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ORIGINAL ARTICLE  
Year : 2014  |  Volume : 57  |  Issue : 2  |  Page : 187-190
Cytopathic effects of toxogenic strains of Helicobacter pylori on different cell lines


1 Department of Botany and Microbiology, College of Sciences, King Saud University, Riyadh, Kingdom of Saudi Arabia
2 Department of Clinical Laboratory, College of Applied Medical Sciences, Riyadh, Kingdom of Saudi Arabia
3 Department of Clinical Microbiology, Christian Medical College and Hospital, Vellore, India
4 Department of Microbiology, Periyar University, Salem, Tamil Nadu, India

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Date of Web Publication19-Jun-2014
 

   Abstract 

Purpose: Many virulence factors are involved in the pathomechanism of infection caused by Helicobacter pylori. Toxins such as vacuolating cytotoxin, encoded by the vacA gene and the immunogenic protein cagA, encoded by the cagA gene (cytotoxin-associated gene) are major factors conferring the property of virulence. The current study is aimed at isolation of H. pylori and separation of its toxin from antral biopsies of patients. Materials and Methods: The following cell lines were used to demonstrate the cytopathic effect (CPE) of the separated toxin: African green monkey kidney (Vero), baby hamster kidney, human lung carcinoma (LLC-MK2), and human epithelial. Results: H. pylori was isolated from 27 out of 45 patients (60%) selected for the study. CPE of H. pylori toxin was highly significant on Vero cells than other cell lines used as it reached a high dilution titer of toxin (1/16) in 13 isolated strains (48.15%). No significant difference in CPE of toxin in different dilutions was detected among other cell lines used in different groups. H. pylori toxin could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis as a distinct band with a molecular weight ranging between 66 and 97 kDa and closely related to 87 kDa. Conclusion: H. pylori vacuolating cytotoxin plays a vital role in the pathogenesis of gastroduodenal diseases (gastritis, gastric ulcer, duodenal ulcer, and gastric cancer). The Vero cell lines were found to be the most suitable form of tissue culture when compared with other cell lines used in our study for demonstrating the activity of H. pylori toxin.

Keywords: cagA gene, cell lines, cytopathic effect, Helicobacter pylori, vacuolating cytotoxin

How to cite this article:
Gowda KL, Marie MM, John J, Pradeep CS, Dabwan KM, Sangeetha G. Cytopathic effects of toxogenic strains of Helicobacter pylori on different cell lines. Indian J Pathol Microbiol 2014;57:187-90

How to cite this URL:
Gowda KL, Marie MM, John J, Pradeep CS, Dabwan KM, Sangeetha G. Cytopathic effects of toxogenic strains of Helicobacter pylori on different cell lines. Indian J Pathol Microbiol [serial online] 2014 [cited 2020 Aug 13];57:187-90. Available from: http://www.ijpmonline.org/text.asp?2014/57/2/187/134660



   Introduction Top


Helicobacter pylori is a spiral Gram-negative, microaerophilic bacterial parasite that inhabitates in gastric epithelium. [1] The close association of H. pylori with gastric inflammatory disease, peptic ulcer disease, atrophic gastritis, gastric adenocarcinoma, mucosa associated lymphoid tissue lymphoma is well-recognized, [2] but interestingly, not all the H. pylori infected individuals develop peptic ulcer or gastric cancer. A significant number of patients have milder form of disease, like inflammation in the stomach and duodenum. [3]

Many virulence factors are involved in the pathomechanism of infection caused by H. pylori. They include various enzymes (e.g., urease, catalase, lipase, phospholipase and proteases), toxins such as vacuolating cytotoxin, encoded by the vacA gene and the immunogenic protein CagA, encoded by the cagA gene (cytotoxin-associated gene), localized in the H. pylori pathogenicity island (PAI). Not all but some strains of H. pylori are known to produce an extracellular cytotoxin that causes vacuolization of tissue-cultured in various mammalian cells. [4],[5] Cytotoxin-producing H. pylori strains were isolated more frequently from patients with peptic ulcer disease than from patients with chronic gastritis. [6],[7]

Production of cytotoxin and the presence of the PAI are thought to be the main virulence factors of H. pylori. Not all strains are able to produce cytotoxin and CagA protein, but those strains with the ability to do so seem to be more virulent. Such strains are involved in the process of ulcerogenesis. [4]

There is an association with cagA-positive H. pylori strains and the risk of peptic ulcer disease, the development of atrophic gastritis and carcinoma of the stomach. [5] This cytotoxin has been reported to be produced by approximately 50% of H. pylori strains isolated from cases where it has been found to be associated with peptic ulceration. The more severe forms of H. pylori disease such as peptic ulcer and gastric cancer are associated with infection with a subset of strains that express the cagA antigen. [6] Although 50% of H. pylori strains are nontoxigenic, all strains possess the gene (vacA) that encodes the vacuolating cytotoxin. [4]

H. pylori producing toxin can be detected by its cytotoxic effect on rabbit kidney, baby hamster kidney (BHK-21), human amnion and green monkey kidney cell lines. HeLa cells are often used for examining vacuolating cytotoxin activity, but may not be sensitive enough to detect minimal changes. [7] In our present study, we aimed to find the relation between H. pylori infection and gastritis, gastric ulcer, duodenal ulcer and gastric carcinoma, and to detect the toxic effect of H. pylori through its cytopathic effect (CPE) on the following cell lines: African green monkey (Vero), BHK-21, human epithelial (HEP-2) and human lung carcinoma (LLC-MK2).


   Materials and methods Top


To carry out the study, 45 patients suffering from dyspeptic symptoms and/or signs (such as vomiting, heart burns, and epigastric pain) with positive endoscopic findings were selected from Endoscopy Unit at Al Eman Hospital, Riyadh. The study was permitted by Institution ethical committee and consent was obtained from all the patients. All patients included in this study were undergoing an upper gastrointestinal endoscopy using fiberoptic endoscope (Penetax FG 34X). All patients had no antibiotics administration 4 weeks prior to endoscopy. According to the upper gastrointestinal endoscopy findings, patients were classified according to their diagnosis, into four) groups: Group I: Patients with gastritis (14), Group II: Patients with gastric ulcer (10), Group III: Patients with duodenal ulcer (15) and Group IV: Patients with gastric cancer. [6] Under complete aseptic conditions, two antral biopsies were taken from each patient (biopsies in gastritis were taken from intact mucosa and avoiding the lesser curvature.

Isolation of H. pylori

One of the biopsy was directly inoculated into Christensen urea agar for direct urease test. The other biopsies were inoculated into transport media (1 ml of Burcella broth Biolife with 25% glycerol). Under complete aseptic conditions, biopsies were grounded using sterile glass rods and then inoculated into Dent's agar in which Colombia agar was used as a base and supplemented with 7% human blood. The plates were incubated at 37°C under microaerophilic conditions (using Oxoid microaerophilic gas pack jar) and inspected within 3-4 days. [8] Any growth was identified as H. pylori based on the colonial morphology, Grams' stain, catalase test, oxidase test, and rapid urease test. [8]

H. pylori toxin separation

H. pylori cultures were transferred to  Brucella More Details broth with 10% fetal calf serum (FCS) and incubated microaerophilically for 2 days with continuous shaking on a rotatory shaker. Cultures were harvested by centrifugation and broth culture filtrates were obtained (from culture supernates) by passing through 0.22 µm syringe membrane filters (Costor, Cambridge, M.A.)

Maintenance of cell lines

The following cell lines kindly supplied from virus isolation section, Naval Medical Research Unit-3, Cairo were used for performing cytotoxicity assays:

  1. African green monkey kidney (Vero) at passage level of 135;
  2. BHK-21 at passage level of 70,
  3. HEP-2 at passage level of 10,
  4. Human lung carcinoma (LLC-MK2) at passage level of 14.


Cells were seeded into 24-wells cell culture plates and incubated for 2 days at 37°C, in the presence of 5% CO 2 . After the formation of monolayer confluent sheet of cells,

the culture medium was decanted and then two-fold dilution of H. pylori broth filtrate and 1 ml of minimal essential medium with 10% FCS was added to the wells, and incubated at 37°C for 48 h in the presence of 5% CO 2 . Cells were then observed using an inverted microscope to determine their cytotoxicity. The titer of cytotoxic activity was taken as the highest dilution that caused vaculation (cytotoxic changes) in more than 25% of the cells. [9]

Determination of the molecular weight of H. pylori toxin

The broth culture filtrate was applied onto a vertical sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Culture medium for H. pylori was used as a negative control.


   Results Top


H. pylori was isolated from 60% patients (27 out of 45) of which 20 were males and seven were females. Among this, H. pylori was isolated from 22 patients (68.8%) in smoker group and the rest were from the non-smoker group (38.5%).

The CPE of H. pylori toxin on Vero cell line had a high titer in all groups of patients (1/16). In Group IV, the CPE of toxin on Vero cell was higher (60%) than the other groups (37.5% in group I, 50% in Group II, and 50% in Group III). The difference of CPE on Vero cells was non-significant among the different titers of each group [Table 1].
Table 1: Cytopathic eff ect of H. pylori toxin on Vero cell line

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The CPE of H. pylori toxin on BHK-21 reaches a high titer (up to 1/16) [Table 2]. CPE was seen higher in the Group IV (60%) than other groups (0.0% in Group I, 33.33% in Group II, and 50% in Group III).
Table 2: Cytopathic eff ect of H. pylori toxin on BHK-21 cell line

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[Table 3] and [Table 4] shows that the CPE of H. pylori toxin on LLC-MK-2 and on HEP-2 cells reached a titer of and respectively and it was higher in Group IV (60%) than other groups (0.0% in Group I, 16.67% in Group II, and 37.5% in the Group III). Difference of CPE among groups was found to be insignificant in all the cell lines.
Table 3: Cytopathic effect of H. pylori toxin on LL C-MK

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Table 4: Cytopathic effect of H. pylori toxin on HEP-2 cell line

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As shown in [Table 5], the CPE of H. pylori toxin on Vero cells was highly significant when compared to the other cell lines used (BHK-21, LLC-MK2, and HEP-2) and reached a titer of 1/16 (48.15%) compared to other cell lines at the same dilution (33.33%) on BHK-21, and 0.0% on LLC-MK2 and HEP-2. There is a correlation between cytopathic findings of BHK-21 and that of LLC-MK2 [Table 6]. By electrophoresis of H. pylori culture filtrate a band was found close to 97 kDA region.
Table 5: Correlation between tissue cell lines as regards to toxin effect of H. pylori

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Table 6: Correlation matrix

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   Discussion Top


H. pylori is a highly host and tissue specific organism. The organism resides exclusively in gastric mucosa and it can be found in remote areas that underwent metaplastic changes in which gastric epithelial cells are present. Toxigenic strains of H. pylori cause progressive vacuolation in mammalian cells. [10],[11] It has been speculated that the phenomenon of vacuole formation is associated with the gastric pathologies, such as gastric ulcer and gastritis induced by H. pylori infection. [7]

Isolation of H. pylori in males (66.66%) was higher than females (46.66%); also H. pylori infection was higher in smokers (68.75%) than non-smokers patients (38.4%), but this association was not statistically significant, [12],[13] except gastric cancer patients where it was significantly higher among smokers.

In our study, the rate of H. pylori infection was higher in gastric carcinoma group (83.3%) than gastric ulcer patients (60%), patients with gastritis (57.1%) and patients with duodenal ulcer (53.3%), but these were not statistically significant. All smokers' patients with gastric carcinoma were positive for H. pylori infection, while it was 71.4% in patients with gastric ulcer, 62.5% in patients with gastritis, and 58.3% in patients with duodenal ulcer. [14] H. pylori was significantly common in ulcer patients than in non-ulcer patients. H. pylori was found in 66% of patients with gastric ulcer and in 77% of patients with duodenal ulcer.

In our study, the CPE of H. pylori toxin was more on Vero cell lines when compared to other cell line and reached a high dilution of toxin (1/16) in 13 isolated strains (48.15%). This effect was more in strains isolated from patients with gastric carcinoma (60%) than other groups. This finding is consistent with those obtained in a previous investigation. [14] Thus, cytotoxin of H. pylori affects the Vero cell line, but there is no association between the positivity of the strains and the risk of any particular H. pylori disease. [15]

The CPE of H. pylori toxin on BHK-21 cell lines reached a high dilution of toxin (1/16) in (33.33%) isolated strains and this effect was more in gastric carcinoma isolated strains (60%) than other groups. [16] The CPE of H. pylori toxin on LLC-MK2 and HEP-2 cell lines reached a low dilution of toxin, and respectively. The CPE effect of H. pylori toxin on LLC-MK2 cell line at the high dilution was more in strains isolated from gastric carcinoma (60%) but its effect on HEP-2 was more on gastric ulcer isolated strains (66.67%). This is supported by the finding that the cytotoxin-producing strains of H. pylori and severity of mucosa damage in the gastrointestinal tract. [17]

In vitro, approximately 50-60% of H. pylori isolates produce vaculating cytotoxin in cultured epithelial cells. Vaculating cytotoxin, which induces vaculation in cultured eukaryotic cells, was secreted by 50% of H. pylori isolated strains. [14],[17],[18]

By electrophoresis of H. pylori culture filtrate a band was found between 66 kDa and 97 kDa and was close to 87 kDa. This molecular weight corresponds to the same molecular weight of H. pylori toxin. It is supported by Schmitt and Hass as they are found that a mature cytotoxin domain of Vac A gene product of approximately 87-94 kDa. [14],[19]

Based on the results obtained so far, we can conclude that vacuolating cytotoxin of H. pylori has an important role in the pathogenesis of gastritis, gastric ulcer, duodenal ulcer and gastric carcinoma and that it is more in gastric carcinoma. Four cell lines were used in our study and the Vero cells were the most suitable tissue culture for demonstration of H. pylori toxin than the other cell lines used. BHK-21 and LLC-MK2 cell lines can be used together for demonstration of H. pylori toxin as there is a significant correlation between them.


   Acknowledgment Top


The authors extend their appreciation to the Research Center at the College of Applied Medical Sciences and the Deanship of Scientific Research at King Saud University for their financial assistance for this work and support.

 
   References Top

1.Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1983;1:1273-5.  Back to cited text no. 1
    
2.Amini M, Karbasi A, Khedmat H, Jeihounian M. Helicobacter pylori eradication and histopathological esophagitis in dyspeptic patients. Trop Gastroenterol 2010;31:175-9.  Back to cited text no. 2
    
3.Shanjana A, Archana A. Cytotoxic isolates of Helicobacter pylori from peptic ulcer diseases decrease K+-dependent ATPase activity in HeLa cells. BMC Gastroenterol 2003;3:31.  Back to cited text no. 3
    
4.Radosz-Komoniewska H, Bek T, Józ´wiak J, Martirosian G. Pathogenicity of Helicobacter pylori infection. Clin Microbiol Infect 2005;11:602-10.  Back to cited text no. 4
    
5.El-Omar EM, Carrington M, Chow WH, McColl KE, Bream JH, Young HA, et al. Interleukin-1 polymorphisms associated with increased risk of gastric cancer. Nature 2000;404:398-402.  Back to cited text no. 5
    
6.Ghiara P, Marchetti M, Blaser MJ, Tummuru MK, Cover TL, Segal ED, et al. Role of the Helicobacter pylori virulence factors vacuolating cytotoxin, CagA, and urease in a mouse model of disease. Infect Immun 1995;63:4154-60.  Back to cited text no. 6
    
7.Wang XM, Kojima T, Satoh K, Taniguchi Y, Tokumaru K, Saifuku K, et al. The value of LYM-1 cells for examining vacuole formation and loss of cell viability induced by culture supernates of Helicobacter pylori. J Med Microbiol 1997;46:705-9.  Back to cited text no. 7
    
8.Piccolomini R, Di Bonaventura G, Festi D, Catamo G, Laterza F, Neri M. Optimal combination of media for primary isolation of Helicobacter pylori from gastric biopsy specimens. J Clin Microbiol 1997;35:1541-4.  Back to cited text no. 8
    
9.Konishi H, Ishibashi M, Morshed MG, Nakazawa T. Cytopathic effects of Helicobacter pylori on cultured mammalian cells. J Med Microbiol 1992;37:118-22.  Back to cited text no. 9
    
10.Cover TL, Dooley CP, Blaser MJ. Characterization of and human serologic response to proteins in Helicobacter pylori broth culture supernatants with vacuolizing cytotoxin activity. Infect Immun 1990;58:603-10.  Back to cited text no. 10
    
11.Phadnis SH, Ilver D, Janzon L, Normark S, Westblom TU. Pathological significance and molecular characterization of the vacuolating toxin gene of Helicobacter pylori. Infect Immun 1994;62:1557-65.  Back to cited text no. 11
    
12.Ferreccio C, Rollán A, Harris PR, Serrano C, Gederlini A, Margozzini P, et al. Gastric cancer is related to early Helicobacter pylori infection in a high-prevalence country. Cancer Epidemiol Biomarkers Prev 2007;16:662-7.  Back to cited text no. 12
    
13.Ghoshal UC, Singh R, Chang FY, Hou X, Wong BC, Kachintorn U, et al. Epidemiology of uninvestigated and functional dyspepsia in Asia: Facts and fiction. J Neurogastroenterol Motil 2011;17:235-44.  Back to cited text no. 13
    
14.Tee W, Lambert JR, Dwyer B. Cytotoxin production by Helicobacter pylori from patients with upper gastrointestinal tract diseases. J Clin Microbiol 1995;33:1203-5.  Back to cited text no. 14
    
15.Ozsan M, Tekeli A, Ozden A, Akyön Y, Ozkul A, Gerçeker D, et al. Vacuolating cytotoxic activity of 40 Helicobacter pylori strains isolated from Turkish patients. APMIS 2000;108:293-5.  Back to cited text no. 15
    
16.Kamiya S, Kai M, Ozawa A, Kobayashi H, Shirai T, Harasawa S, et al. Characteristics of vacuolating toxin produced by Helicobacter pylori. Eur J Gastroenterol Hepatol 1994;6 Suppl 1:S23-7.  Back to cited text no. 16
    
17.Figura N, Guglielmetti P, Rossolini A, Barberi A, Cusi G, Musmanno RA, et al. Cytotoxin production by Campylobacter pylori strains isolated from patients with peptic ulcers and from patients with chronic gastritis only. J Clin Microbiol 1989;27:225-6.  Back to cited text no. 17
    
18.Leunk RD, Johnson PT, David BC, Kraft WG, Morgan DR. Cytotoxic activity in broth-culture filtrates of Campylobacter pylori. J Med Microbiol 1988;26:93-9.  Back to cited text no. 18
    
19.Schmitt W, Haas R. Genetic analysis of the Helicobacter pylori vacuolating cytotoxin: Structural similarities with the IgA protease type of exported protein. Mol Microbiol 1994;12:307-19.  Back to cited text no. 19
    

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Correspondence Address:
Mohammed Ali M. Marie
Department of Clinical Laboratory, College of Applied Medical Sciences, King Saud University, P.O. Box 10219, Riyadh - 11433, Kingdom of Saudi Arabia.

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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.134660

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