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Folia Microbiol. 41 (1), 64–70 (2004)
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Effectiveness of Cyanothece spp. and Cyanospira
capsulata Exocellular Polysaccharides as Antiadhesive
Agents for Blocking Attachment of Helicobacter pylori
to Human Gastric Cells
F. ASCENCIOa,b, N.L. GAMAa,b, R. DE PHILIPPISc, B. HOb
aCenter for Biological Research, Marine Pathology Unit, La Paz, Baja California Sur 23 000, Mexico
fax +52 612 125 3625
e-mail [email protected]
bDepartment of Microbiology, National University of Singapore, Singapore 117 597
cDipartimento di Biotecnologie Agrarie, Università degli Studi di Firenze, Firenze, Italy
Received 17 July 2003
ABSTRACT. The effect of cyanobacterial polysaccharides (from Cyanothece spp. and Cyanospira capsulata) on the binding of Helicobacter pylori to gastric epithelial cells was evaluated. The antiadhesive action
on Kato III and HeLa S3 human gastric cell lines was established.
We have recently found (Ascencio et al. 1993) that adhesion of H. pylori to Kato III and HeLa S3
human gastric cell lines is promoted by heparan-sulfate-inhibitable lectin-like protein. Because prevention
of H. pylori binding to gastric epithelial cells could represent a potential target for therapy, the aim of this
study was to evaluate whether exocellular polysaccharides from cyanobacterial strains Cyanothece spp. and
Cyanospira capsulata, having different monosaccharide composition and anionic charge density, can to
inhibit adhesion of H. pylori to or to displace previously bound H. pylori from Kato III and HeLa S3 cell
monolayers. Our findings indicate that exocellular polysaccharides from Cyanothece sp. PE14 and Cyanothece sp. VI 22 inhibited adhesion and displaced cell-bound H. pylori in a dose-dependent manner and that
this property is dependent of the sulfation degree of the polysaccharide preparation.
Cyanobacteria are widely distributed among naturally illuminated terrestrial and aquatic environments, including some types of extreme environments, covering a broad spectrum of physiological properties and tolerance to environmental stress (Whitton 1992; Tandeau de Marsac and Houmard 1993; Agrawal and Misra 2002; Agrawal and Singh 2002; Agrawal and Pal 2003).
Given their ecophysiological properties and their biochemical diversity, cyanobacteria have been
regarded as good candidates for various biotechnological applications and their potential in the conversion
of light energy into useful chemicals for food, pharmaceutical and other industries has been claimed and
assessed (Borowitzka 1999; Cohen 1998; De Philippis et al. 1999; Kaji et al. 2002; ezanka et al. 2003a,b;
Shah et al. 2003).
A new field of possible exploitation of cyanobacteria has arisen in the last decade by the growing
industrial interest toward polysaccharides of microbial origin, which often show advantages over polysaccharides extracted from plants or marine macroalgae (De Philippis and Vincenzini 1998; Nicolaus et al.
1999; Schaeffer et al. 2000). Within the food industry, polysaccharides of microbial origin, such as the
polymers produced by cyanobacteria and microalgae, are potential candidates as nutraceuticals (German et
al. 1999; Liu and Chen 2003); these are clearly not drugs but pharmacologically active substances that can
potentiate, antagonize, or otherwise modify physiological or metabolic functions (Hardy 2000). Potential
applications include their usage as immunostimulators (Sakai 1999), anti-oxidants (Ruperez et al. 2002;
Zhang et al. 2003), and antiviral (Berge et al. 1999; Carlucci et al. 1999; Schaeffer et al. 2000; Zhu et al.
2003), or anticancer agents (Itoh et al. 1993; Riou et al. 1996).
On the other hand, the human gastric pathogen H. pylori can bind to several receptors on the surface of gastric epithelial cells, including Lewis X and T blood antigens, or sulfated glycoconjugates (Ascencio et al. 1993; Borén et al. 1993; Namavar et al. 1998; Saitoh et al. 1991; Su et al. 1998). The ability to
adhere to gastric celis may be essential for sustained infection and H. pylori-induced diseases (Guruge et al.
1998). In this respect, we have recently found that adhesion of H. pylori to Kato III and HeLa 53 human
2004
CYANOBACTERIAL POLYSACCHARIDES BLOCKING ATTACHMENT OF H. pylori 65
gastric cell lines is promoted by a heparan sulfate protein binding (Guzman-Murillo et al. 2001), and that
binding of H. pylori to heparan sulfate is effectively blocked by heparin oligosaccharides (Ascencio et al.
1993) or, as reported Wadström et al. (1997), binding is also inhibited by sulfated polysaccharides such as
fucoidan and carrageenan O.
Prevention of H. pylori binding to gastric epithelial cells could thus represent a potential target for
therapy. This hypothesis has been tested recently in H. pylori infected rhesus monkeys, using 3´-sialyllactose sodium salt. This oligosaccharide occurs naturally in human and cow milk, is recognized by H. pylori, and has been shown to inhibit H. pylori-mediated hemagglutination and the adhesion of the bacteria to
human epithelial cells in vitro. Administration of this adhesion molecule analog resulted in cure of the infection in a few animals, suggesting this approach warrants further studies (Mysore et al. 1999).
Glycosaminoglycan-binding microbial proteins may mediate adhesion of microbes to eukaryotic
cells, which appears to be a primary mechanism in mucosal infections, such as H. pylori infections (Guruge
et al. 1998).
Glycosaminoglycan-binding microbial proteins are also involved in secondary effects, such as adhesion to cerebral endothelium in cerebral malaria or to synovial membranes in arthritis caused by Borrelia
burgdorferi (Wadström and Ljungh 1999). The aim of this study was to evaluate whether exocellular polysaccharides from cyanobacteria strains Cyanothece spp. and Cyanospira capsulata (having different monosaccharide composition and anionic charge density) are capable to inhibit adhesion of H. pylori to or to
displace previously bound H. pylori from Kato III and HeLa S3 cell monolayers.
MATERIALS AND METHODS
Bacteria and growth conditions. H. pylori strains associated with gastrooesophagal reflux disease
(GERD), duodenal ulcer, and cancer were isolated at the Department of Microbiology of the National University of Singapore from gastric biopsy samples obtained at the National University Hospital, Singapore.
Taxonomic identification was based on Gram staining, cell morphology and positive reaction for catalase,
oxidase and urease activity, and then confirmed by API ZYM kit (BioMériux, France), and by PCR detection of UreC (Monteiro et al. 1997). Strains 51932 (CagA– VacA–, Ure+, noncytotoxic) and 49503 (Cag+
VacA+, Ure–, cytotoxic) were obtained from the American Type Culture Collection (Mannasas, VA). All
strains were cultured on GAB-CAMP agar medium supplemented with 8.5 % lyzed human blood (80 °C,
20 min), 10 % inactivated horse serum (56 °C, 30 min), 0.05 % cysteine hydrochloride, 0.35 % IsoVitaleX,
and the following antibiotics (in Pg/mL): vancomycin 6, nalidixic acid 20, ketoconazole 3, and incubated for
2–3 d at 37 °C under microaerophilic conditions (5 % O2, 10 % CO2, 85 % N2). Stock cultures were stored
at –80 °C in trypticase soy broth (TSB) containing 15 % glycerol.
For cell-adhesion, H. pylori strains were grown on GAB-CAMP agar plates (see above), harvested,
and washed twice in PBS. Bacterial cell suspensions were adjusted to a concentration of 1 CFU/pL (i.e.
109 CFU/mL) and then labeled with (+)-biotin N-hydroxysuccinimide ester (‘N-biotin’) (Harlow and Lane
1988).
Kato III and HeLa S3 cell lines. Kato III (a gastric adenocarcinoma cell line) and HeLa S3 cells
(derived from a human epithelioid carcinoma) were obtained from the American Type Culture Collection
(Mannasas, USA). Both cells were grown in 75-cm2 tissue culture flasks with RPMI-1640 medium, containing 40 Pg/mL gentamicin, 2 mmol/L L-glutamine, and supplemented with 15 % fetal calf serum (FCS). The
flasks were incubated (37 °C, 95 % humidity, 5 % CO2) and cells were washed with phosphate buffer saline
without Mg2+ and Ca2+ (pH 7.2) and then treated with trypsin (0.25 %) in Hank’s modified balanced salt
solution (5 min, 37 °C) to release the cells from the plastic surface. The trypsinized cells were resuspended
in fresh cell medium and then seeded into new tissue culture flasks. The cells were transferred every 5 d for
Kato III and every 3 d for HeLa S3 cells. For cell adhesion, 100 PL (cell concentration 40/nL, i.e. 4 × 107
cells per mL) of the Kato III and HeLa S3 cell suspensions in RPMI 1640 medium containing 15 % FCS
were plated onto 96-well tissue-culture plates and incubated at 37 °C until the formation of a semiconfluent
cell monolayer.
Polysaccharide preparation. Cyanobacterial strains isolated from hypersaline environments were
photoautotrophically grown in enriched seawater or in Zarrouk medium (De Philippis et al. 1998).
Exopolysaccharide separation was done according to De Philippis et al. (1993). Briefly, cells were
removed from the culture medium by centrifugation (14 000 g, 10 min, 10 °C). Soluble polysaccharides
were obtained from the supernatants by addition of two volumes of 2-propanol; the precipitate was harvested and dried for 3 h at 50 °C.
66 F. ASCENCIO et al.
Vol. 49
Enzyme-linked, biotin–streptavidin bacterial-adhesion assay. Adhesion of H. pylori to both carcinoma cells was done according to Guzman-Murillo et al. (2001). Briefly, semiconfluent cell monolayers
(0.5–1.0 × 105 cells per well) grown on 96-well tissue culture plates were washed 3 times in RPMI-1640
medium without antibiotics and incubated (1 h, 25 °C) with a suspension of biotin-labeled H. pylori cells in
RPMI-1640 medium without antibiotics (1 CFU/pL). Then the plates were washed 3 times with PBS containing 500 ppm (V/V) Tween-20 to remove nonadhering bacteria. One hundred PL of horse-radish peroxidase-conjugated streptavidin (Boehringer Mannheim, Germany) diluted 1 : 2000 in PBS was added to each
well and the plate was incubated (90 min, 25 °C). After washing the plates 3 times with PBS–Tween,
100 PL of 1,2-benzenediamine-HCl was added to each well, and the plates were incubated for additional
20 min at 22 °C in the dark. The reaction was stopped by the addition of 100 PL of 2 mol/L H2SO4 and
color development was measured at 495 nm. Results of adhesion of biotin-labeled H. pylori are expressed in
absorbance units.
Inhibition assays were done by preincubating Kato III and HeLa S3 cell monolayers for 1 h at
37 °C with 100 PL of soluble cyanobacterial exocellular polysaccharides, proteoglycans and glycoproteins
adjusted to different concentration (1000 to 1 Pg/mL) in PBS.
Detachment. H. pylori was first allowed to bind to Kato III and HeLa S3 semiconfluent monolayers
for 90 min at 37 °C (see above). After washing the 96-well tissue culture plates with PBS Tween 20 (to
remove nonadhering bacteria), 100 PL of soluble cyanobacterial exocellular polysaccharides, adjusted to
different concentration (1000 to 1 Pg/mL) in PBS, was added to the 96-well tissue culture plates; they were
washed with PBS Tween 20 after 1-h incubation at 37 °C, and residual bacteria were quantified (see above).
Statistical analysis. Student’s t-test was used to assess the difference between means in binding and
inhibition assays. Comparison involving more than two groups were performed with ANOVA. Values
are expressed as percentage calculated from mean values from at least triplicates.
RESULTS
Inhibition of adhesion of H. pylori strain 51932 to Kato III cells. Prior to the standard bacterial
adhesion assay, the reference strain 51932 (previously shown to adhere to both carcinoma cell lines; Guzman-Murillo et al. 2001), was preincubated with the various cyanobacterial exocellular polysaccharides.
The strongest inhibition (60 %) of adhesion of H. pylori to Kato III cell monolayers was shown by the
highly sulfated polysaccharides produced by strains Cyanothece sp. VI22 and VI13 (Table I).
Table I. Inhibition of adhesion of H. pylori strain 51932 to Kato III by various cyanobacterial polysaccharidesa
Exocellular
polysaccharide from
Cyanospira capsulata
Cyanothece ET 5
Cyanothece CE 9
Cyanothece PE14
Cyanothece VI 13
Cyanothece VI 22
Cyanothece 16Som2
Inhibition
%
Sulfate
content
36
30
31
35
60
65
43
–
–
tr
tr
++
++
++
Exocellular
polysaccharide from
Fucoidan
Carrageenan
Dextran sulfate
Heparin
Lactoferrin
Inhibition
%
Sulfate
content
0
53
44
51
52
?
?
?
?
?
a++ = abundantly present; tr = traces; – = absent.
The inhibition of adhesion obtained with some of the exocellular cyanobacterial polysaccharides was
higher than the inhibition shown by preparations of commercially available carrageenan O, dextran sulfate,
heparin, or even with the sialic acid-rich glycoprotein from cow milk, lactoferrin (Table I).
Inhibition of adhesion of different H. pylori strains Kato III and HeLa S3 cells with exocellular
polysaccharides from Cyanothece sp. PE14 and VI 22. Subsequently, the inhibition tests were performed
with various H. pylori strains on both carcinoma cell lines, using one of the most effective and one of the
less effective cyanobacterial exocellular polysaccharides. Preincubation of different H. pylori strains with the
polysaccharides produced by Cyanothece spp. (100 Pg/mL) significantly (p 0.05) reduced adhesion of the
bacterium to both carcinoma cell lines, especially to Kato III cell monolayers in the presence of the exocellular polysaccharide from Cyanothece sp. VI 22 (Table II). However, there was no statistically significant
CYANOBACTERIAL POLYSACCHARIDES BLOCKING ATTACHMENT OF H. pylori 67
2004
(p 0.05) difference between the inhibition of binding of a particular H. pylori strain caused by cyanobacterial polysaccharide or carrageenan O.
Inhibition and displacement kinetics. Exocellular polysaccharides of Cyanothece sp. PE14 and
Cyanothece sp. VI 22 inhibited adhesion in a dose-dependent manner (Table III). Adhesion to HeLa S3 cells
was inhibited by 73 % with Cyanothece PE14 polysaccharide at 100 Pg per well, whereas the same concentration of carrageenan O inhibited the adhesion to Kato III cells by 81 %, or to HeLa S3 cells by 50 %.
Neither the polysaccharide from Cyanothece VI 22 nor dextran sulfate inhibited adhesion of strain 51932 to
both carcinoma cell monolayers; in contrast, a higher concentration of polysaccharide inhibited the adhesion
to both cell monolayers to a lesser extent. (Concentration >100 Pg per well could not be evaluated since
these polysaccharides form a gel in the solution.)
Table II. Inhibitory activity of Cyanothece spp. exocellular polysaccharides on the adhesion of different H. pylori strains to Kato III
and HeLa S3 cell monolayers (numbers of strains, triplicates)
Kato III; inhibition range, %
HeLa S3; inhibition range, %
Inhibitor
0–20
Cyanothece sp. PE14
Cyanothece sp. VI 22
Carrageenan O
2
1
18
21–40
41–60
61–80
2
1
5
7
2
4
19
11
2
81–100
0
15
1
65
85
10
0–20
21–40
41–60
61–80
81–100
8
8
22
15
6
1
7
10
3
0
4
0
0
2
2
30
45
10
Table III. Inhibition of adherence and adhesion of H. pylori strain 51932 to Kato III and HeLa S3 cell monolayersa
Inhibition, %
Inhibitorb
Cyanothece sp. PE14
Cyanothece sp. VI 22
Carrageenan O
Dextran sulfate
aTriplicates, SD 8 %.
Kato III
Adhesion, %
HeLa S3
Kato III
HeLa S3
1
10
100
1
10
100
1
10
100
1
10
100
28
31
25
16
36
33
47
45
23
32
81
68
29
46
13
1
39
24
31
0
37
17
50
1
95
100
86
86
36
67
72
100
22
63
18
100
100
87
100
100
62
59
73
100
19
41
29
100
b1, 10, 100 Pg per well.
On the other hand, exocellular polysaccharides of Cyanothece PE14 and VI 22, as well as carrageenan O, induced detachment of previously bound H. pylori strain 51932 from both carcinoma cell monolayers, in a dose-dependent manner (Table III). Displacement of cell-bound H. pylori from both carcinoma cell monolayers was more effective with the polysaccharide of strain PE14, as compared with that of
strain VI 22 or carrageenan O. However, displacement of cell-bound H. pylori from Kato III cells by the
polysaccharide of strain PE14 reached a steady state at polysaccharide concentration of 40 Pg per well,
while displacement of H. pylori bound to HeLa S3 cells needed a concentration of 100 Pg per well.
DISCUSSION
Increased antibiotic resistance has begun to impair our ability to cure human gastric mucosal infections such as those produced by H. pylori and related bacterial infections. Therapies that disrupt the ability
of H. pylori to colonize the gastric mucosa, impair its living conditions, limit its ability to garner essential
nutrients, or minimize its ability to evade the immune response, may have great therapeutic potential (Opekun et al. 2000).
Because of the affinity of H. pylori for glycosidic moieties on mucosal epithelial cells, it was examined whether different glycoconjugates can block adhesion, colonization of H. pylori on the gut, and thus
inhibit the infection process. Simon et al. (1997) found that 3´-sialyllactose can block adhesion of H. pylori
to the duodenum-derived cell line HuTu-80. Furthermore, Mysore et al. (1999) found that an antiadhesive
therapy using 3´-sialyllactose can cure or decrease H. pylori colonization in some rhesus monkeys. How-
68 F. ASCENCIO et al.
Vol. 49
ever, Opekun et al. (2000) found no measurable effect on H. pylori density, nor on the severity of the
inflammatory response in human volunteers when an oral therapy of 3´-sialyllactose was applied; neither
a low nor a high dose of recombinant human lactoferrin showed suppressive or beneficial effects with
regard to elimination of H. pylori infection.
Slomiany et al. (1999), using the Sprague–Dawley rat model of H. pylori lipopolysaccharide-induced gastritis, suggested that sulglycotide (i.e. an antiulcer agent derived by proteolysis from pig duodenum
mucin and the chemical esterification of its saccharide chains with sulfo groups) suppresses H. pylori-induced mucosal inflammatory responses by upregulating constitutive nitric-oxide synthase (cNOS) and interfering with the events by NOS-2 and caspase-3.
Certainly, a number of factors in the application or delivery systems are important for the efficacy
of any potential antiadhesive or nutraceutic agent. Similarly, the methods used to measure any cure must also
be taken into consideration before we can accept or discard any antiadhesive or nutraceutic agent against
mucosal pathogens such as H. pylori.
Various natural products have been proposed as potential therapeutic agents against H. pylori infections. Belogortseva et al. (2000) reported the inhibition of H. pylori hemagglutination by polysaccharides
from roots of Panax ginseng; Wang et al. (2000) reported the usefulness of the anti-oxidant-rich microalgae
Haematococcus pluviali in inhibiting an experimentally induced H. pylori infection in a BALB/c mouse model. Shibata et al. (2003) found that cladosiphon fucoidan inhibited H. pylori attachment to porcine gastric
mucin. In addition, in an in vivo experiment, H. pylori-induced gastritis and prevalence of H. pylori infected
Mongolian gerbils were markedly reduced by fucoidan in a dose-dependent manner (Shibata et al. 2003).
We believe a therapy that combines antioxidants (Wang et al. 2000) and antiadhesive agents, such
as the sulfated polysaccharides of microalgal or algal origin (Wadström et al. 1997; Guzman-Murillo and
Ascencio 2000) or the cyanobacterial polysaccharides described here, may act more effectively against the
microbe. In addition, sulfated polysaccharides, besides showing an antiadhesive activity towards H. pylori
on gastric cell, can also stimulate the nonspecific immune system of the host.
The cyanobacterial sulfated polysaccharides tested by us did not cause any cytotoxic effect on the
carcinoma cells (data not shown) as has been evaluated for sulfated polysaccharides isolated from the red
algae Bostrychia montagnei and Porphyra columbina or from the brown algae Laminaria brasiliensis and
Sargassum stenophyllum (Stevar et al. 2001). An antioxidant-rich algal meal as proposed by Wang et al.
(2000), but supplemented with cyanobacterial or microalgal exocellular sulfated polysaccharides as an antiadhesive and immunostimulant agent, may represent a potential nutraceutical for H. pylori infections, and
clearly, a putative therapeutic alternative to the currently available multidrug treatments.
On the other hand, Icatlo et al. (2000), using an established mouse model of H. pylori infection,
demonstrated that the use of famotidine (a histamine H2 receptor antagonist) dextran sulfate significantly
reduced the group mean titer of H. pylori in the gastric mucosa of precolonized mice in terms of both
mucus-resident and epithelial-adherent populations and eradicated the bacterium in 50 %. This drug combination holds potential for incorporation as an adjunct to the standard multidrug therapy for H. pylori
infections.
Our results clearly demonstrate that cyanobacterial sulfated polysaccharides are bound by almost
all the H. pylori strains tested as compared with dextran sulfate or other glyconjugates, which are recognized
by H. pylori to a lesser extent. In particular, application of this novel approach to the clinical setting bears
on the overall cost-effectiveness of chemotherapy as it may abbreviate the treatment time and aid in the
elimination of all H. pylori strains resistant to certain antimicrobial drugs (Icatlo et al. 2000).
Finally, it is also important to stress that production of exocellular polysaccharides of cyanobacterial or microalgal origin for using as antiadhesive agents may be promising, considering that the cultivation of cyanobacteria or microalgae under controlled conditions can guarantee a constant and homogeneous
production of biological agents, because it is not dependent on the climatic variations as is the case with
production of chemicals from natural macroalgal stocks (Hernández-Guerrero et al. 2000).
This work was supported by the National University of Singapore (NUS). The first author was on a sabbatical scholarship
from NUS and the Mexican Council of Science and Technology (CONACyT). The second author was on a graduate scholarship from
CONACyT.
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