissue (MALT) lymphoma (3). The histopathological hallmarks of H. pylori-induced disease are a massive inflammatory cells infiltration of the lamina propria and erosion of the gastric epithelium.

H. pylori infection would cause continuous gastric inflammation in infected persons. During the infection, binding of H. pylori to gastric epithelial cells, in particular through BabA and by strains harboring the cag pathogenicity island, results in the production of IL-8 and other chemokines by epithelial cells. The recruitment of neutrophils represents an acute inflammation and correlates directly with tissue damage. The chronic phase of H. pylori-induced gastritis is associated with the adaptive lymphocyte response. Lymphocyte recruitment is facilitated by up-regulation of VCAM-1 and ICAM-1 for extravasation. Macrophages respond by producing proinflammatory cytokines involved in the activation of the recruited T cells. The cytotoxin VacA- and Fas-mediated apoptosis induced by TNF-α leads to disruption of the epithelial barrier, facilitating translocation of bacteria and leading to further activation of macrophage. Cytokines produced by macrophages can also alter the secretion of mucus, contributing to H. pylori-mediated disruption of the mucus layer. The density of the H. pylori becomes the major factor for the whole process of the infection (1). However, H. pylori is not effectively controlled by macrophages; it exhibits a novel mechanism of virulence that allows these organisms to retard phagocytosis and disrupt membrane trafficking and phagosome maturation (4). A homotypic phagosome fusion was induced to form a megasome that contained H. pylori to delay the bacteria clearance in macrophage (5–7).

Autophagy is an evolutionarily conserved lysosomal pathway involved in cytoplasmic homeostasis to control the turnover of long-lived proteins. Autophagy can be stimulated in response to different situations of stress, such as starvation, oxidative stress, infection, or anti-cancer cytotoxic drugs, or lectins (8–10). Several viruses have taken advantage using components of the autophagic pathway to facilitate their own replication (11, 12). On the other hand, autophagic degradation is also an innate immunity effector against intracellular bacteria (13–15). In this study, we reported that H. pylori could induce autophagic vesicle formation, which is associated with the multiplication of the H. pylori in the infected macrophages.

Materials and Methods

Helicobacter pylori Strains and Culture.

The Helicobacter pylori clinical isolates (HP238, HP250, HP46A and HP46B) were obtained from the Department of Pathology, National Cheng Kung University Hospital. ATCC43504 and J99 were derived from the American Type Culture Collection (ATCC). H. pylori bacteria were grown on CDC anaerobic 5% sheep blood agar plates (BBL, Becton-Dickinson, USA) under microaerophilic conditions (5% O₂, 10% CO₂, 85% N₂) and 85% humidity in a Nuaire incubator (Plymonth, Minnesota, USA) at 37°C. Fresh plates were started from glycerol stocks and subcultured every 48 hrs. Bacteria from plates were suspended with phosphate buffer saline (PBS), and concentrations were determined using OD₆₀₀ = 1 as 2 × 10⁸ confirmed by viable plate counting.

Determination of Viable H. pylori in Macrophages.

THP-1 cells were cultured in RPMI1640 medium with 10% fetal bovine serum with 0.05 mM 2-mercapto-ethanol. U937 cells were in RPMI1640 medium with 5% fetal bovine serum. Raw264.7 cells were cultured in DMEM medium with 5% fetal bovine serum. No PMA, retinoic acid, or vitamin D3 was added. The cells were grown at 37°C in a 5% CO₂ incubator. Resident mouse peritoneal macrophages were harvested from 6- to 8-week-old C3H/HeN mice peritoneum with Hank’s BSS medium. After 2 hrs of 37°C incubation, non-adherent lymphocytes were removed by medium washing and adherent macrophages were used for infection. For infection, cells were co-cultured with bacteria at cell:bacteria (m.o.i.) = 1:10. The bacteria-cell mixtures were centrifuged at 400 g for 10 mins at 4°C. Then cells and bacteria were incubated at 37°C for 60 mins. Gentamicin at 100 μg/ml in 2% FBS cell medium was added to kill extracellular bacteria for 60 mins, and the cells were washed three times with PBS. At various time points post infection (p.i.), cells were permeated with 0.5% saponine for 10 mins at room temperature, and then plated on CDC plates with serial dilution to determine the viable bacteria. Colonies were grown and counted after 4–5 days culture.

Microscopy Observation.

Cells (2 × 10⁵) were seeded on glass coverslips each well in 24-well plates and infected with H. pylori at m.o.i. of 1:5 at 37°C for 60 mins after bacteria were centrifuged on to cells at 400 g for 10 mins at 4°C. The H. pylori was harvested from CDC plates after 48 hrs propagation, and concentration was adjusted with PBS into OD₆₀₀ = 1 for cell infection. Infected cells were washed three times with RPMI1640, and were then incubated with medium containing 100 μg/ml gentamicin in 2% FBS at 37°C for 60 mins. At various time points post infection, the cells were fixed with 3% paraformaldehyde, pH 7.4, at room temperature for 10 mins, and then washed with PBS for 3 times. A stable fluorescent dye, PKH2-GL (Sigma, USA), was used for bacterial staining. Anti-calnexin and anti-COX 4 (Abcam, Cambridge, UK) primary antibodies, followed by TRITC-labeled secondary antibody, were used for ER and mitochondria staining. Prepared cells were mounted with mounting medium and photographed using Olympus FV1000 confocal microscopy and Workstation of data analysis system. On the autophagic analysis, anti-LC3 II antibody (ABGENT, USA) was used for autophagy detection. Rabbit anti-H. pylori antiserum (DAKO, California, USA) was used to stain the presence of the bacteria. For ultrastructural analysis, H. pylori infected cells at different time points were fixed with 4% glutaraldehyde and postfixed in 1% OsO₄. The cells were observed under transmission electron microscopy (Hitachi 7000, Japan).

Western Blot Analysis.

The cells were harvested at different times post infection, and lysed in lysis buffer (25 mM Tris, 137 mM NaCl, 10% (v/v) glycerol, 0.5% (w/v) sodium deoxycholate, 2 mM EDTA) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 30 μg/ml aprotonin, 1 mM sodium orthovanadate). Lysates were clarified by pipetting and vortexing at 4°C, 30 mins. Insoluble material was removed by 12,000 rpm centrifugation. Protein concentrations were determined by Bio-Rad protein detection kit. Equal amounts of protein were loaded on a 12% SDS-PAGE, transferred to nitrocellulose membrane. Anti-Beclin 1 (Santa Cruz, CA), LC3 II (ABGENT, USA) and GAPDH (Abcam, Cambridge, UK) antibodies were used to detect protein expression patterns. After incubation with peroxidase-conjugated secondary antibodies, the blots were visualized by enhancing chemiluminescence reagents (PerkinElmer Life Sciences, Boston, MA).

Construction of the cagA and vacA Mutants.

The cagA and vacA mutants were constructed by using insertional mutagenesis. The PCR fragments from cagA sense (5′-GATATAGCCACTACCACCACCG-3′) and anti-sense (5′-TAACCCATTACCGACTAGGGT-3′) primers, and vacA sense (5′-GAGGATATCAAACGCCTCCCATTTACC-3′) and antisense (5′-CGCGGATCCCCAGTGTGCCAATATGAGTC-3′) primers were ligated into the pZErO-2 (Invitrogen, CA, USA) and pBluescript II SK (Stratagene, CA, USA) to construct the plasmids pMW302 and pMW356, respectively. A chloramphenicol acetyltransferase (cat) gene was digested from a plasmid, vector 78, with Hinc II and inserted into the blunt-end site of plasmids pMW302 and pMW356, respectively. These plasmids were transformed into HP238 by natural transformation and chloramphenicol(20 μg/mL) was used as a selection marker. The cagA and vacA mutants were further confirmed by the Southern blotting with specific probe.

Results

Multiplication of H. pylori in Human Monocytic Cell Lines.

It was reported that H. pylori could induce homotypic phagosome fusion and form large communal vacuoles after incubation with macrophage (3). Although the megasome formation was delayed, the internalized H. pylori was eventually killed. However, we are surprised to have found that the H. pylori isolated from Taiwan would multiply in the macrophage cell line THP-1. In the infection assay, H. pylori bacteria were incubated with THP-1 cells for 1 hr, and gentamycin was used to kill the extracellular H. pylori. At various time points post infection (p.i.), THP-1 cells were lysed, and the viable H. pylori bacteria were re-cultured and plate-counted on CDC plate. The colony forming units (CFU) of H. pylori were found to increase at least 10-fold at 12 hrs p.i., compared with the number that was only internalized (2 hrs) (Fig. 1A). The H. pylori bacteria were associated with THP-1 cells because they were recovered from the pelleted THP-1 cells after centrifugation. The viability of the H. pylori-THP-1 cells gradually decreased after 24 hrs post infection (data not shown). The multiplication of H. pylori in macrophage was dependent on the cell lines and isolates used. H. pylori can multiply in another human U937 macrophage cell line (Fig. 1B ), but not in murine macrophage Raw264.7 cell line (Fig. 1C ) or mouse resident peritoneal macrophage (Fig. 1D ). The multiplication in THP-1 cells was not restricted to the particular strain HP238 used. Other clinical isolate of strain HP250 could also multiply in THP-1 cells (Fig. 2 ). However, we did not find the multiplication for the strain HP46A, HP46B, J99, or ATCC43504 in THP-1 cells. They were eliminated gradually after internalization. During the 1 hr-incubation, the internalized H. pylori in THP-1 cells varied, and the number of the recovered H. pylori depended on the strain used: ATCC43504 >HP238 >HP250 >HP46A >HP46B >J99. But only HP238 and HP250 strains can undergo multiplication. The HP46A, HP46B, and J99 strains were completely killed in 6 hrs p.i.. For the standard strain ATCC43504, the killing was slower and delayed, but we could not detect any viable H. pylori left at 24 hrs p.i. or afterward. Based on its ability to replicate in the macrophage, the H. pylori can be considered as a type of intracellular bacterium.

The Induction of H. pylori-Associated Vacuole in Infected THP-1 Cells.

The multiplication of H. pylori in THP-1 cells was further characterized. To trace its multiplicative site, the H. pylori was green fluorescent labeled with PKH2 dye, while the ER, mitochondria, lysosome, or nucleus was stained with anti-calnexin Ab, lyso-tracker, mito-tracker, or DAPI, respectively. As shown in Figure 3 with the confocal microscopic observation, the H. pylori was found to reside and multiply in the cytoplasm, no co-localization of the green H. pylori was found with the staining of the ER (Fig. 3A ), lysosome (Fig. 3B ), or mitochondria (Fig. 3C ). The green H. pylori accumulated in some areas of the cytoplasm at 6 hrs post infection, and the internalized H. pylori had high fluorescent intensity of FITC in the cytoplasm. But along the time of infection at 12 hrs, the intensity of the green fluorescent H. pylori decreased, and spread out in the whole cytoplasm. Moreover, the stain of the microbial DNA by DAPI, smaller than the nucleus of THP-1 cell, was found to have occurred in the cytoplasm, with increasing number at 12 hrs p.i. This indicates that the multiplication of the H. pylori occurs in the cytoplasm. We further used electron microscopy to examine this multiplication. The formations of H. pylori-associated vacuoles in THP-1 cells were observed. Double-layer vesicles that contained H. pylori were found at 6 hrs p.i. Two dividing H. pylori were observed in the same vacuole (Fig. 4A ). When a more detailed kinetic examination at 6, 12, and 24 hrs p.i. was performed, more vacuoles were seen with disrupted cellular structure. Some multiple layer vesicles or onion-like structures became prominent. At 24 hrs p.i., intact H. pylori was less abundant, and less electron-dense H. pylori (a sign of bacterial death) in vacuole was observed (Fig. 4B ). There were many membranous structures around the H. pylori-containing vacuole (Fig. 4C ).

Autophagy Was Induced Post H. pylori Infection in THP-1 Cells.

The formation of double-membrane vesicles in H. pylori-infected THP-1 cells suggested that H. pylori-infection might induce autophagy in macrophages. The anti-LC3-II antibody was used to detect the distribution of LC3-II protein in H. pylori-infected THP-1 cells. The H. pylori infection caused the LC3-II punctate aggregations, which were co-localized with H. pylori in the cytoplasm (Fig. 5 ). More LC3 punctuates were observed at 6 hrs and 12 hrs p.i. than at 2 hrs p.i. We used m.o.i. = 5 to infect the THP-1; H. pylori will replicate in every THP-1 cell, therefore, every infected cell has LC3 punctate formation. Furthermore, Western blot analyses were performed to detect autophagic markers. At 6 hrs p.i., mild LC3-II conversion was detected, but at 24 hrs p.i., LC3-I was up-regulated compared with the mock control in infected THP-1 cells. None of Beclin 1 (Fig. 6 ), BNIP3, or Atg5 (data not shown) was found to increase post infection. We further used the common autophagy inhibitor, 3-MA, and autophagy stimulator, rapamycin, to evaluate the effect of autophagy on the H. pylori infection. In the permissible THP-1 cells, more multiplication of H. pylori was observed at 6, 12 and 24 hrs p.i. in the presence of 3-MA (Fig. 7 ). On the contrary, the autophagy inducer, rapamycin, inhibited the multiplication of H. pylori at 6 and 12 hrs p.i. in THP-1 cells. However, in the resistant murine macrophage cell line Raw264.7, the clearance of H. pylori was not affected by 3-MA or rapamycin treatment (data not shown). This suggests that autophagic vesicles are induced after H. pylori infection in THP-1 cells, and that the vesicle formation was associated with the multiplication. The observations that 3-MA treatment enhanced while rapamycin inhibited the multiplication of H. pylori suggest that autophagosome formation seems to participate for the clearance of H. pylori.

The Mutation of VacA or CagA Affects the Multiplication or Survival of H. pylori in Macrophages.

H. pylori would multiply in THP-1 cells whereas it is cleared in Raw264.7 cells. The role of VacA or CagA was further evaluated in these different responses. The CagA and VacA mutants are internalized with a lower efficiency than the wild type. In THP-1 cells, the viable H. pylori CagA or VacA mutants are barely detectable at 2 hrs p.i.; however, they still can be recovered from the infected cell at 6 hrs p.i., suggesting that the H. pylori mutants are still multiplying in THP-1 cells. But they were then quickly eliminated at 12 or 24 hrs p.i. (Fig. 8B ). On the other hand, in the resistant Raw264.7 cells, the internalized CagA and VacA mutants at 2 hrs p.i. were lower than the wild type and were cleared faster than the wild type (Fig. 8A ). These data suggest that both CagA and VacA are virulent factors for H. pylori and contribute to the multiplication or survival in macrophages.

Discussion

H. pylori is not efficiently controlled by phagocytes because it exhibits several mechanisms of virulence that cause the evasion of the osponization, actively retard phagocytosis or disrupt membrane trafficking and phagosome maturation after internalization of the microorganism (4). In this study, we further reported that some Taiwanese clinical isolates of H. pylori can escape the killing of the phagolysosome and replicate in the autophagic vesicle.

Autophagy has been found to be a component of the innate cellular immune responses against not only intra-cellular but also for extracellular microorganisms (13–15). After phagocytosis of the microorganism, the autophagosomes will be formed to degrade the ingested bacterium by the lysosomal killing mechanism. However, the intracellular bacteria such as Legionella pneumophila, Listeria mono-cytogenes, Mycobacterium, and Shigella have developed different mechanisms to evade the autophagic cellular surveillance in macrophage. Although the double-membrane-bound compartments that bear autophagic markers were induced, its maturation into autolysosomes will be arrested or delayed. The subversion or avoidance of the autophagy by a microorganism will favor its own replication (9, 16, 17). Listeriolysin O (LLO), a pore-forming virulence factor, is essential for L. monocytogenes to escape from phagocytic killing. Cheryl L. Birmingham et al. revealed that autophagy restricts L. monocytogenes replication to late endosome under conditions when LLO expression is impaired within macrophage (18). In our study, the H. pylori infection induced autophagosome formation that provides a place for its multiplication site in the permissive THP-1 cells. The autophagosome formation will have different outcomes depending on the virulence of the H. pylori or the host defense status. Why different macrophages respond differently to H. pylori infection is not clear. It might be related to the cell line used because murine macrophage cell lines especially the Raw264.7, are known to have high inducible NO synthase. NO is known to play an important antimicrobial role in innate immunity (19, 20). But, the human or mouse origin is not critical because we found that mouse bone-marrow derived dendritic cells can also support the replication of H. pylori and induce autophagy (unpublished data).

Vaculoating toxin (VacA) is a 95 kDa secreted exotoxin. VacA was initially characterized and purified on the basis of its ability to induce the formation of intracellular vacuoles in tissue culture cells. It can insert itself into the epithelial cell membrane and forms a hexameric anion-selective, voltage-dependent channel through which bicaronate and organic anions can be released. VacA can also target to the mitochondria membrane, and can induce the release of cytochrome c and apoptosis (21). VacA can also induce the proliferation of acidic vacuoles that contain late endosomal and lysosomal proteins that are hallmarks of autophagosomes (22). H. pylori strains possessing different alleles of vacA differ in their ability to express active toxin. Those strains expressing higher toxin levels are correlated with more severe gastric disease. The cytotoxin-associated gene A (CagA) is an important virulence factor. Initial studies on different isolates of H. pylori showed that strains carrying vacA and cagA are more virulent and cause more severe pathology. These isolates are referred to as type I strains, while the less virulent strains are type II. Both type I and type II strains express the vacA gene and the vast majority of them produce active cytotoxin, though in variable amounts. The major difference resides in the expression of CagA. Most type I H. pylori strains contain the cag pathogenicity island (cag-PAI), a 37-kb genomic fragment composed of 29 genes. Most of the cag genes are probably involved in the assembly of secretory machinery that translocates the 120 kDa protein CagA into the cytoplasm of gastric epithelial cells by type IV secretion system. Proteins encoded by the island are involved in two major processes: the induction of IL-8 production by gastric epithelial cells and the CagA translocation. After entering the epithelial cell, CagA is phosphorylated and binds to SHP-2 tyrosine phosphatase, leading to a growth factor-like cellular response. H. pylori can induce homotypic phagosome fusion and form a large communal or multivesicular vacuoles in human monocytes or epithelial cells, which is independent of VacA or CagA (23–25). In our study, we found that CagA or VacA mutant would compromise the ability to multiply in the susceptible THP-1 cells, or enhance the clearance in the resistant Raw264.7 cells.

The best characterized adhesin for H. pylori to bind to epithelial cells is the BabA, which is a 78-kDa outer-membrane protein that interacts with the fucosylated Lewis B blood group antigen. BabA2 of H. pylori could effectively bind to Lewis B antigen in the stomach, and the bacterial adhesion to gastric mucosa could be highly facilitated to enhance the colonization (26). Another SabA protein performs the binding to sLex glycosphingolipids for membrane close attachment and apposition (27). In our study, the ingestion of various H. pylori in macrophages varied, which probably reflects the difference of adhesive between the cell membrane of H. pylori and the phagosome of the macrophage. The other components, such as the membrane lipid or cholesterol composition, might relate to the internalization of the bacterium. Excessive cholesterol promotes phagocytosis of H. pylori by phagocytes and enhances T cell response, whereas cholesterol glucosylation abrogates phagocytosis and promotes immune evasion (28). Another intracellular pathogen-recognition molecule, NOD1, was found to recognize the peptidoglycan of H. pylori by the type IV secretion system of cag pathogenicity island (29). Although type I H. pylori was defined as the more virulent strain and delayed entry followed by homotypic phagosome fusion (6, 30), catalase and urease are reported to play important roles in avoiding phagocytic killing (31, 32). Rittig et al. reported that human mono-nuclear phagocytes and epithelial cell lines treated with different vacA and cag genotypes and the homotypic phagosome fusion were frequently induced by all live H. pylori strains, independent of VacA or Cag status (7). In our study, VacA or CagA participates in the multiplication or survival in macrophages because these mutants have lower levels of replication and are cleared in a much quicker manner. Different regions or countries have unique characteristics of the isolated H. pylori. There were reports of more virulence on the Taiwanese isolated H. pylori. The HP238 and HP250 isolates were derived from gastric adenocarcinoma and mucosa-associated lymphoid-tissue (MALT) lymphoma, respectively, whereas the HP46A, HP46B were derived from patients with mild gastritis (unpublished observation). The genotypes of H. pylori isolates in Taiwan have several unique features that are different from the rest of the world. Most of Taiwan’s H. pylori are cagA ⁺, vacA ⁺, babA2 ⁺. The fate of H. pylori is dependent on the isolates used for the infection. The macrophage THP-1 cell line used in this study cannot solely support the replication of H. pylori HP238. We also found that gastric epithelial AGS cell line, as well as mouse bone marrow-derived dendritic cells, can support the replication of H. pylori HP238 and induce autophagy post infection (unpublished data). Moreover, the percentage of iceA ⁺ is also higher than in Western countries, and is associated with the severity of H. pylori-induced disease (33). Genetic polymorphism exists between Taiwanese and Western strains (34, 35). Distinct vacA alleles and cagA were found in geographic distribution of H. pylori genotypes. The East Asia H. pylori isolate from gastric carcinoma had a distinct tyrosine phosphorylation sequence at the region on the repeat sequence of Western CagA, which conferred stronger SHP-2 binding and morphologically transforming activities than the Western CagA. The presence of distinctly structured CagA proteins in Western and East Asian H. pylori isolates may underlie the striking difference between the different findings in our study and the literature report that only delay the phagosome maturation. C.C. Chen and her colleagues also reported that the Taiwan H. pylori isolates from different diseases (gastritis, ulcer, or cancer) bear different abilities to induce cyclooxygenase-2 or cyclin D1 with the highest from gastric cancer (36–39). This virulence is not related to iceA, vacA, babA2, cagA, or hrgA. This raises an interesting question about the unique feature of particular isolates from gastric cancer or MALT lymphoma, and the oncogenesis of H. pylori. Our finding about the resistance to macrophage killing and multiplication in macrophage by HP238 and HP250, but not HP46A or HP45B, provides another unidentified virulent gene in isolates from H. pylori-associated cancer patients. To reveal this micro-heterogeneity of various isolates might provide important information why this microorganism causes so broad of a spectrum of diseases.

Figure 1.
The infection of H. pylori in macrophages. Macrophages were infected with HP238 at m.o.i. = 10 for 1 hr. The extracellular bacteria were killed with gentamycin. The internal H. pylori was quantitated after lysis of the macrophages at various time points (2, 6, 12, or 24 hrs) post infection. The recovered viable H. pylori were determined as CFU on CDC plate. (A) THP-1, (B) THP-1 and U937 cells, (C) Raw264.7, (D) mouse peritoneal macrophage.

Figure 2.
The infection of THP-1 cell with various strains of H. pylori. THP-1 cells were infected with various strains of H. pylori, HP238 (•), HP250 (○), HP46A (▾), HP46B (▿), and standard strains, J99 (▪) and ATCC43504 (□) at m.o.i. = 10 for 1 hr. The extracellular bacteria were killed with gentamycin. The internal H. pylori were quantitated after lysis of the THP-1 cells at various time points (2, 6, or 12 hrs) post infection. The recovered viable H. pylori were determined as CFU on CDC plate.

Figure 3.
The multiplication of H. pylori in the cytoplasm of THP-1 cells. THP-1 cells were infected with PKH2-labeled HP238 at m.o.i. = 5 for 1 hr. The extracellular bacteria were killed with gentamycin. At 6 hrs or 12 hrs post infection, the infected THP-1 cells were stained with anti-calnexin antibody (A), Lyso-tracker (B), anti-COX-4 antibody (C) and DAPI. The arrow indicates the presence of H. pylori.

Figure 4.
Ultrastructural alterations in H. pylori-infected THP-1 cells. THP-1 cells were infected with HP238 at m.o.i. = 10 for 1 hr. The extracellular bacteria were killed with gentamycin. At 6, 12, and 24 hrs post infection, infected cells were collected for EM examination. The (A) shows dividing bacteria in double-layer vesicle at 6 hrs p.i.. The enlarged pictures in ‘b’ and ‘c’ were derived from the squares of ‘a’. The arrow indicates dividing H. pylori while the triangle indicates double membrane formation. M, mitochondria; N, nucleus. The (B) shows the location of H. pylori in THP-1 cells at various time points. The enlarged pictures in blow panel were derived from upper panel in (B). The closed triangles show the double membrane structure containing H. pylori-like organisms in infected cells, and the arrows indicate the onion-like structure.

Figure 5.
H. pylori infection in THP-1 cells induces LC3 aggregation around the multiplicative bacteria. THP-1 cells were infected with HP238 at m.o.i. = 5 for 1 hr. At 2, 6 and 12 hrs post infection, the infected THP-1 cells were collected for immunofluorescent staining assay as described in Materials and Methods. The arrow indicates the location of H. pylori stained by anti-H. pylori antibodies.

Figure 6.
H. pylori infection induces LC3-II conversion in THP-1 cell. THP-1 cells were infected with HP238 at m.o.i. = 10 for 1 hr. At 6, 12 and 24 hrs post infection, the infected cells were collected for Western blot analysis to detect LC3-II conversion. Mock groups were treated without H. pylori and collected at various time points to compare with infection groups.

Figure 7.
The effects of 3-MA or Rapamycin on the multiplication of H. pylori in THP-1 cells. THP-1 cells were infected with HP238 at m.o.i. = 10 for 1 hr. After infection and gentamycin treatment, infected THP-1 cells were then treated with PBS (HP alone), 10 mM 3-MA or 200 nM rapamycin with complete media. The internal H. pylori was quantitated after lysis of the macrophages at various time points (2, 6, 12 and 24 hrs) post infection. The recovered viable H. pylori were determined as CFU on CDC plate. * P value < 0.05.

Figure 8.
The effect of Vac A or Cag A mutation on the fate of H. pylori in macrophages. Raw264.7 cells (A) and THP-1 cells (B) were infected with HP238, HP238 vacA::cm or HP238 cagA::cm mutants at m.o.i. = 10 for 1 hr. The extracellular bacteria were killed with gentamycin. The internal H. pylori was quantitated after lysis of the macrophages at various time points (2, 6, 12, or 24 hrs) post infection. The recovered viable H. pylori was determined as CFU on CDC plate.

Footnotes

This work was supported by grant NSC94-2320-B006–008, NSC95-2320-B006-063-MY2 from the National Science Council, Taiwan.

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