ENVIRONMENTAL FACTORS AFFECT THE SURVIVAL AND EXPRESSION OF VIRULENT CYTOLETHAL DISTENDING TOXIN OF Campylobacter jejuni

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1 ENVIRONMENTAL FACTORS AFFECT THE SURVIVAL AND EXPRESSION OF VIRULENT CYTOLETHAL DISTENDING TOXIN OF Campylobacter jejuni Deepika Prasadi 1 and K. N. Prasad *2 1. Allenhouse Institute of Technology, Kanpur (INDIA) 2. Department of Microbiology, Sanjay Gandhi postgraduate institute of Medical Sciences, Lucknow (INDIA). Received July 02, 2010 Accepted October 05, 2010 ABSTRACT Several environmental factors regulate the expression of virulence genes under certain conditions and the expression of bacterial virulence determinants. Cytolethal distending toxin (CDT) is an important virulence determinant secreted by Campylobacter jejuni (C. jejuni), a leading cause of gastrointestinal bacterial pathogen worldwide. Therefore, the aim of the study was to investigate the effect of selected environmental stress factors such as temperature, ph and proteolytic enzymes on cytotoxicity of culture supernatants containing CDT activity of 10 C. jejuni strains [5 CDT (-ve) and equal number of randomly selected CDT (+ve) C. jejuni strains] using Hep-2 and HeLa cells. Additionally, after stress exposure, adhesion, invasion and intracellular survival of C. jejuni were also studied. Key Words : Campylobacter jejuni, Cytolethal distending toxin, Stress, Cytotoxicity, Epithelial cell line INTRODUCTION Campylobacter is recognized as one of the leading cause of food-borne bacterial enteritis worldwide 1. Infections in humans are usually acquired through the consumption of under cooked poultry or contaminated drinking water 2. While Campylobacter infections are quite common and often severe, relatively little is known about mechanism of pathogenesis. C. jejuni * Author for correspondence may encounter a series of unfavorable conditions outside the host, such as poultry processing, passage through the human gastro-intestinal tract and colonization of the human intestinal tract. Several environmental factors such as temperature, ph and enzymes regulate the expression of virulence genes under certain conditions and the expression of bacterial virulence determinants. The production of several Campylobacter toxins has also been reported 3,4. The bestcharacterized of the toxins attributed to 413

2 Campylobacter spp. is cytolethal distending toxin (CDT) 5. The effect of environmental stress factors on culture supernatant of C. jejuni containing CDT activity is not yet clear. Some of the mechanisms which are proposed to explain the survival of C. jejuni encountering stress have been reported. C. jejuni may use a stringent response during nutrient depletion 6. C. jejuni can enter into the viable but non-culturable state and change from spiral rod to coccoid shape in response to starvation and stress may induce the virulence gene 7. A recent study showed significantly impaired culturability of isolates due to heat shock and low nutrient medium. C. jejuni isolates also exhibited decline in the adhesion and invasion ability 8. Adherence of C. jejuni to host epithelial cells has also been shown to play an important role in colonization 9 and may increase the local concentration of secreted bacterial products 4, 10. Adherence and invasion into epithelial cells are considered essential steps in producing cellular damage and intestinal disease 11. Therefore, adhesion, invasion and intracellular survival of C. jejuni need to be further studied. Therefore the present study was planned to investigate the effect of selected environmental stress factors such as temperature, ph and proteolytic enzymes on cytotoxicity of C. jejuni strains on Hep-2 and HeLa cell line. In addition to this, we aimed to determine the adherence and invasion properties on C. jejuni isolates. MATERIAL AND METHODS Bacterial strains, culture media and growth conditions A total of 49 Campylobacter strains (41 C. jejuni, 7 C. coli and 1 C. lari) isolated from the rural community of Lucknow district, India. The strains were cultured on blood 414 free charcoal cefaperazone deoxycholate agar (CCDA) and sheep blood (7%) agar containing cefaperazone (30 mg/l), vancomycin (5 mg/l) and amphotericin B (2 mg/l). The plates were incubated at 37 ºC under micro-aerophilic conditions using anoxomat system and examined after 48 h and finally after 72 h. Ten C. jejuni strains [5 CDT negative and 5 randomly selected CDT positive C. jejuni strains] were subjected to characterization of CDT under stress conditions and for the adherence and invasion assay on Hep-2 and HeLa cell line. Cell lines and preparation of bacteria free culture supernatant and lysate Hep-2 and HeLa (Human cervical adenocarcinoma) cell lines were used for assaying the characterization of CDT under stress conditions as well as for the adherence, invasion and cytotoxicity assay of C. jejuni strains. The cell lines were maintained in Eagle s minimum essential medium (MEM) with 10% fetal calf serum (FCS). Cell-free bacterial culture supernatants from all the strains were prepared according to the method described by Florin & Antillon (1992) 12 with minor modifications. Briefly, each of five CDT (+ve) and CDT (-ve) strains was harvested from CCDA plates into MEM cell culture medium. The bacterial suspension was 0.125, which was measured by optical density. Bacterial strains suspended in MEM tissue culture medium were lysed by sonication (4x30 s bursts with 30 s intervals between each burst). Unlysed bacteria and cell debris were then removed by centrifugation at 10,000 r.p.m. for 20 min at 5ºC and filter (0.22 µm) sterilized. Finally the concentrated supernatant was collected. Hep-2 and HeLa cells were cultivated into 24-well tissue culture plates (Nunc) at a density of 2x10 4 cells per well in 0.5 ml

3 medium.0.5 ml of each dilution of culture filtrates was added to the Hep-2 and HeLa cells and incubated for 3 days at 37ºC in an atmosphere of 5% CO 2.For the demonstration of morphological changes, the cells were examined under inverted microscope every 24h up to 72 h. Characterization of CDT under stress conditions The cell-free culture supernatant containing CDT activity of these 10 C. jejuni strains under stress conditions were characterized as follows: Effect of temperature on CDT Cell free bacterial culture supernatant of CDT positive C. jejuni strains were incubated at different temperatures such as 25 ºC, 37 ºC, 45 ºC, and 60 ºC (30 min and 1 h) for 24, 48 and 72 h and then cytotoxic effect was examined on Hep-2 and HeLa cell lines. Effect of ph on CDT The ph of the culture medium was evaluated to determine if the ph of the culture medium influenced toxin production, 15 ml brucella broth with Campylobacter growth supplements was prepared in 7 different 50 ml flasks and the final ph of each flask was adjusted (with either 5 mol/l HCl or 2 mol/l NaOH) at a difference of 10 log ph, starting from 4.0 to The 50 ml flasks were incubated with C. jejuni culture and incubated for 48 h in micro-aerophilic conditions. Supernatants were collected by centrifugation and concentrated to 1 ml with a dialyzed membrane (Sigma, St. Louis, MO, USA). Then the ph was neutralized by washing the sample with PBS (ph 7.5), until the final range was between ph 7 and 8. Now the cytotoxicity assay on Hep-2 and HeLa cells were performed as described previously Effect of proteolytic enzymes (Trypsin and Papain) on CDT The effect of proteolytic enzymes such as trypsin and papain on cytotoxic activity was tested by the modified methods of Guerrant et al (1987) 14. Solution of 1% (w/v) trypsin and 1% (w/v) papain (Sigma, St. Louis, MO, USA) were prepared in 10 mm/l Tris-HCl, ph 8.0. Culture supernatant of the CDT positive C. jejuni strains were mixed with trypsin and papain and incubated at 37 ºC for 30 min and 1 h. The proteolytic enzymes were heat inactivated by boiling for 10 min and the contents were cooled to room temperature and tested for cytotoxicity on cell lines. Adherence, invasion and intracellular survival in Hep-2 and HeLa cell line Hep-2 and HeLa cells were grown in MEM media supplemented with 2% FCS, 2 mm L- glutamine (Gibco-BRL, London, UK). The cells were grown routinely in tissue culture plates at 37 ºC in a 5% CO 2 humidified atmosphere. For all experimental assays, 24- well tissue culture plates (Nunc, Roskilde, DK) were seeded with approximately 5.0 x 10 4 cells/ml and incubated until semiconfluent monolayers were obtained. The cells were washed twice with phosphatebuffer saline (PBS) and incubated with MEM containing 2% FCS. Suspension of CDT positive and CDT negative C. jejuni strains were prepared in PBS (ph 7.0) from cultures grown on CCDA under micro-aerophilic conditions at 37ºC for 48 h. The suspension was centrifuged at 10,000g for 10 min at 4ºC. The bacterial pellet was resuspended in MEM with 2% FCS and inoculums were adjusted to bacteria/ml. The adherence and invasion assays were done by methods described earlier 15,16. The inoculums (0.5 ml) were

4 added to the HeLa cell monolayer in duplicate and the plates were incubated for 3 h to allow adhesion and invasion to occur. Following this period, the monolayers were washed 3 times with MEM without antibiotic to remove unbound bacteria. For measurement of bacterial invasion, medium containing gentamicin (250 µg/ml) was added for 3 h to kill extracellular bacteria. Monolayers were lysed with 0.01% Triton X- 100 and the released intracellular bacteria were enumerated by counting the CFU on the plates, multiplied by the dilution factor. For intracellular survival analysis, doubling dilutions of culture filtrates were prepared in MEM and 0.5 ml of each dilution was added to the HeLa cells and incubated for 72 h at 37 ºC. The cells were examined under an inverted microscope every 24 h up to 72 h for demonstration of morphologic changes. CDT activity titre was defined as the reciprocal of the highest dilution that produced distension in >50% of the cells. Statistical analysis The adherence, invasion and intracellular survival assays were done by using the Kruskal-Wallis test. All the statistical analysis was performed with SPSS statistical software, version 12.0 (Chicago, IL, USA). RESULTS AND DISCUSSION Characterization of cytolethal distending toxin under stress conditions Effect of temperature on CDT The effect of temperatures on the culture supernatants of C. jejuni strains containing CDT activity was tested after incubation of HeLa cells for 24, 48 and 72 h. Maximum CDT activity was observed at temperature 37ºC and 45ºC even after exposure for 60 min (Fig.1). Fig. 1 : Effect of temperature on CDT activity of C.jejuni culture supernatants at 24, 48 and 72h. 416

5 Effect of ph on CDT The effect of a range of ph was tested on the culture supernatant of C. jejuni strains containing CDT. The C. jejuni generally grows between the ph ranges 5-10; however the maximum cfu/ml count was obtained at ph 7.6 because the normal ph range of brucella broth is 7.6 and this bacterium grows best at this ph. CDT activity on HeLa cells was observed only in culture supernatants of C. jejuni grown at ph 6, 7 and 8; culture supernatants of C. jejuni strains grown at ph 4, 5, 9 and 10 did not yield any cytotoxic effect on HeLa cells. The effect of ph on CDT activity of C. jejuni strains are shown in (Fig. 2). % cytotoxicity ph=6.0 ph=7.0 ph= h 48 h 72 h Time Points Fig. 2 : Effect of different ph on CDT activity of C. jejuni culture supernatants at 24, 48 and 72h. Effect of proteolytic enzymes (trypsin and papain) on CDT The effect of proteolytic enzymes, such as trypsin and papain when added to the culture supernatant of CDT positive strains and the strains were cultured as described above. The effect of proteolytic enzymes completely abolished the cytotoxic activity within 1 h of incubation at 37 ºC. Modulation of expression of C. jejuni virulence in response to environmental stresses To determine whether temperature stress, ph or enzymes interfere with the C. jejuni- host cell association, adherence and invasion properties were studied in an in vitro cell 417 culture model using Hep-2 and HeLa cells in comparison to CDT negative strains. We also followed the effects of selected stress conditions on the intracellular growth/ survival of C. jejuni strains. CDT activity of C. jejuni culture supernatant was confirmed by distension and rounding of Hep-2 cells in all five CDT positive strains, CDT titers in these strains ranged from 1 in 32 to 1 in 64. None of the CDT negative isolates showed CDT activity. Consistent result was found when we use HeLa cell line. Enteric Campylobacter are interacted with a wide variety of stress conditions during life in their host and especially extra-host environment. Therefore,

6 their ability to adapt to the changing and unfavorable environment is essential for cell survival. In the present study, the characterization of supernatant containing CDT activity was assessed due to its consistent results in the cytotoxicity assay in sufficient amount to provide a constant response. A number of environmental factors regulate the expression of virulence genes only under certain conditions and the expression of bacterial virulence determinants is modulated by changes in environmental conditions; hence we investigated the effects of environmental conditions such as temperature, ph and proteolytic enzymes on culture supernatants of C. jejuni containing CDT activity. Heat stability was a characteristic feature of this toxin as the cytotoxic activity remained unaffected after exposure of CDT at 60ºC for 60 min. Similarly the cytotoxic activity was maximal in culture supernatant when the strain was grown at ph 7.0. CDT activity of culture supernatant was sensitive to the proteolytic action of trypsin and papain strongly suggests that the proteinaceous nature of this toxin was required for cytotoxicity. Loss of the protein portion of the cytotoxin resulted in the loss of cytotoxicity. Similar observations were made by Lee, et.al. (2000) 13, they reported that the effect of a range of temperature was tested on crude fraction containing the cytotoxin of C. jejuni When the cytotoxin of C. jejuni was stored at room temperature and tested on CHO cells, cytotoxic activity was retained for 5d before complete loss of cytotoxicity. However, this was significantly reduced to 2d when the crude fraction was heated to 60ºC. The resistance of the crude fraction to heat to 100ºC foe 30 min suggests the presence of a heat-stable cytotoxin. The 418 effect of different ph values on culture filtrates of C.jejuni strains in brucella broth were tested for cytotoxicity with CHO cells, the cytotoxic activity was only detected between ph 6 and 10. The effects of proteolytic enzymes (trypsin and papain) significantly affected the cytotoxicity, which clearly indicated that the cytotoxin was proteinaceous by nature since cytotoxicity was completely lost within 30 min incubation at 37ºC. Lee, et. al. (2000) 13 had also reported that trypsin inactivated the cytotoxin more rapidly than pepsin. The survival and infectivity of Campylobacter depends on how rapidly it can adapt to acidic conditions in the stomach and gastrointestinal tract of the host 17. A study by Reid et al (2008) 17 was observed that gene expression was highest when Campylobacter was exposed to ph 5.5. The study of ph stress is important for understanding survival mechanisms and adaptation of Campylobacter to conditions in the human gastrointestinal tract 18 Further we also assessed the adherence, invasion and intracellular survival of CDT (+ve) and CDT (-ve) C. jejuni strains. CDT (-ve) strains showed significantly lower adherence and invasion on Hep-2 and HeLa cells than the CDT (+ve) C.jejuni strains as well as all the five CDT (+ve) isolates showed CDT activity at higher dilutions (> 1 in 32) whereas none of the CDT (-ve) isolates showed CDT activity. According to the Biswas et al (2006) 19, there was no difference in adhesion to HeLa cells of cdt mutants compared to wild-type, but they showed the reduced level of invasion. In the present study, all the CDT(+ve) strains showed cytotoxicity in both the cell lines, while none of the CDT (-ve) strains did so. The cytotoxicity assay of our study was

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