Download The Sialic Acid Binding Adhesin (SabA) of Helicobacter pylori

Egyptian Journal of Medical Microbiology Volume 24 / No. 4 / October 2015 35-40
ORIGINAL ARTICLE
The Sialic Acid Binding Adhesin (SabA) of Helicobacter pylori
Isolated from Egypt is Associated with Gastric Cancer
1
Shymaa Enany*
1
Department of Microbiology and Immunology, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt
ABSTRACT
Key words:
H. pylori,
Gastric cancer,
SabA.
Background: Helicobacter pylori (H. pylori) expressed outer membrane proteins
(OMPs) that assist in bacterial adherence to the gastric epithelium promoting successful
colonization. One of these OMPs is the blood group antigen binding adhesin A (BabA)
which bind to the fucosylated Lewisb blood group antigen (Leb) on the surface of gastric
epithelial cells. Another OMPs is the sialic acid binding adhesin (SabA) that mediates H.
pylori binding the specific sialyl dimeric Lewisx glycosphingolipid (Lex) on the gastric
epithelium. A lot of discrepancies about the correlation between the presence of both
babA and sabA genes and the apparent clinical outcome of H. pylori infection were
reported. Objectives: The present study was to disclose the relationship between the
presence of these genes and the clinical outcomes in Egyptian H. pylori patients.
Methodology: Forty three H. pylori strains were isolated from patients with different
clinical findings. Polymerase chain reaction (PCR) for detecting the presence of babA
and sabA genes was performed using different sets of primers for detecting different
regions of the gene. Further bioinformatics analysis for the sabA product was done using
KEGG and Pfam websites. Results: evincing striking correlation between sabA presence
and the gastric cancer disease. However, we could not find any correlation between
presence of babA and the associated diseases. Conclusions: SabA is one of the H. pylori
OMPs adhesins involving in increasing the risk of H. pylori associated gastric cancer in
H. pylori Egyptian patients.
INTRODUCTION
Outer membrane proteins (OMPs) are expressed in
Helicobacter pylori (H. pylori) and are essential for
bacterial adherence to the gastric epithelium facilitating
its gastric colonization, the establishment of the
infection, and the delivery of the virulence factors to the
epithelial cells1.
The H. pylori OMPs differ significantly from other
Gram negative bacteria in having multiple lower
abundance OMPs and no major predominate OMPs,
however, they are like other Gram negative bacteria
OMPs in being
consisted of phospholipids and
lipopolysaccharide 2,3.
They consisted of five major families; the largest
family (Family 1) is adhesins, while the other four
families are porins, iron transporters, flagellum
associated proteins, and proteins with unknown
function3.
*Corresponding Author:
Shymaa Enany
Department of Microbiology and Immunology,
Faculty of Pharmacy, Suez Canal University, Ismailia, 41522, Egypt.
Email: [email protected]. Phone: +201224297176.
Egyptian Journal of Medical Microbiology
Family 1 consisted of 21 members Hop and 12
members Hor, family 2 comprised 8 Hof, family 3
consisted of 4 Hom, family 4 composed of 6 iron
regulated OMPs, and family 5 comprised 3 efflux pump
OMPs2. Most expressed H. pylori adhesins which
promote specific binding to the host gastric epithelium
receptor are included in family 1; Hop members 4. Due
to these adhesins, it was found that H. pylori is the most
successful bacterium that colonizes the mucus layer of
the gastric epithelium with a prevalence of its infection
reached up to 100% and around 60% in developing and
developed countries; respectively4.
These OMPs include the blood group antigen
binding adhesin A (BabA), the sialic acid binding
adhesin (SabA), the adherence associated lipoproteins A
and B (AlpA and AlpB), and the outer inflammatory
protein A (OipA). Association of these OMPs with
clinical outcomes was variably reported all over the
world 5-8.
Blood group antigen binding adhesin A (BabA) is
the major adhesion in H.pylori that mediates the binding
of the bacterium to the fucosylated Lewisb blood group
antigen (Leb) on the surface of gastric epithelial cells
which is considering the dominant antigen in the gastric
mucosa4. Although European researchers previously
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Enany / The Sialic Acid Binding Adhesin (SabA) of Helicobacter pylori, Volume 24 / No. 4 / October 2015 35-40
indicated that babA gene is of high clinical relevance
and would be a useful marker to identify patients who
are at higher risk for specific H. pylori related diseases7,
Asian group proved that presence of babA is not of high
clinical relevance in Japan and that Japanese strains are
different from those infecting Western populations 9.
The second most characterized adhesion of H.
pylori is the sialic acid binding adhesin (SabA) that
mediates the binding of the bacterium to the sialyl
dimeric Lewisx glycosphingolipid (Lex) expressed on
gastric epithelium4. The correlation between the
presence of SabA and the related H. pylori diseases is
also a matter of controversy. Epidemiological studies
showed that in Asia SabA presence has little influence
on H. pylori clinical outcomes 10, 11 and in European and
North American countries SabA status has close
relationship with gastric cancer 12; that inconsistent
result is mostly attributed to the mutable geographic
variations.
Therefore, in this study we investigated the
presence of sabA and babA genes in the clinical H.
pylori isolates from Ismailia, Egypt and tried to find the
correlation between their presence and the clinical
outcomes of the patients.
METHODOLOGY
H. pylori culture
Forty three H. pylori strains were selected from our
previous master work as following: 10 from normal (N),
10 from gastric ulcer (GU) patients, 10 from duodenal
ulcer (DU) patients, 10 from gastritis (G) patients, and 3
from gastric cancer (GC) patients 13. They were isolated
from antral biopsy specimens of patients with dyspeptic
symptoms and clinically indicated for upper
gastrointestinal
endoscopy.
All
endoscopic
examinations were carried out using Video endoscope
PENTAX EMP-3500. The selected strains were recultured on H. pylori selective ready plates containing
trimethoprim, polymyxin B, vancomycin, and
amphotericin B (Nissui Pharmaceuticals, Tokyo, Japan)
at 37°C in a microaerophilic atmosphere. Single
colonies were sub-cultured on 5% sheep blood agar
(Becton Dickinson, Tokyo, Japan) and then harvested
for storage at −80°C to be ready for subsequent
reactions.
Detection of urea, sabA, and babA genes by PCR
Genomic DNA was extracted from the colonies by
boiling method and DNA density was determined by
spectrophotometer. The extracted DNA was used for all
PCR reactions. Since in our previous study, we
confirmed the presence of subunit of H. pylori urease
gene (ureA) by PCR in 20 samples out of 150 from
them 7 only were included in this study, we performed
PCR for detecting ureA gene in all the selected strains
including here according to He et al and Enany et al 13,14.
Primers used were included in table 1.
The presence of sabA and babA in H. pylori clinical
isolates was analyzed by PCR using two sets of primer
pairs for sabA gene separately and one set for babA
gene as listed in table 1. PCR reactions were performed
in a final volume of 25 μL containing 12.5μL 2x
EmeraldAmp ® PCR Master Mix(Takara, Japan),
500ng from genomic DNA as a template, and 10μ molar
from each primer using a thermal cycler (Takara,
Japan). PCR amplification was performed under the
following condition: an initial denaturation for 5 min at
98°C; 30 cycles of 1min at 98°C, 1min at 60°C, and
1min at 72°C; and a final extension at 72°C for 10min
15
. The resulting PCR product was analyzed on 1.5%
agarose gel with ethidium bromide.
Table 1: The primers list used in this study
Target gene
Primer sequence (5`-3`)
F-CCCAATGGTAAATTAGTT
ureA
R-CTCCTTAATTGTTTTTAC
F-CCGCTAGTGTCCAGGGTAAC
sabA (set 1)
R-CGCGCTGTAAGGGTTATTGAAC
F-TTTTTGTCAGCTACGCGTTC
sabA (set 2)
R-ACCGAAGTGATAACGGCTTG
F-AATCCAAAAAGGAGAAAAAGTATGAAA
babA
R-TGTTAGTGATTTCGGTGTAGGACA
Analysis of SabA protein pathway
The pathway of SabA; the product of the sabA gene
was analyzed using the free Kyoto Encyclopedia of
Genes
and
Genomes
(KEGG)
database
(http://www.genome.jp/kegg/). The entry used was
exported
from
the
UniProt
database
(http://www.uniprot.org/uniprot/) for both reviewed
36
PCR product size
411
Reference
1330
16
622
15
833
17
16
(Swiss-Prot); manually annotated and unreviewed
(TrEMBL); computationally analyzed with a gene name
(ordered locus name) of HPV225_0734.
Confirmation of SabA family and domain
For confirming the role of SabA protein as one of the
OMPs in H. pylori, we detected the protein family and
its domains using both CD search of NCBI's interface
Egyptian Journal of Medical Microbiology
Enany / The Sialic Acid Binding Adhesin (SabA) of Helicobacter pylori, Volume 24 / No. 4 / October 2015 1-8
(http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)
and Pfam database (http://pfam.sanger.ac.uk/) using the
previously mentioned exported protein query sequence.
RESULTS AND DISCUSSION
Forty three strains from normal, peptic ulcer, gastritis,
and gastric cancer cases were included in this study. We
examined them for the presence of ureA gene and we
detected its presence in all the tested strains that showed
a clear positive band at 411 bp (Figure 1). Since the
fragment of urease A gene was amplified, we
confirming that all the included samples in this study
were pure H.pylori whereas He et al and Vinette et al
reported that ureA gene is conserved and specific to H.
pylori 14, 18.
Fig. 1: Agarose gel electrophoresis of the PCR amplification of ureA gene. Lane 1: Low molecular weight marker,
Lane 2-17: DNA extracted from different H. pylori strains included in this study
For the analysis of babA gene, we showed 40 out of
43 positive samples for babA gene (93%). PCR showed
a positive band at 833 bp as shown in figure 2. The
correlation between the presence of babA gene and the
clinical outcomes in our study was shown in table 2. We
could not determine any correlation between the babA
presence and the associated disease. Its presence was
proved in almost all our isolates even in 9 out of 10 with
normal findings. Although it has been reported before
that the presence of babA gene is associated with sever
gastric diseases 7, it is also found that babA could not be
used as a biomarker for peptic ulcer and gastric cancer
diseases6. Some studies have suggested H. pylori
expressing BabA and OipA are associated with an
increased risk of peptic ulcer and gastric cancer in
European countries 7, 8. Other studies from Brazil and
Thailand showed that BabA is not a biomarker for
peptic ulcer disease or gastric cancer5,6. These
conflicting data about babA and the prediction of the
clinical outcomes was attributed to the geographic
different origin of the isolated H. pylori strains 1.
Fig. 2: Agarose gel electrophoresis of the PCR amplification of babA gene. Lane 1: Low molecular weight marker,
Lane 2-17: DNA extracted from different H. pylori strains included in this study
Egyptian Journal of Medical Microbiology
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Enany / The Sialic Acid Binding Adhesin (SabA) of Helicobacter pylori, Volume 24 / No. 4 / October 2015 35-40
On the other hand, we found that sabA gene was
associated with the gastric cancer disease in all tested H.
pylori isolated from gastric cancer patients using two
sets of primers for detecting different regions of sabA
gene. PCR showed positive bands corresponding to
1330 bp and 622 bp which are the product sizes of both
set 1 and set 2 of sabA gene detecting primers in the
group of gastric cancer, while no band appeared by both
primers sets in the remaining groups (Figure 3).
Fig. 3: Agarose gel electrophoresis of the PCR amplification of sabA gene. (3A): Amplification for sabA1 region; Lane
1: High molecular weight marker, Lane 2-4: DNA extracted from H. pylori strains isolated from gastric cancer patients,
Lane 5-17: DNA extracted from H. pylori strains isolated from patients with other different symptoms.
(3B): Amplification for sabA2 region. Lane 1: Low molecular weight marker, Lane 2-4: DNA extracted from H. pylori
strains isolated from gastric cancer patients.
Our results were in accordance with a previous
study which showed that sabA binds to the carbohydrate
structure Lex antigen expressed on the gastric epithelium
and thus associated with an increased gastric cancer
risk1. As well, European study provided evidence before
that SabA protein may enhance H. pylori pathogenecity
in the human gastric epithelium 11. They explained that
association by the alteration in the glycosylation
patterns in the gastric mucosa during H. pylori infection
which in turn promotes bacterial attachment 11. On
contrary, in Asia, SabA presence showed no relation
with the gastric cancer diseases 10, 11. The prevalence
rate of gastric cancer was not statistically different
between patients infected with SabA positive and SabA
negative H. pylori19. These variations among the
relationship between SabA presence and the clinical
outcomes again were attributed to the geographical
differences. Furthermore, SabA is regulated by phase
variation; its expression can rapidly be switched on/off
to adapt changes in the gastric media 1 and thus not all
strains will produce functional proteins. This will
increase the ambiguity about the relationship between
the presence of sabA gene and the clinical findings of
the patients.
Table (2): The prevalence of sabA and babA genes in each clinical group of H. pylori patients.
Gene
N
GU
DU
G
GC
0
0
0
0
3
Positive sabA (set 1)
0
0
0
0
3
Positive sabA (set 2)
9
10
10
8
3
Positive babA
Our analysis of the pathways of SabA protein using
KEEG revealed that Sab A recognized the sialyl-Lewisx
(sLex) which acts as a functional receptor for H. pylori
adherence as illustrated in figure 4. It was reported
before that H. pylori gastric cancer is associated with
the replacement of the non sialylated Lewis antigen by
the sialylated Lewis antigen (sLex) 11. One of the chronic
inflammatory responses after H. pylori infection is the
elevation of the gastric mucosal sialylation, however
38
this level is remaining low either in the healthy or in the
individuals after eradication of H. pylori. The
identification of sialyl dimeric Lex glycosphinogolipid
as a receptor for H. pylori for the first time was reported
by a group of researchers in 2002 and they showed that
H. pylori infection induced Lex antigen formation in
gastric epithelium. They identified this sialyl dimeric
Lex binding bacterial adhesion as a 70 K OMP and
named it SabA11.
Egyptian Journal of Medical Microbiology
Enany / The Sialic Acid Binding Adhesin (SabA) of Helicobacter pylori, Volume 24 / No. 4 / October 2015 1-8
Fig. 4: The biological pathway of SabA in H. pylori using Kyoto Encyclopedia of Genes and
Genomes (KEGG) database
Precise identification of this adhesin was done
using the freely available bioinformatics Pfam website.
Pfam is a large collection of protein families, each
represented by multiple sequence alignments and hidden
Markov models (HMMs). It is useful for identifying
functionally conserved regions. Our Pfam search results
showed that SabA protein was confirmed to be belonged
to the family seems confined to Helicobacter. It is
predicted to be an outer membrane protein based on its
pattern of alternating hydrophobic amino acids similar
to porins. It showed Pfam family named HP_OMP.
Figure (2) illustrated the arrangement of the Pfam
domains that were found on the exported sequence of
SabA protein.
REFERENCES
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Fig. 5: Pfam domains arrangements in SabA protein.
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CONCLUSION
We could say that SabA is one of the H. pylori
OMPs adhesins involving in increasing H. pylori
associated diseases risk. In the Egyptian isolated H.
pylori strains there was a strong correlation between the
presence of sabA gene and the H. pylori associated
gastric cancer and there was no relationship between
babA gene existence and the clinical outcomes of H.
pylori.
Egyptian Journal of Medical Microbiology
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Egyptian Journal of Medical Microbiology