Immunosuppressive Proteins Isolated from Spiral and Coccoid Cytoplasmic Solutions of Helicobacter pylori
The aim of this study was to investigate the antiproliferative proteins that probably have a role in Helicobacter pylori evade of immune response and cause chronic infection disease and also to see if coccoid form had a role in its chronicity. H. pylori strain VacA s2/m2 positive and CagA negative, from a gastric biopsy of a patient with peptic ulcer disease, was isolated and cultured in brucella agar. Both spiral and coccoid forms were harvested and ruptured by sonication. The cytoplasmic solutions of both forms were collected and their fractions obtained by gel chromatography and preparative polyacrylamide gel electrophoresis. The fractions were analyzed by MTT assay for their antiproliferative activity. We isolated two proteins with a significant dose dependent antiproliferative activity that analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one of them that was urease positive showed two bands with 61 and 27 kDa, which is resumed to urease of H. pylori, another consist of 57 and 63 kDa. Helicobacter pylori secret some proteins like urease that inhibit immune cells proliferation response against its antigens.
Received: November 04, 2010;
Accepted: January 11, 2011;
Published: February 09, 2011
Helicobacter pylori is a gram negative and spiral bacterium that has
infected more than 50% of worlds population. H. pylori infection
is related to gastric ulcer and MALT lymphoma (Kimmel et
Although, H. pylori are noninvasive bacteria, it induces chronic infection
(Marshall, 2002). H. pylori infection is almost
asymptomatic and less than 10% of the infected individuals have ulcer or gastric
associated diseases. These patients are in high risk of stomach cancer. Relationship
between H. pylori infection and gastric cancer is so significant that
World Health Organization (WHO) has organized H. pylori as a type I carcinogen.
It is postulated that long-term inflammation induced by H. pylori has
an important role in gastric cancer development (Bah et
Helicobacter pylori releases factors that are responsible for colonization
and pathogenesis (Franco et al., 2008). It has
been shown by Ibraghimov and Jacques (2000) that H.
pylori secrets a protein that inhibits epithelial and lymphocyte cell
lines proliferation and that its effects is dose dependent. It was also shown
that urease, VacA and CagA have cytotoxic effects on cells. In the stomach,
mostly spiral forms of H. pylori were found and coccoid forms were in
the more damaged regions of gastric mucosa (Saito et
It has been shown that chronic inflammation promotes cancer (Shacter
et al., 2002). Despite local and systemic response to H. pylori
infection (Bhat et al., 2005), H. pylori
induces chronic gastric inflammation. Epidemiological studies have shown that
gastric cancer and MALT lymphoma are related to H. pylori infection (Feldman,
2001). H. pylori is acquired in childhood (Mitchell
et al., 2003), but gastric cancer is seen mostly after the six decade
of life. It is postulated that chronic infection with H. pylori is necessary
for gastric cancer (Feldman, 2001).
-It has been shown that lymphocytes from H. pylori, that infected individuals
sometimes shows a decrease in proliferation, in response to helicobacter
antigens in vitro. This had revealed that H. pylori might down
regulate the cellular immune responses toward itself (Windle
et al., 2005). It is possible that H. pylori secrets proteins
that inhibit immune response to eradicate the infection locally (Ibraghimov
and Jacques, 2000). The aim of this study was to isolate the cytoplasmic
proteins of H. pylori responsible for the inhibition of T cells proliferation.
In this study, we isolated different fractions of both spiral and coccoid forms of H. pylori and evaluated their antiproliferative activity.
MATERIALS AND METHODS
Bacterial isolation and culture conditions: The H. pylori strain
was isolated from gastric biopsy of a patient suffering from a gastric ulcer.
Present study was performed in 2006 and it took 7 mounts. The cells were grown
on brucella agar (Merck, Germany) supplemented with 10% sheep blood, vancomycin
(10 mg mL-1), nystatin (1 mg mL-1) and trimethoprim (5
mg mL-1) and incubated for 48 h in CO2 incubator (10%
CO2, 5% N2 and 85% O2) (Ramarao
and Meyer, 2001). Bacterial cells were harvested after the second and 13th
days of incubation and suspended in PBS (pH 7.2). To assess the relative percentages
of coccoid and bacillary forms, Gram-staining was used for each suspension.
The Gram staining showed that the suspension of 2nd day culture, contained only
spiral forms and 13th day culture contained coccoid forms.
Preparation of cytoplasmic solutions: The cells were collected and washed
three times with 20 mL of PBS. The packed cells were frozen at -80°C before
use. The cells were sonicated on ice (eight times for 30 sec each time). Unbroken
cells and debris were precipitated by centrifuge at 4,000 x g for 10 min at
4°C. The supernatants were collected and centrifuged at 45,000 x g for 20
min at 4°C. The pellets contained cell walls with outer membrane protein
(Ge et al., 2001) and the supernatants contained
Chromatography: The cytoplasmic solutions obtained from spiral and coccoid forms were concentrated with freeze-drying and then dialyzed against PBS for three times. The concentrated solutions of spiral and coccoid forms (7 and 3 mg mL-1) were applied in gel filtration column using S-200 and eluted with sodium phosphate buffered saline, pH 7.2, (0.02 M sodium phosphate, 0.15 M NaCl). Optical density was measured at 280 nm and fractions with maximum OD were pooled.
Preparative PAGE: Fractions with antiproliferative activity obtained
by chromatography were used in preparative PAGE to isolate proteins with antiproliferative
effect. A piece of the gel was cut from length and stained by silver stain.
According to the stained gel, the protein bands were cut from wide of the remained
gel and cut into pieces in eluting solution (0.1 M Tric-HCl; pH 7) and then
centrifuged at 3200 x g for 15 min after shaking. The supernatants were collected
and stored at 4°C for 30 min. Proteins were precipitated by acetone and
the solution was stirred magnetically for 15 min at 20°C. After being centrifuged
at 3200 x g, the acetone was removed and the proteins were diluted in PBS. The
isolated proteins were dialyzed against PBS and prepared for proliferation test.
The concentration of proteins was determined by Bradford assay (Kruger,
2002). The presence of urease activity in the stomach biopsies and the isolated
proteins was determined with a Christensen urease medium. A rapid color change
suggests the presence of urease activity.
Cell viability assay: The antiproliferative effect of the fractions
on Jurkat cell lines, a CD4+ leukemia T-cell line, were determined
by MTT assay described by Young et al. (2005).
The cells were maintained in the culture medium in 10 mL culture flasks at 37°C
in a 7% CO2 humidified atmosphere. Cell viability was assessed by
trypan blue and the exclusion was over 90%. The cells were seeded in 96-well
microtiter plates at a density of approximately 4x106 cells/well
containing 100 μL RPMI 1640 (Gibco, UK), 10% heat-inactivated Fetal Calf
Serum (FCS), 100 U L-1 penicillin and 100 mg L-1 streptomycin
(Gibco, Scotland). Each fraction was added in various concentrations and some
wells were leaved without samples as controls. All MTT assays were measured
in triplicate. 10 μL of the MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium-bromide,
5 mg mL-1; Sigma) solution was added to each well and incubated for
4 h until a purple precipitate was visible. The developed crystals were dissolved
in 100 μL of 0.04 M HCl in isopropanol and the OD was read in a microplate
spectrophotometer at a wavelength of 570 nm.
The stimulation index was calculated by dividing OD at 570 nm with sample by OD at 570 nm without samples. Student's t-test was utilized for the statistical analysis of the samples. Differences between the treatment means were considered statistically significant if p<0.05.
RESULTS AND DISCUSSION
Isolation of antiproliferative proteins from cytoplasmic solutions:
After chromatography of cytoplasmic solutions, various fractions were pooled
as S1, S2 and S3 for spiral forms and C1, C2 and C3 for coccoid forms (Fig.
1a, b). The fractions with antiproliferative activity (S2 and C2) were applied
in preparative PAGE for isolation of proteins with antiproliferative activity.
The preparative PAGE (Fig. 2) was cut in 6 pieces (P1, P2,
P3, P4, P5 and P6) from length in both S2 and C2. P5 band was not seen in preparative
PAGE of C2. Both P4 and P5 consisted of one band in preparative PAGE. The P5
from S2 with urease activity showed 61 and 27.5 kDa bands in SDS-PAGE, P4 showed
two bands of 63 and 57 kDa (Fig. 3a, b).
Antiprolifrative activity of the isolated proteins: After gel chromatography, the fractions were analyzed by MTT assay for their antiproliferative activity (Fig. 4a). The cells treated with S2 and C2 showed lower OD than negative controls (p<0.05). No statistical difference was observed between other fractions.
||Cytoplasmic solutions of (a) spiral and (b) coccoid forms
were applied in gel filtration column, using S-200. The 1-17, 18-30 and
31-56 fractions were pooled as S1, S2 and S3 for Spiral forms and 1-11,
12-17 18-23 as C1, C2 and C3 for coccoid forms, respectively
||PAGE analysis of spiral and coccid forms fractions 2, S2 and
C2, obtained by gel chromatography. Lane 1. C2, Lane 2 and 3. S2. The preparative
PAGE of S2 and C2 were cut in 6 pieces (P1-P6), from its wide
The P1 to P6 fractions obtained by preparative PAGE were analyzed by MTT assay (Fig. 4b). The P4 and P5 fractions showed significant dose dependent antiproliferative activity on Jurkat cells.
CagA, VacA and urease are antiproliferative proteins (Takagi
et al., 2000) but VacA s2/m2 does not have the cytotoxic effect that
is due to defect in N-terminal of this protein. For inhibiting the interference
of CagA and VacA cytotoxic effect the selected strain was urease positive, VacA
s2/m2 positive and CagA negative.
||SDS-PAGE analysis of fractions isolated from preparative PAGE
(a) Lane 1. P4 isolated from S2, lane 2. P4 isolated from C2, (b) lane 1.
P5 isolated from preparative PAGE of S2
Spiral forms had two proteins with antiproliferative activity (p<0.05):
P5 with urease activity in SDS-PAGE showed two bands, 61 and 27.5 kDa, the other
(P4) showed two bands of 63 and 57 kDa. It is likely that P5 protein and its
subunits that were shown in SDS-PAGE are similar to urease protein of H.
pylori (Figueroa et al., 2002). It has
been shown that in coccoid forms, urease mRNA does not exist (Bah
et al., 2001). We have shown that in coccoid forms, only P4 was expressed
and urease was not expressed.
The isolated protein (P4) showed a dose dependent antiproliferative activity that is necessary be cloned for more recognition and sequencing.
Relation between H. pylori and gastric cancer is so certain that WHO
has classified H. pylori in class I carcinogens (Romero-Gallo
et al., 2008). Chronic inflammation alone is not sufficient to induce
||In vitro influence of different concentrations of H.
pylori fractions on proliferation of Jurkat T cell was estimated by
MTT assay. Experiments were performed in triplicate and at least 3 separate
experiments, the Means±SD of the values are indicated. (A) S1, S2
and S3 from spiral forms and C1, C2 and C3 from coccoid forms, obtained
by gel filtration (10, 20 and 30 μg mL-1) and (B) P1, P2,
P3, P4, P5 and P6 obtained by preparative PAGE (75, 100, 125 ng mL-1)
were utilized in MTT assay. Untreated control cells were given the value
of 1 (100% SI). *Indicates a significant difference in comparison with control
as shown by Student t-test, at p<0.05
H. pylori requires a direct effect of such mutagens to induce tumorogenes.
H. pylori induces progression of carcinoma, but it may have direct effect
on gastric to induce carcinoma.
Helicobacter pylori has a role in development of gastric carcinoma.
It is postulated that H. pylori induces mutation in cells and inhibits
proliferation of immune cells and also induces initiation and progression of
carcinoma (Leon-Barua et al., 2006).
In this study it was shown that both spiral and coccoid forms of H. Pylori have two antiproliferative proteins that one of them had urease activity and the other showed two bands of 63 and 57 kDa. It is recommended to evaluate the effect of the unknown protein on immune response since it perhaps have a role in the chronic infection and also stomach cancer.
The financial support by the Research Council of Pasteur institute of Iran is gratefully acknowledged.
Kimmel, B., A. Bosserhoff, R. Frank, R Gross, W. Goebel and D. Beier, 2000.
Identification of immunodominant antigens from Helicobacter pylori
and evaluation of their relativities with sera from patients with different gastroduodenal pathologies. Infect Immune., 68: 915-920.
Marshall, B., 2002.
Helicobacter pylori: 20 Years on. Clin. Med., 2: 147-152.PubMed |
Bah, E., D.M. Parkin, A.J. Hall, A.D. Jack and H. Whittle, 2001.
Cancer in the Gambia: 1988–97. Br. J. Cancer, 84: 1207-1214.
Franco, A.T., E. Johnston, U. Krishna, Y. Yamaoka and D.A. Israel et al
Regulation of gastric carcinogenesis by Helicobacter pylori
virulence factors. Cancer Res., 68: 379-387.Direct Link |
Ibraghimov, A. and P. Jacques, 2000.
The immune response against Helicobacter pylori
: A direct linkage to the development of gastroduodenal disease. Microbes Infect., 2: 1073-1077.PubMed |
Saito, N., K. Konishi, F. Sato, M. Kato, H. Takeda, T. Sugiyama and M. Asaka, 2003.
Plural transformation-processes from spiral to coccoid Helicobacter pylori and its viability. J. Infect., 46: 49-55.PubMed |
Shacter, E. and S.A. Weitzman, 2002.
Chronic inflammation and cancer. Onchology, 16: 217-226.Direct Link |
Bhat, N., J. Gaensbauer, R.M. Peek, K. Bloch, K.T. Tham, M.J. Blaser and G. Perez-Perez, 2005.
Local and systemic immune and inflammatory responses to Helicobacter pylori
strains. Clin. Diagnostic Lab. Immunol., 12: 1393-1400.CrossRef |
Feldman, R.A., 2001.
Would eradication of Helicobacter pylori infection reduce the risk of gastric cancer. Alimentary Pharmacol. Therapeutics, 15: 2-5.PubMed |
Mitchell, A., M.J.S. Terezinha, J.B. Leah, A.M.L. Aldo and L.G. Richard, 2003.
Age-specific Helicobacter pylori
seropositivity rates of children in an impoverished urban Area of Northeast Brazil. J. Clin. Microbiol., 41: 1326-1328.CrossRef |
Windle, H.J., Y.S. Ang, V.A. Morales, R. McManus and D. Kelleher, 2005.
Human peripheral and gastric lymphocyte responses to Helicobacter pylori NapA and AphC differ in infected and uninfected individuals. Gut, 54: 25-32.CrossRef |
Ramarao, N. and T.F. Meyer, 2001. Helicobacter pylori
resists phagocytosis by macrophages: Quantitive assessment by confocal microscopy and fluorescence-activated cell sorting. Infect. Immun., 69: 2604-2611.Direct Link |
Ge, Z., P. Doig and J.G. Fox, 2001.
Characterization of proteins in the outer membrane preparation of a murine pathogen, Helicobacter bilis
. Infect. Immun., 69: 3502-3506.CrossRef |
Kruger, N., 2002.
The Bradford Method for Protein Quantitation: Biomedical and Life Sciences. The Protein Protocols Hand Book Part I. John Wiley and Sons Ltd., New York, pp: 15-21
Young, F.M., W. Phungtamdet and B.J.S. Sanderson, 2005.
Modification of MTT assay conditions to examine the cytotoxic effects of amitraz on the human lymphoblastoid cell line, WIL2NS. Toxicol. In vitro
, 19: 1051-1059.PubMed |
Takagi, A., S. Watanabe, M. Igarashi, J. Koike and K. Hasumi et al
The effect of Helicobacter pylori
on cell proliferation and apoptosis in gastric epithelial cell lines. Aliment. Pharmacol. Ther., 14: 188-192.PubMed |
Figueroa, G., G. Faundez, M. Troncoso, P. Navarrete and M.S. Toledo, 2002.
Immunoglobulin G antibody response to infection with coccoid forms of Helicobacter pylori. Clin. Diagn. Lab. Immunol., 9: 1067-1071.CrossRef |
Romero-Gallo, J., E.J. Harris, U. Krishna, M.K. Washington, G.I. Perez-Perez and Jr. R.M. Peek, 2008.
Effect of Helicobacter pylori eradication on gastric carcinogenesis. Lab Invest., 88: 328-336.PubMed |
Leon-Barua, R., S. Recavarren-Arce, E. Chinga-Alayo, C. Rodriguez-Ulloa and D.N. Taylor et al
., 2006. Helicobacter pylori
-associated chronic atrophic gastritis involving the gastric body and severe disease by Vibrio cholerae
. Trans. Roy Soc. Trop. Med. Hyg., 100: 567-572.Direct Link |