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Helicobacter pylori infection is associated with risk for chronic gastritis (CG), gastric ulcer (GU), duodenal ulcer (DU), and gastric cancer (GC). The H. pylori Cag type IV secretion system (TFSS) translocates the virulence factor cytotoxin-associated gene A protein into host cells and plays an important role in initiating gastric carcinogenesis. The CagL and CagI proteins are components of the TFSS. The Arg-Gly-Asp (RGD) motif of CagL, and the six most distal C-terminal amino acids (Ser-Lys-Ile-Ile-Val-Lys, and Ser-Lys-Val-Ile-Val-Lys) of CagL and CagI are essential for TFSS adhesion to host cells. Additionally, the CagL variant Tyr58Glu59 was previously shown to be associated with GC patients.
Results
We isolated 43 H. pylori isolates from 17 CG, 8 GU, 8 DU, and 10 GC patients in Southeast Asia. Total DNAs were extracted and sequenced with MiSeq. H. pylori strain ATCC 26695, which was isolated from CG patients, was used as a reference. We examined the full sequences of H. pylori cagL and cagI using whole-genome sequencing (WGS), and analyzed whether single nucleotide variants and amino acid changes (AACs) correlated with adverse clinical outcomes. Three isolates were excluded from the analysis due to cagPAI rearrangements. CagL RGD motifs were conserved in 39 isolates (97.5%). CagL-Glu59 and Ile234 in the C-terminal motif were more common in 10 H. pylori isolates from GC patients (p < 0.001 and p < 0.05, respectively). When 5 Vietnamese isolates from GC patients were excluded, CagL-Glu59 still remains significant (p < 0.05), but not Ile234. CagL-Tyr58 was seen in only one isolate. The CagI C-terminal motif was completely conserved across all 40 isolates, and there were no significant AACs in CagI.
Conclusions
Using WGS, we analyzed genetic variants in clinical H. pylori isolates and identified putative novel and candidate variants in uncharacterized CagL and CagI sequences that are related to gastric carcinogenesis. In particular, CagL-Glu59 has the possible association with GC.
The online version of this article (doi:10.1186/s13099-017-0165-1) contains supplementary material, which is available to authorized users.
H. pylori
Helicobacter pylori
WGS
whole-genome sequencing
SNVs
single nucleotide variants
AACs
amino acid changes
TFSS
type-IV secretion system
cag
cytotoxin-associated gene
CG
chronic gastritis
GU
gastric ulcer
DU
duodenal ulcer
GC
gastric cancer
Background
The infection rate for the Gram-negative bacterium Helicobacter pylori is around 50% worldwide [1, 2]. H. pylori infection increases the risk of chronic gastritis (CG), gastric ulcer (GU), duodenal ulcer (DU), and gastric cancer (GC). Nevertheless, the exact molecular action to the development of these adverse clinical outcomes remains not well-defined. Especially, in the East Asia, since the infection of cytotoxin-associated gene A (cagA) positive H. pylori is nearly 100%, their correlation to the different clinical outcomes could not be fully assessed [3‐5].
Most H. pylori strains (so-called type I strains) contain the cag pathogenicity island (cagPAI), a chromosomal region that includes about 37,000 bp and 28 genes [3, 4]. Genes encoded in the cagPAI allow H. pylori bacteria to translocate its major virulence protein cytotoxin associated gene A (CagA) into host gastric epithelial cells using a type-IV secretion system (TFSS) [5, 6]. The role of the H. pylori TFSS and CagA translocation was examined in previous sequential studies that showed Src-mediated phosphorylation of CagA tyrosines is important for H. pylori virulence [5, 7, 8]. In East Asia in particular, nearly all H. pylori infections are CagA positive, which complicates assessment of how clinical H. pylori isolates are associated with disease outcomes [9‐11]. Moreover, the mechanisms by which H. pylori expresses and regulates its TFSS injection apparatus when adapting to human epithelial cell receptors are unclear.
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A recent study identified integrin α5β1 expressed on gastric epithelial cells as the putative host receptor for H. pylori TFSS [12]. The H. pylori CagL protein was found to be an adhesion target on the injected pilus surface for binding to host integrin α5β1 through the CagL Arg-Gly-Asp (RGD) motif [13]. Initial CagL-integrin binding properly induced to locate the bacterial TFSS prior to CagA translocation as well as to activate host tyrosine kinase [12, 14]. This interaction between the H. pylori TFSS and host integrin α5β1 can activate the NF-kB proteins and several important pro-inflammatory cytokines that resulted in more adverse clinical outcomes, such as gastric carcinogenesis.
CagI is another H. pylori protein, but its function is less clear [12, 15]. CagI has no sequence similarities to any other TFSS components, or to other known proteins [16, 17]. Although an isogenic cagI mutant has been examined, there were conflicting reports about whether CagI is required for TFSS function [3, 18]. Based on H. pylori transcriptome evidence [19], cagI is certainly part of an operon containing cagPAI genes involved in the TFSS, but the actual contribution of CagI to clinical phenotypes is unknown.
Here, we used whole-genome sequencing (WGS) to analyze genetic variants of 43 H. pylori isolates from patients in Southeast Asia who had different clinical disease. Using the WGS data, we examined whether CagL and/or CagI amino acid changes (AACs) correlated with adverse clinical outcomes such as GC.
Results
Characteristics of clinical H. pylori isolates
We previously performed WGS on 19 H. pylori clinical isolates that we deposited under accession number DRA001250 (see “Methods”). Here we undertook WGS of 24 new clinical H. pylori isolates, and analyzed a total of 43 H. pylori whole genome sequences (Table 1). The 43 isolates were from 17 chronic gastritis (CG), 8 gastric ulcer (GU), 8 duodenal ulcer (DU) and 10 gastric cancer (GC) patients whose diagnosis was based on endoscopy results. The 43 H. pylori isolates we analyzed also had different geographic origins in that 31, 7, and 5 isolates were isolated from Japanese, Chinese, and Vietnamese patients, respectively.
Table 1
Characteristics of clinical H. pylori isolates and sequencing results
Strain
Diagnosis
Isolation
Total reads (before trimming)
Total reads (after trimming)
Total consensus length (bp)
Total consensus coverage (%)
Average coverage (fold)
Quality control
1
174
CG
Okinawa
3,004,008
3,003,954
1,534,120
91.98
165.1
Yes
2
177
CG
Okinawa
2,619,844
2,591,582
1,656,888
99.34
101.2
Yes
3
179
CG
Okinawa
2,200,712
2,190,051
1,518,618
91.05
123.6
Yes
4
189
CG
Okinawa
2,010,838
1,989,271
1,651,152
99.00
79.5
No
5
194
CG
Okinawa
6,936,714
6,929,125
1,553,487
93.14
407.5
No
6
S1
GU
Kobe
2,271,944
2,271,917
1,534,031
91.98
144.7
Yes
7
S2
CG
Kobe
5,962,628
5,962,520
1,542,099
92.46
333.4
Yes
8
S4
CG
Kobe
4,640,490
4,640,409
1,536,143
92.10
255.4
Yes
9
S8
CG
Kobe
10,869,516
10,869,208
1,563,806
93.76
669.4
Yes
10
S13
CG
Kobe
3,873,394
3,873,195
1,526,445
91.52
258.2
Yes
11
S16
CG
Kobe
6,782,392
6,782,004
1,555,964
93.29
437.9
Yes
12
S17
GC
Kobe
5,700,038
5,699,682
1,554,277
93.19
318.4
Yes
13
S22
GU
Kobe
6,597,254
6,596,851
1,557,252
93.37
416.5
Yes
14
S23
CG
Kobe
5,064,464
5,040,754
1,514,281
90.79
340.9
Yes
15
S26
CG
Kobe
6,692,038
6,691,708
1,543,795
92.56
432.7
Yes
16
F21
GC
Fukui
3,757,854
3,745,628
1,511,942
90.65
263.0
Yes
17
F23
CG
Fukui
5,765,092
5,733,420
1,514,387
90.80
387.6
Yes
18
F24
DU
Fukui
4,672,544
4,648,960
1,527,146
91.56
323.5
Yes
19
F28
DU
Fukui
7,184,480
7,122,238
1,537,656
92.19
365.6
Yes
20
F32
GC
Fukui
5,230,564
5,200,093
1,531,123
91.80
268.2
Yes
21
F44
DU
Fukui
3,887,082
3,846,981
1,663,639
99.75
135.5
Yes
22
F51
DU
Fukui
5,849,012
5,816,015
1,503,522
90.15
276.7
No
23
F52
DU
Fukui
4,431,904
4,412,519
1,531,591
91.83
310.2
Yes
24
F57
GC
Fukui
6,401,956
6,362,361
1,526,712
91.54
405.4
Yes
25
F65
CG
Fukui
4,248,748
4,202,979
1,664,897
99.82
164.9
Yes
26
F75
GU
Fukui
5,186,826
5,148,984
1,528,778
91.66
330.2
Yes
27
F79
GU
Fukui
3,036,516
3,036,231
1,521,709
91.24
185.2
Yes
28
F94
GU
Fukui
3,267,624
3,247,625
1,512,244
90.67
220.0
Yes
29
F214
GU
Fukui
5,329,874
5,285,297
1,533,936
91.97
341.5
Yes
30
F215
GU
Fukui
4,441,566
4,411,519
1,535,097
92.04
293.0
Yes
31
F229
GU
Fukui
4,130,184
4,105,896
1,514,484
90.80
281.8
Yes
32
HZ2
CG
Hang Zhou
3,953,756
3,926,283
1,511,585
90.63
261.8
Yes
33
HZ11
CG
Hang Zhou
5,990,624
5,972,745
1,517,201
90.97
422.0
Yes
34
HZ21
GC
Hang Zhou
6,697,176
6,676,385
1,530,926
91.79
406.4
Yes
35
HZ34
CG
Hang Zhou
6,245,816
6,203,622
1,547,241
92.77
374.0
Yes
36
HZ53
DU
Hang Zhou
6,631,022
6,593,830
1,556,114
93.30
402.0
Yes
37
HZ67
GC
Hang Zhou
3,398,652
3,388,149
1,526,962
91.55
245.5
Yes
38
HZ82
CG
Hang Zhou
3,984,240
3,968,256
1,532,373
91.88
276.1
Yes
39
VN8
GC
Ho Chi Minh
5,876,658
5,850,475
1,537,665
92.19
403.1
Yes
40
VN17
GC
Ho Chi Minh
6,810,950
6,786,447
1,520,811
91.18
465.3
Yes
41
VN19
GC
Ho Chi Minh
4,071,510
4,060,588
1,521,512
91.23
284.2
Yes
42
VN24
GC
Ho Chi Minh
4,241,646
4,226,765
1,533,944
91.97
294.1
Yes
43
VN27
GC
Ho Chi Minh
4,315,490
4,298,304
1,547,804
92.80
283.4
Yes
Okinawa is an island southwest of the main island of Japan. Kobe and Fukui are located on the main island of Japan. Hang Zhou is located in eastern China and Ho Chi Minh is in southern Vietnam. The 24 strains shown in italics were newly sequenced in this research
CG chronic gastritis, GU gastric ulcer, DU duodenal ulcer, GC gastric cancer
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Sequence reads mapping to ATCC 26695 and quality check
The total reads for the 43 H. pylori isolates ranged from 1.99 to 10.87 million (Table 1). Sequencing data were mapped to the genome of the H. pylori strain ATCC 26695, which was isolated from CG patients, as a reference. Total consensus length (bp) ranged from 1,503,522 to 1,664,897, and total consensus coverage (%) ranged from 90.15 to 99.82%. Average coverage (fold) ranged from 79.5 to 669.4-fold.
Following the initial quality check, we focused on the 28 genes in the cagPAI region (Additional file 1: Table S1). Among the 43 isolates, strain ID 189 had lower coverage (under 100-fold) in the cagPAI region, strain ID 194 had no genes in the cagPAI region, and strain ID F51 carried the cagA gene alone. Due to these major sequence differences in the cagPAI region, we excluded data for these three isolates, which were all from Japanese patients, such that 40 clinical H. pylori isolates were subjected to further analysis. Of these 40 isolates, 15, 8, 7, and 10 were from CG, GU, DU, and GC patients, respectively, and 28, 7, and 5 isolates were derived from Japanese, Chinese, and Vietnamese patients, respectively. CagA motifs of 40 clinical isolates were different (Additional file 2: Table S2).
After the quality check, the average coverage of the remaining 40 isolates ranged from 99.6- to 361.4-fold for cagL, and from 105.4- to 416.3-fold for cagI (nearly over 100-fold). Consistent with our earlier report, the WGS data in this study had high sequencing coverage, and were of sufficiently high quality to allow detection of SNVs in the H. pylori genome [20].
CagL variants in patients with different clinical disease outcomes
We translated the CagL nucleotide sequences into amino acid sequences (residues 1–237) with Genomics Workbench 8.5.1, and analyzed CagL variants based on clinical disease outcomes. Table 2 lists CagL variants, and the partial alignments of CagL amino acid changes (AACs) and their locations are shown in Fig. 1. In particular, we characterized AACs present in 10 clinical H. pylori isolates derived from GC patients.
Table 2
The number of CagL variants in GC and non-GC isolates
Residue
Reference
Variant
GC (n = 10)
(%)
non-GC (n = 30)
(%)
p value (GC versus non-GC)
4
Leu
Phe
1
10
2
6.7
NS
19
Met
Va1
1
10
0
0
NS
28
Lys
Arg
0
0
2
6.7
NS
32
Ser
Arg
0
0
2
6.7
NS
Asn
2
20
0
0
NS
35
Gln
Lys
10
100
29
96.7
NS
41
Val
Ala
2
20
0
0
NS
54
Pro
Ser
0
0
1
3.3
NS
58
Asn
Asp
8
80
30
100
NS
Tyr
1
10
0
0
NS
59
Glu
Lys
2
20
25
83.3
0.001
Asn
1
10
1
3.3
NS
60
Met
Ile
9
90
30
100
NS
Thr
1
10
0
0
NS
62
Glu
Lys
8
80
30
100
NS
Gln
1
10
0
0
NS
65
Ala
Ser
0
0
6
20
NS
78
Asp
Asn
0
0
1
3.3
NS
88
Ala
Thr
3
30
0
0
<0.05
98
Val
Met
0
0
2
6.7
NS
101
Lys
Asn
3
30
0
0
<0.05
108
Glu
Asp
1
10
1
3.3
NS
122
Asn
Lys
9
90
28
93.3
NS
Glu
0
0
1
3.3
NS
141
Gly
Ala
5
50
0
0
<0.001
142
Lys
Glu
5
50
0
0
<0.001
144
Lys
Gln
1
10
0
0
NS
154
Glu
Asp
0
0
2
6.7
NS
158
Thr
Ala
1
10
1
3.3
NS
162
Ala
Thr
0
0
1
3.3
NS
167
Ile
Val
0
0
1
3.3
NS
171
Ala
Thr
0
0
1
3.3
NS
181
Val
Ile
2
20
0
0
NS
201
Asn
Asp
3
30
24
80
<0.05
203
Val
Ile
1
10
3
10
NS
210
Glu
Lys
8
80
27
90
NS
216
Arg
Ile
10
100
28
93.3
NS
221
Ser
Asn
2
20
4
13.3
NS
223
Arg
Gln
0
0
1
3.3
NS
234
Ile
Val
3
30
23
76.7
<0.05
Statistical analysis was performed by Fisher’s exact test. Residue 234 is a part of the C-terminal motif in the distal six amino acids of CagL
GC gastric cancer, NS not significant
Fig. 1
Partial alignment of CagL sequences from 40 isolates from patients with different clinical outcomes. A partial alignment of CagL sequences (aa 1–150 and 201–237) is shown. The 40 clinical isolates included 15 from chronic gastritis (CG), 8 from gastric ulcer (GU), 7 from duodenal ulcer (DU) and 10 from gastric cancer (GC) patients. The amino acid sequence of the H. pylori reference strain ATCC 26695 is shown on the top line. Tyr58, Glu59, RGD motifs (76–78), Ala141, Glu142, Asn201, and C-terminal motifs of Ser-Lys-Ile-Ile-Val-Lys (232–237) are marked in grey blocks. Sequences of 10 isolates from GC patients are indicated in red
More recently, the CagL variants Tyr58 and/or Glu59 (CagL-Y58E59) was found to occur at significantly higher rates in H. pylori isolates from Taiwanese GC patients. CagL-Tyr58Glu59 can induce higher integrin α5β1 expression levels in the upper stomach and increase inflammation in the corpus [21]. Consistent with this report, we found that CagL-Glu59 occurred at a significantly (p < 0.001) higher rate (7/10, 70.0%) in H. pylori isolates from GC patients compared to that for 30 H. pylori isolates from non-GC patients (4/30, 13.3%). Shown in Table 3, this association between CagL-Glu59 and clinical outcome was still significant with the exception of 5 Vietnamese isolates from GC patients (p < 0.05). The remaining 26 isolates from non-GC patients had Lys59 (K59), and all 15 isolates from DU and GU patients had the CagL-Lys59 variant. In contrast, the reference H. pylori strain ATCC 26695 carried CagL-Glu59.
Table 3
Seven variants of CagL in GC and non-GC isolates without 5 Vietnamese isolates
Residue
Reference
Variant
GC (n = 5)
(%)
non-GC (n = 30)
(%)
p value (GC versus non-GC)
59
Glu
Lys
1
20.0
25
83.3
<0.05
88
Ala
Thr
1
20.0
0
0.0
NS
101
Lys
0
0
0
0
ND
141
Gly
0
0
0
0
ND
142
Lys
0
0
0
0
ND
201
Asn
Asp
3
60.0
24
80.0
NS
234
Ile
Val
2
40.0
23
76.7
NS
Statistical analysis was performed by Fisher’s exact test. Residue 234 is a part of the C-terminal motif in the distal six amino acids of CagL
GC gastric cancer, NS not significant, ND not determined
Meanwhile, CagL-Tyr58 was present in only one isolate (HZ67) from a GC patient, and its frequency was not significant. Aspartic acid was the most commonly present amino acid at position 58 (Asp58), and occurred in 38 of 40 isolates (95.0%). The remaining isolate (F32) had CagL-Asn58, as did the reference strain ATCC 26695. Only one isolate (HZ67) among the 43 tested had a CagL sequence with both Tyr58 and Glu59.
The C-terminal motifs that include the most distal amino acids of both CagL and CagI are functionally important for the TFSS [22]. In CagL, the sequence of this motif is Ser-Lys-Ile-Ile-Val-Lys (232–237). In this study, we found that Ile234 occurred at a significantly (p = 0.018) higher rate in GC patients (7/10 isolates, 70.0%) relative to that for non-GC patients (23.3%, 7/30). However, Ile234 lost significance with the exception of 5 Vietnamese isolates from GC patients (Table 3). The other five residues showed no significant amino acid sequence differences among the disease outcomes tested.
For CagL, Ala141 and Glu142 variants occurred in all 5 isolates isolated from Vietnamese GC patients. Asp201 had a significantly (p = 0.006) lower frequency in GC patients (3/10 isolates, 30.0%) compared to that for isolates from non-GC patients (24/30, 80.0%). However, Ala141, Glu142, and Asp201 variants lost significance when 5 Vietnamese isolates were excluded (Table 3). Notably, the Arg-Gly-Asp (RGD) motif was well conserved in 39 of 40 isolates (97.5%), but there were no significant differences among disease outcomes.
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CagI variants in patients with different clinical disease outcomes
We also translated CagI nucleotide sequences into amino acid sequences (1–381), and analyzed rates and locations of CagI variants based on clinical disease outcomes (Table 4; Fig. 2).
Table 4
The number of CagI variants in GC and non-GC isolates
Residue
Reference
Variant
GC (n = 10)
(%)
non-GC (n = 30)
(%)
p value (GC versus non-GC)
1
Val
Glu
1
10
0
0
NS
2
Lys
Met
1
10
0
0
NS
3
Cys
Phe
1
10
0
0
NS
Tyr
0
0
1
3.3
NS
5
Leu
Lys
1
10
0
0
NS
6
Ser
His
1
10
0
0
NS
7
Ile
Val
0
0
1
3.3
NS
10
Phe
Phe
0
0
1
3.3
NS
15
Gly
Ser
0
0
1
3.3
NS
17
Ser
Phe
9
90
28
93.3
NS
21
Thr
Thr
3
30
7
23.3
NS
22
Glu
Gly
1
10
8
26.7
NS
23
Val
Ala
8
80
26
86.7
NS
29
Pro
Ser
9
90
28
93.3
NS
36
Ala
Val
0
0
1
3.3
NS
40
Ala
Val
0
0
1
3.3
NS
57
Ser
Asn
1
10
2
6.7
NS
65
Ala
Val
1
10
1
3.3
NS
70
Glu
Gln
5
50
25
83.3
NS
78
Met
Ile
9
90
27
90
NS
94
Gly
Ser
10
100
30
100
NS
102
Gly
Ala
0
0
1
3.3
NS
109
Val
Ile
2
20
12
40
NS
125
Lys
Asn
9
90
27
90
NS
152
Ile
Met
7
70
19
63.3
NS
165
Glu
Gln
1
10
0
0
NS
179
Thr
Ala
1
10
0
0
NS
182
Glu
Ala
0
0
1
3.3
NS
190
Ser
Asn
0
0
1
3.3
NS
195
Ala
Thr
0
0
4
13.3
NS
196
Gln
Lys
8
80
26
86.7
NS
203
Ile
Val
1
10
0
0
NS
207
Ala
Thr
0
0
1
3.3
NS
213
Lys
Glu
1
10
1
3.3
NS
214
Gly
Asp
0
0
1
3.3
NS
221
Val
Ala
3
30
3
10
NS
222
Ala
Thr
0
0
1
3.3
NS
238
Ala
Asp
3
30
9
30
NS
Thr
0
0
1
3.3
NS
243
Ala
Thr
0
0
1
3.3
NS
Val
0
0
1
3.3
NS
246
Ala
Glu
5
50
19
63.3
NS
Val
0
0
1
3.3
NS
262
Ile
Val
2
20
14
46.7
NS
294
Met
Lys
9
90
30
100
NS
304
Ser
Asn
9
90
30
100
NS
319
Gly
Glu
9
90
30
100
NS
346
Asn
Ser
0
0
1
3.3
NS
368
Thr
Ala
0
0
1
3.3
NS
Met
1
10
0
0
NS
Statistical analysis was performed by Fisher’s exact test
GC gastric cancer, NS not significant
Fig. 2
Partial alignment of CagI sequences from 40 isolates from patients with different clinical outcomes. A partial alignment of CagI sequences (aa 101–120, 221–270, and 361–380) is shown. The 40 clinical isolates included 15 from chronic gastritis (CG), 8 from gastric ulcer (GU), 7 from duodenal ulcer (DU), and 10 from gastric cancer (GC) patients. The amino acid sequence of the H. pylori reference strain ATCC 26695 is shown on the top line. Val109, Ile262, and the Ser-Lys-Val-Ile-Val-Lys (376–381) C-terminal motif are marked by grey blocks. The results for 10 isolates from GC patients are indicated in red
As with CagL, the C-terminal motif of Ser-Lys-Val-Ile-Val-Lys (376–381) in CagI is functionally essential for the TFSS. In our analysis, all 40 H. pylori isolates from both GC and non-GC patients had the same motif, which had a completely conserved sequence.
Valine at CagI amino acid residue 109 (Val109) was frequent in H. pylori isolates from both GC patients (8/10, 80.0%) and non-GC patients (18/30, 60.0%). Isoleucine at position 262 (Ile262) was similarly frequent in GC patients (8/10, 80%) and non-GC patients (16/30, 53.3%), and the difference in rates was not significant. There were no other AACs associated with clinical outcome in the CagI sequence.
Phylogenetic implications of H. pylori CagL and CagI diversity
Phylogenetic trees were conducted using MEGA7 [23]. In general, CagL sequences showed no characteristic clusters around disease outcomes (Fig. 3a), although there was a cluster among the five Vietnamese isolates (Fig. 3b). Meanwhile, CagI sequences had no characteristic clusters for either region or disease outcome (Fig. 3c, d).
Fig. 3
Phylogenetic tree of 40 clinical isolates based on CagL and CagI sequences. Neighbor-Joining tree analysis of concatenated CagL (a, b) and CagI (c, d) sequences for 40 isolates is shown. Each Neighbor-Joining method tree was made using MEGA7 software. Open square, open circle, filled circle, and filled triangle symbols correspond to isolates derived from gastric cancer patients, Japanese isolates, Chinese isolates, and Vietnamese isolates, respectively
Using the advantages provided by whole-genome sequencing (WGS), we analyzed candidate and novel variants of CagL and CagI proteins in 40 clinical H. pylori isolates from patients in Southeast Asia. We showed that CagL from H. pylori isolates derived from GC patients carried several specific amino acid changes (AACs), but we detected no significant changes in the CagI amino acid sequence.
Whole-genome sequencing technology was recently applied to clarify the pathogenicity and evolution of H. pylori, as well as to identify its virulence factors [24, 25]. Using WGS, we and others detected potential mutations throughout the H. pylori genome and identified variants when sequence changes were present [20, 24, 25]. Here, we used WGS technology to detect novel variants in uncharacterized cagPAI genes associated with H. pylori pathogenicity.
cagPAI is a 37 kb segment of H. pylori DNA that contains 28 genes [3, 4], and is found in about 60% of Western isolates, whereas nearly all East Asian isolates are cagPAI positive [26]. We analyzed cagPAI integrity and showed the rearrangement of this island in three Japanese isolates (189, 194 and F51). Although cagPAI was most intact in Japanese isolates, it was disrupted in isolates isolated throughout the world [27]. Since the pathogenic role of the cagPAI is well defined as a whole or in part, we excluded the three isolates that had cagPAI rearrangements.
Several Cag proteins have been detected as constituents of the H. pylori cag TFSS apparatus and have important roles in CagA translocation [14, 15, 22]. The CagL and CagI proteins have been previously characterized [16, 17], so in this study we used WGS to screen 40 clinical H. pylori isolates for CagL and CagI variants, and analyzed the relationship between amino acid sequence and clinical outcomes. Consistent with a previous report [21], we detected complete RGD motifs in CagL sequences from all isolates. These data highlight the importance of the RGD motif for CagL function in the TFSS. We also checked whether other AACs in CagL and CagI were correlated with clinical outcomes.
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We further confirmed that the frequency of the candidate variant CagL-Glu59 in GC patients significantly differed from that seen for isolates from non-GC patients. This association of Glu59 was still significant with the exception of 5 Vietnamese isolates, which was the half of all GC isolates (5/10). However, the frequency of Tyr58 was not significantly different, which is in contrast to a previous study that showed the CagL-Tyr58Glu59 variants were more common in H. pylori isolates from GC patients [21]. CagL-Tyr58Glu59 variants have strong binding affinity for integrin α5β1 and also promote increased expression of this integrin, and significantly enhances CagA translocation and phosphorylation relative to wild type CagL [28]. However, these results contrasted with those shown by Tegtmeyer et al. [29]. Our data support the importance of CagL-Glu59 variant, and imply that Glu59 could be incorporated into strategies to screen clinical H. pylori isolates. However, the current study is rather small and limited to the patients in Southeast Asia. These results require validation with larger isolates in Southeast Asia and the other samples in Western countries.
The C-terminal motif in CagL and CagI consisting of six amino acids (Ser-Lys-Ile-Ile-Val-Lys, and Ser-Lys-Val-Ile-Val-Lys, respectively) is important for TFSS function [22]. However, whether these sequences were conserved among genomes of clinical H. pylori isolates was unclear. Here, we showed that the CagI C-terminal motif was completely conserved. Although the CagL C-terminal motif was also well conserved, we found a significant difference at position 234 of CagL among H. pylori isolates derived from GC and non-GC patients. However, Ile234 lost significance with the exception of 5 Vietnamese isolates from GC patients. Future studies on additional H. pylori isolates could validate whether CagL-Ile234 could serve as a marker that indicates an increased risk for gastric carcinogenesis.
Conclusions
We analyzed genetic variants of H. pylori using WGS, which has significant advantages over other approaches that examine only a fraction of the genome at any one time. WGS identified several putative novel variants of CagL and CagI sequences from previously uncharacterized H. pylori isolates. These variants, particularly in CagL-Glu59, have the possible effect on the TFSS activity and the relevance with clinical outcomes.
Methods
H. pylori samples
Forty-three H. pylori clinical isolates were obtained from gastric epithelium biopsy tissues taken during upper gastroduodenal endoscopy procedures performed at Okinawa Prefectural Chubu Hospital, Kobe University Hospital, and Fukui University Hospital in Japan, as well as Zhejiang University Hospital in China and Cho Ray Hospital in Vietnam. All patients gave written informed consent for use of their samples in this study, which was performed according to the principles of the Declaration of Helsinki. The major reference strain, ATCC 26695 (NC_000915), was isolated from CG patients in the United Kingdom [30], and its sequence served as the reference sequence.
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H. pylori culture
Gastric biopsy specimens were first inoculated onto trypticase soy agar II (TSA-II)-5% sheep blood plates (Becton, Dickinson and Company: BD) and cultured under microaerophilic conditions (O2 5%; CO2 5%; N2 90%) at 37 °C for 3–5 days. Then, one colony was picked from each primary culture plate, and seeded onto a Columbia Helicobacter pylori agar plate containing vancomycin (10 mg/l), trimethoprim (5 mg/l), amphotericin B (5 mg/l), and polymyxin B (2500 units/l), and cultured under the same conditions. A colony was picked from this second plate, seeded onto a TSA-II plate, and cultured under the same conditions. Several colonies were picked from the third plate, transferred into Brucella Broth medium (2 ml) containing 10% fetal calf serum, and cultured for 18 h under the same conditions.
A portion of each culture was stored at −80 °C in 0.01 M phosphate-buffered saline (PBS), pH 7.4, containing 20% glycerol. H. pylori DNA was extracted from bacterial pellets prepared from liquid cultures using the protease–phenol–chloroform method. The extracted DNA was suspended in 100 μl distilled water and stored at 4 °C.
Whole-genome sequencing (WGS)
Total DNA of H. pylori isolated from patients and the reference strain ATCC 26695 were sequenced. The bacterial DNA concentration of each sample was measured with a Qubit dsDNA HS assay kit (Q32851; Invitrogen, Carlsbad, CA) and the concentration of each sample was between 250 and 320 pg/μl.
A DNA library of H. pylori isolates was prepared using a Nextera XT DNA Sample Prep Kit (Illumina, Carlsbad, CA), which was used according to the manufacturer’s instructions to uniformly shear the DNA into 500 bp fragments and add unique adapter sequences to the fragments. The resulting DNA library was run on a MiSeq sequencer (Illumina) with a reagent kit (300 cycle, paired-end). Fluorescence images were analyzed using MiSeq Control Software, and FASTQ-formatted sequence data were generated using MiSeq Reporter Analysis.
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Sequence read mapping and single nucleotide variant (SNV) detection
For the analyzed DNA sequence data, read qualities having a Q30 value above 80% were selected according to recommendations by Illumina. After a quality check and data trimming, the sequence reads were assembled with Genomics Workbench 8.5.1 (CLC bio, Aarhus, Denmark). The read mapping module was termed as CLC Assembly Cell 4.0, which was based on an uncompressed Suffix-Array representing the entire reference genome in a single data structure (White paper on CLC read mapper; October 10, 2012). Sequence reads were mapped against the ATCC 26695 genome (NC_000915) as a reference, and single nucleotide variants (SNVs) were identified with Fixed Ploidy Variant Detection modules with default parameters and minor modifications to the mapping algorithm. Variant detection of the software was set to 1.
To exclude false-positive variants that resulted from sequencing errors, we selected variants that were present in >90.0% of mapped reads with a minimum coverage of 100. Insertions, deletions, and successive multi nucleotide variants were also excluded due to the previously reported complexity involved in detecting true variants [18].
Phylogenetic analysis
We constructed a phylogenetic tree from CagL and CagI sequences of H. pylori isolates using Molecular Evolutionary Genetics Analysis version 7.0 (MEGA7) [23]. Evolutionary history was inferred using the Neighbor-Joining tree [31]. Trees were drawn to scale, wherein branch lengths are shown in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The analysis involved 40 isolates, and the CagL and CagI sequences included 237 and 381 amino acids, respectively.
Statistical analysis
Differences in the number of amino acid changes (AAC) in CagL and CagI in clinical outcomes and regions in Southeast Asia were compared using the Fisher’s exact test. A difference associated with a p value <0.05 was considered to be significant. The SPSS statistical software package version 23.0.0.0 (SPSS, Inc., Chicago, IL) was used for all statistical analyses.
Nucleotide sequence accession number
Sequence reads of 19 Japanese clinical isolates and ATCC 26695 were previously deposited in the DNA Data Bank of Japan Sequence Read Archive (http://www.ddbj.nig.ac.jp/index-e.html) under accession number DRA001250. Sequence reads of 5 Vietnamese clinical isolates were deposited under accession number DRA002946, whereas 7 Chinese isolates and an additional 12 Japanese isolates were deposited under DRA004713.
Authors’ contributions
HO, TT, AI, and RO conceived and designed the research. HO, AI, RO, and KY collected samples and performed experiments. HO, TT, AI, and RO analyzed the data and prepared figures, interpreted results of the experiments, and drafted the manuscript. HO and TT edited the manuscript. SN, MY, and TA supervised the study. All authors read and approved the final manuscript.
Acknowledgements
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its additional files. Sequence reads for the 43 H. pylori clinical isolates and ATCC 26695 were deposited in the DNA Data Bank of Japan Sequence Read Archive (http://www.ddbj.nig.ac.jp/index-e.html).
Funding
This study was supported by a Grant-in-Aid (No. 26460212) to T. T., a Grant-in-Aid (No. 15H06404) to A. I., a Grant-in-Aid for Young Scientists (B) (No. 15K19092) to K. Y., and a Grant-in-Aid (No. 16H05835) to T. A. from the Japan Society for the Promotion of Science (JSPS).
Publisher’s Note
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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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