TY - JOUR
AU - Krogfelt, K.A.
AB - Abstract In a human gastric biopsy specimen, 30% of adhering Helicobacter pylori strain AF4 (cagA and VacA positive) was associated with adhesion pedestals. In an AGS cell assay, only a few percent of this type I strain was found to be associated with adhesion pedestals. Nevertheless, a larger proportion of the type I strain was found to invade AGS cells (P<0.03) and to attach with depressions in the AGS cell membrane (P<0.03) than a type II strain (cagA and VacA negative). Incubation of AGS cells and H. pylori without adding fetal calf serum (FCS) to the culture medium increased actin accumulations (FITC-phalloidin stained) beneath adhering H. pylori, and decreased H. pylori invasion of AGS cells significantly (P<0.01). However, no increase in the number of adhesion pedestals was observed by electron microscopy. Proteinase K treatment of FCS eliminated the H. pylori invasion promoting effect (P<0.01). Our results suggest differences in the ability of H. pylori to induce adhesion pedestals in human gastric epithelial cells and in AGS cells, but a correlation between adhesion pedestal formation in vivo and H. pylori invasion in vitro can be speculated. In addition, H. pylori invasion into AGS cells was found to be mediated by proteins in FCS. Adhesion, Invasion, Fetal calf serum, Strain type, Host cell, Helicobacter pylori 1 Introduction Helicobacter pylori is accepted as a major cause of duodenal and gastric ulcers [1,2], and a relationship to distal gastric adenocarcinoma and to gastric mucosa associated lymphoid tissue (MALT) lymphoma has been suggested [3–5]. H. pylori attaches to gastric epithelial cells lining the antrum of the stomach. Electron microscopic studies have revealed distinct H. pylori ultrastructural adhesion patterns, which have been classified as: (i) binding through filamentous strands; (ii) adhering to microvilli; (iii) abutting on the cell membrane; (iv) associating with adhesion pedestals; (v) occupying depressions in the cell membrane; and (vi) invading the cell [6]. Studies have described H. pylori adhesion to adhesive pedestals in human biopsy specimens [7,8], as well as in vitro using a human gastric cancer cell line (AGS cells) and primary cultured human gastric cells overlaid with H. pylori[9,10]. Actin accumulation in AGS cells beneath adhering H. pylori has been linked to the induction of adhesion pedestals, and a comparison with similar changes induced by attaching and effacing Escherichia coli has been made [9–11]. H. pylori is generally not considered an invasive enteropathogen [11]. Nevertheless, intracellular H. pylori have been observed in human gastric biopsy specimens on several occasions [6,12–21]. The aim of the present study was to compare the ultrastructural adhesion pattern in vivo and in vitro of an H. pylori type I strain (vacuolating cytotoxin activity in vitro (VacA) and cytotoxin associated gene A (cagA) positive), and to compare in vitro the type I strain with a type II strain (cagA and VacA negative). Another aim was to explore if fetal calf serum (FCS) interfered with H. pylori adhesion, examined by electron microscopy and FITC-phalloidin staining, and invasion of AGS cells. 2 Materials and methods 2.1 Strains H. pylori strain AF4 isolated from a patient with a gastric ulcer [22], induced adhesion pedestals in vivo according to electron microscopic evaluation of an antral gastric biopsy from the patient. (Three additional biopsies were taken from the patient and the H. pylori cultured from these biopsies were genotypically identical to AF4 according to randomly polymorphic DNA typing confirming that this patient was only infected with one H. pylori strain.) AF4 was positive for the cagA gene by PCR (cagA gene expression was confirmed by SDS–PAGE), and exhibited cytotoxic effect with vacuolation in vitro [23]. An H. pylori type II strain (American Type Culture Collection 51932, cagA and VacA negative), a non-invasive eae-A positive E. coli strain D1826 [24] and an invasive Salmonella typhimurium C17 [25] were chosen as controls of in vitro adhesion and invasion. All strains were kept in ox broth with 10% glycerol (Statens Serum Institut, Copenhagen, Denmark) at −80°C until use. 2.2 Adhesion assay and preparation for electron microscopy AGS cells (American Type Culture Collection 1739, Rockville, MD, USA) were grown to confluence in tissue culture flasks 25 cm2 (Nunc, Naperville, IL, USA) in modified Eagle's minimal essential medium (MEM) (Statens Serum Institut, Copenhagen, Denmark) and overlaid, after removal of growth medium and washing, with H. pylori, taken from 2 days old cultures. H. pylori was cultured on 7% lysed horse blood agar plates (chocolate agar plates, Statens Serum Institut, Copenhagen, Denmark). The plates were incubated under microaerobic conditions at 37°C. 800 µl of a 5×108 bacteria ml−1 suspension was added to 3 ml MEM both with and without the addition of 10% fetal calf serum (FCS) (Bodinco BV, Alkmaar, The Netherlands). The multiplicity of infection (MOI) (the ratio of bacteria per cell) was ~100:1. Thereafter, the bacterial suspensions were incubated with the AGS cells for 3 h. E. coli and S. typhimurium were treated according to standard procedures for the actin phalloidin staining test [26]. Cells with adhering bacteria were fixed in 3% glutaraldehyde for 2 h, centrifuged and embedded in Noble agar (Difco, Augsburg, Germany). Small cubes with visible clusters of cells were fixed overnight in 3% glutaraldehyde. The specimens were postfixed for 90 min in 1% OsO4 (w/v) and en bloc stained in 2% (w/v) uranyl acetate for another 60 min. Procedures for dehydration and embedding in epon were according to a standard protocol [27]. The human gastric biopsy specimen from the patient infected with H. pylori strain AF4 was fixed using the same procedure. Transmission electron microscopy (TEM) was carried out with a Philips 201C electron microscope at 60 kV, and AGS cells and human gastric epithelial cells with adherent H. pylori were studied by a magnification of 30 000×. Adherent and ingested bacteria were counted and classified. 2.3 Adhesion assay and preparation for immunofluorescence and actin staining AGS cells were grown in Permanox plastic chamber slides (Nunc, Naperville, IL, USA) to 80% confluence in modified MEM supplemented with 10% FCS and antibiotics. Before adding H. pylori, AGS cells were washed in serum free MEM. Bacteria were grown for 2 days on chocolate agar plates (Statens Serum Institut, Copenhagen, Denmark), harvested and suspended in modified MEM both with and without the addition of 10% FCS. 500 µl of a 107 bacteria ml−1 suspension were added to each well (MOI~100:1). After 3 h incubation the wells were aspirated and washed three times with PBS, followed by fixation of the cells in 3% formalin for 15 min. Cells were then treated with 0.1% Triton X-100 for 4 min. Actin condensation was revealed by FITC-phalloidin staining [28]. The presence of H. pylori was demonstrated by additional staining with rabbit anti-H. pylori polyclonal antibodies (Biomeda Corp, Foster City, CA, USA) and swine anti-rabbit IgG rhodamine conjugate (DAKO, Glostrup, Denmark). Specimens were examined in a Leica DMRB epi-illumination microscope (Leica A/S, Herlev, Denmark). Photos were scanned and formatted using Adobe Photoshop 3.0 for Macintosh and printed on a Kodak XLS 8600 PS printer. 2.4 Gentamicin invasion assay AGS cells were seeded in 24-well tissue culture plates (Falcon, Franklin Lakes, NJ, USA). Confluent monolayers were infected with 107H. pylori in 400 µl MEM, in 400 µl MEM with 10% FCS and in 400 µl MEM with 10% modified FCS (MOI~30:1). FCS was modified either by (a) boiling for 20 min, (b) treatment with 0.5 mg proteinase K ml−1 for 1 h at 60°C and boiled for 20 min, and (c) treatment with 0.4 U neuraminidase ml−1 for 1 h at 37°C and boiled for 20 min. Infected cells were incubated for 3 h at 37°C. Wells were washed three times with PBS to remove unbound bacteria. To determine the number of cell associated bacteria per well, infected cells were treated with 1 ml of 0.1% digitonin and 20% trypsin in PBS for 10 min to lyse and separate epithelial cells, and CFU were counted by plating serial 10-fold dilutions on chocolate agar plates (Statens Serum Institut, Copenhagen, Denmark). To determine the number of internalized H. pylori, another well of similarly infected cells was incubated for an additional 1.5 h in medium supplemented with 100 µg gentamicin ml−1, and CFU counting was repeated. Invasion and adherence were determined as the mean of at least three experiments (two wells per test). 2.5 Statistics Ultrastructural differences between H. pylori invasion of and adhesion to AGS cells, with and without FCS, were assessed by a chi-square test. Differences between means of invasion and adhesion in gentamicin assays were for assays with different concentrations of FCS evaluated by analysis of variance and for all other assays by post hoc comparisons of means, Scheffé's test. P<0.05 was considered significant. 3 Results 3.1 Adhesion of H. pylori AF4 (type I) in a human gastric biopsy Electron microscopic examination of a gastric biopsy specimen from the gastric ulcer patient infected with H. pylori AF4 (type I) showed that 30% of adhering H. pylori associated with adhesion pedestals (Fig. 1), 28% adhered through filamentous strands, 25% abutted to the cell membrane, 13% adhered to microvilli and 3% made depressions in the cell membrane (n=60). No intracellular H. pylori was found in the gastric epithelial cells in the biopsy. Figure 1 View largeDownload slide Electron micrograph of a gastric antrum biopsy from patient infected with H. pylori AF4 (type I). Representative bacteria adhering to a gastric epithelial cell through an adhesion pedestal, big arrowheads show sheathed flagella, while the small arrowheads indicate vacuoles in the epithelial cell cytoplasm close to the contact zone. Bar=0.3 µm. Figure 1 View largeDownload slide Electron micrograph of a gastric antrum biopsy from patient infected with H. pylori AF4 (type I). Representative bacteria adhering to a gastric epithelial cell through an adhesion pedestal, big arrowheads show sheathed flagella, while the small arrowheads indicate vacuoles in the epithelial cell cytoplasm close to the contact zone. Bar=0.3 µm. 3.2 Adhesion of H. pylori AF4 (type I) and H. pylori ATCC 51932 (type II) to AGS cells Electron micrographs of AGS cells infected with wild-type H. pylori AF4 (type I), cultured from the patient mentioned above, and H. pylori ATCC 51932 (type II) for 3 h were studied, and the different types of adhesion were counted. For both strains only a few percent of adhering bacteria associated with adhesion pedestals (Table 1), incubation for 24 h did not increase the number of bacteria associated with adhesion pedestals (data not shown). No qualitative difference between the ultrastructural adhesion of the type I and the type II strains to AGS cells could be demonstrated. Although the frequency of invasion was higher for the type I strain, 41 of 280 (15%), than for the type II strain, 13 of 168 (8%) (Table 1), P<0.03. In addition more type I H. pylori, 31 of 280 (11%), adhered through depressions in the cell membrane than type II H. pylori, 8 of 168 (5%), P<0.03. Table 1 Effect of fetal calf serum (FCS) on H. pylori association with AGS cells H. pylori strain and experimental condition  No. of AGS cell profiles examined  Number of bacteria/all (%)      Adhering to microvilli  Abutting to the cell membrane  Adhering to pedestals  Depressions in the cell membrane  Intracellular  H. pylori type I  With FCS  61  25/280 (9%)  179/280 (64%)  4/280 (1%)  31/280 (11%)  41/280 (15%)  Without FCS  71  53/318 (17%)  235/318 (74%)  3/318 (1%)  12/318 (4%)  15/318 (5%)  H. pylori type II  With FCS  61  37/168 (22%)  105/168 (63%)  5/168 (3%)  8/168 (5%)  13/168 (8%)  Without FCS  58  49/192 (26%)  135/192 (70%)  4/192 (2%)  1/192 (1%)  3/192 (2%)  H. pylori strain and experimental condition  No. of AGS cell profiles examined  Number of bacteria/all (%)      Adhering to microvilli  Abutting to the cell membrane  Adhering to pedestals  Depressions in the cell membrane  Intracellular  H. pylori type I  With FCS  61  25/280 (9%)  179/280 (64%)  4/280 (1%)  31/280 (11%)  41/280 (15%)  Without FCS  71  53/318 (17%)  235/318 (74%)  3/318 (1%)  12/318 (4%)  15/318 (5%)  H. pylori type II  With FCS  61  37/168 (22%)  105/168 (63%)  5/168 (3%)  8/168 (5%)  13/168 (8%)  Without FCS  58  49/192 (26%)  135/192 (70%)  4/192 (2%)  1/192 (1%)  3/192 (2%)  AGS cells infected with H. pylori strains type I (AF4) and type II (51932) were incubated in MEM with or without supplementation of heat inactivated FCS. Electron micrographs were taken and cell profiles were examined for attaching and invading microorganisms. Intrusion of the bacteria was greater than one third of the diameter of the bacteria. Significantly more bacteria were found in the cytoplasm and significantly more bacteria were associated with depressions in the cell membrane when the medium was supplemented with 10% FCS (P<0.01, chi-square), and under conditions with FCS more strain type I H. pylori were found inside AGS cells and adhering through depressions in the cell membrane than type II H. pylori (P<0.03, chi-square). Significantly fewer AF4 bacteria were binding to microvilli and abutting to the cell membrane when the medium was supplemented with 10% FCS (P<0.01, chi-square). View Large Table 1 Effect of fetal calf serum (FCS) on H. pylori association with AGS cells H. pylori strain and experimental condition  No. of AGS cell profiles examined  Number of bacteria/all (%)      Adhering to microvilli  Abutting to the cell membrane  Adhering to pedestals  Depressions in the cell membrane  Intracellular  H. pylori type I  With FCS  61  25/280 (9%)  179/280 (64%)  4/280 (1%)  31/280 (11%)  41/280 (15%)  Without FCS  71  53/318 (17%)  235/318 (74%)  3/318 (1%)  12/318 (4%)  15/318 (5%)  H. pylori type II  With FCS  61  37/168 (22%)  105/168 (63%)  5/168 (3%)  8/168 (5%)  13/168 (8%)  Without FCS  58  49/192 (26%)  135/192 (70%)  4/192 (2%)  1/192 (1%)  3/192 (2%)  H. pylori strain and experimental condition  No. of AGS cell profiles examined  Number of bacteria/all (%)      Adhering to microvilli  Abutting to the cell membrane  Adhering to pedestals  Depressions in the cell membrane  Intracellular  H. pylori type I  With FCS  61  25/280 (9%)  179/280 (64%)  4/280 (1%)  31/280 (11%)  41/280 (15%)  Without FCS  71  53/318 (17%)  235/318 (74%)  3/318 (1%)  12/318 (4%)  15/318 (5%)  H. pylori type II  With FCS  61  37/168 (22%)  105/168 (63%)  5/168 (3%)  8/168 (5%)  13/168 (8%)  Without FCS  58  49/192 (26%)  135/192 (70%)  4/192 (2%)  1/192 (1%)  3/192 (2%)  AGS cells infected with H. pylori strains type I (AF4) and type II (51932) were incubated in MEM with or without supplementation of heat inactivated FCS. Electron micrographs were taken and cell profiles were examined for attaching and invading microorganisms. Intrusion of the bacteria was greater than one third of the diameter of the bacteria. Significantly more bacteria were found in the cytoplasm and significantly more bacteria were associated with depressions in the cell membrane when the medium was supplemented with 10% FCS (P<0.01, chi-square), and under conditions with FCS more strain type I H. pylori were found inside AGS cells and adhering through depressions in the cell membrane than type II H. pylori (P<0.03, chi-square). Significantly fewer AF4 bacteria were binding to microvilli and abutting to the cell membrane when the medium was supplemented with 10% FCS (P<0.01, chi-square). View Large 3.3 Adhesion pedestals and actin accumulation with and without FCS in AGS cell assays When H. pylori infected AGS cells were stained specifically for H. pylori and actin, clearly visible co-localization occurred only when H. pylori and AGS cells were incubated without FCS (Fig. 2A,B). For both strain AF4 and 51932 the actin accumulation was very subtle compared to cytoskeletal changes in AGS cells induced by an eae-A positive E. coli (data not shown). Actin accumulations (FITC-phalloidin stained) was primarily found under H. pylori bacteria adhering in large groups (Fig. 2A). When AGS cells and bacteria were incubated with FCS, H. pylori was not associated with clearly visible actin accumulations (FITC-phalloidin stained) (Fig. 2C,D). Coincubation of cells and bacteria without addition of FCS to the cell culture medium did not, according to electron micrographs, increase the number of bacteria associated with adhesion pedestals (Table 1). Four clinical H. pylori strains had similar responses to FCS (data not shown). Figure 2 View largeDownload slide Immunofluorescence image of H. pylori AF4 (type I) attached to AGS cells without (A, B, original magnification ×500), and with (C, D, original magnification ×1250) the addition of 10% fetal calf serum (FCS). (A, C) Fields stained for H. pylori (red) and actin (green). (B, D) Fields stained for actin. Accumulation of large amounts of actinous material beneath H. pylori are seen only when cells and bacteria are coincubated without FCS (arrows). Figure 2 View largeDownload slide Immunofluorescence image of H. pylori AF4 (type I) attached to AGS cells without (A, B, original magnification ×500), and with (C, D, original magnification ×1250) the addition of 10% fetal calf serum (FCS). (A, C) Fields stained for H. pylori (red) and actin (green). (B, D) Fields stained for actin. Accumulation of large amounts of actinous material beneath H. pylori are seen only when cells and bacteria are coincubated without FCS (arrows). 3.4 H. pylori invasion of AGS cells with and without FCS 15% of the H. pylori AF4 (type I) bacteria were intracellular according to electron micrographs when FCS was present compared to 5% when FCS was not (Table 1) (P<0.01). A similar pattern with H. pylori 51932 (type II), 8% vs. 2%, indicates that FCS effects invasion. Intracytoplasmic H. pylori bacteria were found in membrane bound vacuoles both with and without FCS added to the culture medium (Fig. 3a). When FCS was not added to modified MEM, adhering bacteria co-localized with large amounts of disorganized filamentous structures within the AGS cells (Fig. 3b). Further, adherence accompanied by depressions in AGS cell membranes was more frequent for both strain types when FCS was added to the medium (P<0.01). Four clinical H. pylori strains had a similar response to FCS, while no invasion increasing effect of FCS was seen on a non-invasive eae-A positive E. coli (0 of 150 (0%) invaded without and 3 of 128 (2%) invaded with FCS), and an invasive S. typhimurium (62 of 78 (79%) without and 37 of 70 (53%) with FCS). Several batches of FCS were evaluated and all batches increased H. pylori invasion into AGS cells (data not shown). Figure 3 View largeDownload slide Electron micrographs from AGS cell cultures incubated for 3 h with H. pylori AF4 (type I) with (a) and without (b) 10% FCS. (a) Thin section of a part of an AGS cell shows intracellular H. pylori (stars). The vacuoles containing the organisms are surrounded by mitochondria (M). Bar=0.3 µm. (b) A representative thin section shows several H. pylori organisms adherent to the surface of an AGS cell. A bundle of actin-like filaments (arrowheads) are seen in the cytoplasm together with a network of microfilaments (arrows), which adhere to a dense zone, close to an adherent H. pylori. N denotes the nucleus of the epithelial cell. Bar=0.3 µm. Figure 3 View largeDownload slide Electron micrographs from AGS cell cultures incubated for 3 h with H. pylori AF4 (type I) with (a) and without (b) 10% FCS. (a) Thin section of a part of an AGS cell shows intracellular H. pylori (stars). The vacuoles containing the organisms are surrounded by mitochondria (M). Bar=0.3 µm. (b) A representative thin section shows several H. pylori organisms adherent to the surface of an AGS cell. A bundle of actin-like filaments (arrowheads) are seen in the cytoplasm together with a network of microfilaments (arrows), which adhere to a dense zone, close to an adherent H. pylori. N denotes the nucleus of the epithelial cell. Bar=0.3 µm. 3.5 Influence of FCS concentration on invasion in gentamicin assays In a gentamicin invasion assay, adding 10% FCS to the test medium increased the proportion of invaded H. pylori AF4 from a mean of 2% to a mean of 12% of adhering bacteria (Fig. 4) (P<0.01). Within the observed time interval of 3 h, the number of adhering bacteria did not change, irrespective of whether bacteria and AGS cells were incubated with 10% FCS or not. To determine the concentration of FCS which gave the maximum H. pylori invasion, AGS cells were infected with H. pylori strain AF4 for 3 h, without FCS, and with 2, 5, 10 and 20% FCS. The invasion was calculated by dividing CFU of intracellular bacteria with CFU of adhering bacteria. An increase in invasion was observed with 2% and 5% FCS added, and the maximum invasion rate was observed with 10% FCS (Fig. 4) (P<0.01). Addition of 20% FCS resulted in both decreasing adhesion and decreasing invasion, data not shown. Figure 4 View largeDownload slide Gentamicin invasion assay. The effects of various concentrations of FCS in MEM on H. pylori invasion of AGS cells. All assays were performed in triplicate. Of 4×106 added bacteria a mean of 4.9×105 (12%) adhered to AGS cells. Means and standard error are shown. P<0.01, P value represents analysis of variance. Figure 4 View largeDownload slide Gentamicin invasion assay. The effects of various concentrations of FCS in MEM on H. pylori invasion of AGS cells. All assays were performed in triplicate. Of 4×106 added bacteria a mean of 4.9×105 (12%) adhered to AGS cells. Means and standard error are shown. P<0.01, P value represents analysis of variance. 3.6 Modification of FCS and invasion in gentamicin assays Proteinase K treatment and subsequent boiling completely eliminated the invasion promoting effect of FCS (Fig. 5) (P<0.01), although no changes were seen in the adhesion of H. pylori to AGS cells (Fig. 6). Neuraminidase treatment of FCS resulted in a decrease of H. pylori adhesion (Fig. 6) (P=0.02), but no significant change was seen in the H. pylori percentage invaded bacteria of cell adherent bacteria (Fig. 5). With boiled FCS (without further enzymatic degradation of FCS) a similar drop in adhesion was seen (Fig. 6) (P<0.01), and again no significant change in invasion could be demonstrated (Fig. 5). Neither vitronectin nor fibronectin induced an increase in H. pylori invasion into AGS cells, data not shown. Figure 5 View largeDownload slide The influence on H. pylori invasion of AGS cells of various modifications of FCS; MEM without FCS (MEM); MEM with 10% FCS (FCS); MEM with 10% FCS boiled 20 min (FCS100); MEM with 10% FCS treated with 0.5 mg proteinase K ml−1 for 1 h at 60°C and boiled for 20 min (pK100); and, MEM with 10% FCS treated with 0.4 U neuraminidase ml−1 for 1 h at 37°C and then boiled for 20 min (n100). Assays were repeated at least three times. The adhesion levels under the different conditions are shown in Fig. 6. Means and standard error are shown. P values assess differences from assay with 10% FCS. NS, no significant difference. Figure 5 View largeDownload slide The influence on H. pylori invasion of AGS cells of various modifications of FCS; MEM without FCS (MEM); MEM with 10% FCS (FCS); MEM with 10% FCS boiled 20 min (FCS100); MEM with 10% FCS treated with 0.5 mg proteinase K ml−1 for 1 h at 60°C and boiled for 20 min (pK100); and, MEM with 10% FCS treated with 0.4 U neuraminidase ml−1 for 1 h at 37°C and then boiled for 20 min (n100). Assays were repeated at least three times. The adhesion levels under the different conditions are shown in Fig. 6. Means and standard error are shown. P values assess differences from assay with 10% FCS. NS, no significant difference. Figure 6 View largeDownload slide The influence on H. pylori adherence to AGS cells of various modifications of FCS. The adherence is expressed as the relative adhesion (%) compared to MEM with 10% FCS (100%), the mean adhesion of which was 4.9×105 (12% of initial inoculum). Modifications of FCS were made as described in Fig. 5. Assays were repeated at least three times. Means and standard error are shown. P values assess differences from assay with 10% FCS. NS, no significant difference. Figure 6 View largeDownload slide The influence on H. pylori adherence to AGS cells of various modifications of FCS. The adherence is expressed as the relative adhesion (%) compared to MEM with 10% FCS (100%), the mean adhesion of which was 4.9×105 (12% of initial inoculum). Modifications of FCS were made as described in Fig. 5. Assays were repeated at least three times. Means and standard error are shown. P values assess differences from assay with 10% FCS. NS, no significant difference. 4 Discussion Adhesion pedestal formation is a known virulence factor of E. coli possessing the eae-A gene [26]. It has been proposed that adhesion pedestals have a similar significance for H. pylori infection of the gastric mucosa [11], although no H. pylori genes have been found to correspond to the E. coli eae-A gene [29]. Type I strains of H. pylori (VacA and cagA positive) are more frequently associated with gastritis, peptic ulcer, and gastric cancer than type II strains of H. pylori (VacA and cagA negative) [30,31]. Therefore, a difference in the ability to produce adhesion pedestals between type I and type II strains could be expected. In our study the type I and the type II strains had the same qualitative ultrastructural adhesion modes in vitro after incubation with AGS cells. Both H. pylori subtypes only rarely associated with adhesion pedestals, below 3% of adhering bacteria this was true for additional four clinical H. pylori isolates and prolonged incubation time did not change this fact. The invasion frequency and the association of adhering bacteria with depressions in the cell membrane were however found to be larger for the type I strain (15% and 11% respectively) than for the type II strain (8% and 5%). Others have very recently reported that type I strains invades AGS cells significantly better than a type II strain [32]. For the type I strain, a marked difference between adhesion pedestal formation in vivo (human gastric biopsy) and in vitro (AGS cells) was seen, 30% and 1% respectively. The general level of adhesion pedestals associated with H. pylori in vivo has previously been reported to be 8% [6]: We found likewise that approximately 8% of all adhering H. pylori, in a study of human gastric biopsy specimens from 13 patients, was associated with adhesion pedestals (unpublished data). This could imply that receptors involved in H. pylori adhesion pedestal formation in vivo are only represented to a minor degree on the surface of AGS cells, or that the outcomes of attachment to common receptors in AGS cells and normal human gastric epithelial cells are different based on other factors in the human host and in the in vitro assay. In E. coli involvement of eae genes in both adhesion pedestal formation and invasion has been described [33], a similar connection between genes involved in H. pylori adhesion pedestal formation and H. pylori invasion could be speculated. In our study, visible actin accumulation (FITC-phalloidin stained) by immunofluorescence light microscopy only occurred in a major degree when H. pylori type I and type II strains were incubated with AGS cells without FCS. Surprisingly, in electron microscopic examination, the number of H. pylori associated with adhesion pedestals did not increase when FCS was not added to the cell culture medium. This indicates that a positive FITC-phalloidin staining test does not necessarily demonstrate the ability of H. pylori to induce adhesion pedestals in AGS cells. In some studies of H. pylori adhesion to AGS cells, actin accumulation, demonstrated by phalloidin staining, has been linked to adhesion pedestals, but no attempt was made at quantifying the pedestals in these studies [9,10]. Even though phalloidin stained actin beneath adhering H. pylori increased as the period of incubation increased, no electron micrographs were presented to evaluate whether a similar increase in adhesion pedestals had occurred [10]. Deletions of several cagA associated genes eliminated the mutants ability to phosphorylate host cell proteins and to induce IL8 production in AGS cells. These mutants were still able to induce accumulations of actin in AGS cells, according to a phalloidin staining test [34]. It has at this point not been documented whether a direct correlation does occur between a positive actin accumulation test and adhesion pedestal formation for H. pylori. Our results would indicate that such a positive correlation does not exist, and that other explanations for the observed actin accumulations will have to be sought for the majority of the adhering H. pylori. Internalization of H. pylori into immortalized human gastric cancer cells has been reported [35–37]. In addition it has, in two separate studies, been shown that actin is involved in H. pylori invasion of AGS cells, since the presence of cytochalasin D, an inhibitor of actin polymerization, diminishes H. pylori invasion [32,38]. Host cell cytoskeletal components provide the machinery for the internalization of many invading enteric bacteria. For some of these invading bacteria, the cytoskeletal changes will be moderate and disappear as soon as the bacteria has invaded the host cell, as seen with Yersinia enterocolitica[39] and Salmonella spp. [40]. On the other hand the adhesion pedestals induced by E. coli strains (EPEC and VTEC) are stable actin-based structures [41,42]. In conclusion, this could imply that the cytoskeletal rearrangements in AGS cells observed beneath adhering H. pylori in the absence of FCS represent unsuccessful attempts of invasion into AGS cells. FCS increased H. pylori invasion of AGS cells, but had no similar effect on neither a non-invasive E. coli nor an invasive S. typhimurium. This would indicate a specific interaction between H. pylori and FCS mediating H. pylori invasion. Additionally, H. pylori was more frequently associated with depressions in the cell membrane when FCS was present in the culture medium. Increased H. pylori invasion of AGS cells was seen with increasing additions of FCS. A maximum was reached with approximately 10% FCS. The outcome of H. pylori adhesion to AGS cells seems to depend on attachment factor(s) in FCS. FCS likewise increases the internalization of Neisseria gonorrheae by Chinese hamster ovary cells and HeLa cells [43,44]; further, invasion of human epithelial cell cultures by a Streptococcus pyogenes serotype N1 was shown to multiply over 50-fold, when FCS was added to the test medium [45]. Modified samples of FCS were used to illuminate which serum factors affected H. pylori invasion into AGS cells. It was shown that proteinase K treatment of FCS completely eliminated the invasion promoting effect of FCS, although boiling of FCS had no significant effect on invasion. The adhesion of the type I strain to AGS cells was not changed by the proteinase K treated FCS. This indicates specific involvement of heat stable FCS protein(s) in H. pylori invasion of AGS cells, and at the same time indicates that involvement of the complement system is unlikely. Neuraminidase treatment of FCS did not change the invasion rate significantly, which makes it unlikely that sialylated structures in FCS are involved in H. pylori invasion. On the other hand our data did suggest interference of sialic acid with H. pylori adhesion to AGS cells, and others have described that sialic acid binding compounds are involved in the binding to epithelial cells of H. pylori[46,47]. The serum factors mediating N. gonorrheae and S. pyogenes invasion of epithelial cells were also found to be proteins, vitronectin and fibrinogen respectively [43,45], preliminary data suggest that neither vitronectin nor fibronectin are involved in H. pylori invasion. Recently a novel strategy has been described for bacterial recruitment of mammalian host proteins [48]. In this study it was shown that bacteria such as Neisseria species, S. pyogenes and H. pylori recruit heparin or related sulfated polysaccharides, which in turn serve as universal binding sites for mammalian heparin binding proteins. For Neisseria and S. pyogenes this was accompanied by increased bacterial invasion of epithelial cells. These data support our observations of H. pylori invasion into AGS cell being mediated by protein compounds in FCS. The fact that H. pylori has been found intracellularly particularly at the edges of duodenal ulcers could suggest that serum from humans may have the same effect as does FCS [12]. If such intracellular H. pylori could impair ulcer healing is presently not known. In conclusion, our results suggest that differences do exist between outcome of H. pylori adhesion to AGS cells and to human gastric epithelial cells. The differences in invasion rate are partly explained by mediation of invasion into AGS cells by protein factors in FCS. Previous reports of H. pylori invasion observed in human gastric biopsy specimens could suggest that variation in the conditions in the human stomach could also induce H. pylori invasion of epithelial cells in vivo. In addition an inverse relationship was found between the accumulation of actin beneath adhering H. pylori and the number of H. pylori having invaded AGS cells under conditions with and without FCS added to the cell culture medium. This suggests that the observed actin accumulations represent unsuccessful attempts of invasion. A higher invasion frequency and a higher amount of bacteria being associated with depressions in the epithelial cell membrane of a type I H. pylori strain than a type II strain could suggest that in the AGS cell assay invasion frequency is of significance for the pathogenic potential of an H. pylori strain. 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TI - Role of strain type, AGS cells and fetal calf serum in Helicobacter pylori adhesion and invasion assays
JO - Journal of the Endocrine Society
DO - 10.1111/j.1574-695X.2000.tb01506.x
DA - 2000-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/role-of-strain-type-ags-cells-and-fetal-calf-serum-in-helicobacter-iTOq0yZH7G
SP - 59
EP - 67
VL - 29
IS - 1
DP - DeepDyve
ER -