A comparative whole genome comparison analysis of Helicobacter pylori from gastric cancer and gastritis in China setting

doi:10.21203/rs.3.rs-3964333/v1

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A comparative whole genome comparison analysis of Helicobacter pylori from gastric cancer and gastritis in China setting

https://doi.org/10.21203/rs.3.rs-3964333/v1

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Background: This study aimed to explore and compare the differences in the genomics and pathogenicity of Helicobacter pylori (H. pylori) strains derived from the gastric cancer (GC) and gastritis (GI) in the Chinese population.

Methods: We sequenced 12 H. pylori from GC and GI patients in china by whole genome sequencing. 20 H. pylori sequencing data from other regions of the world were obtained from the public platform as reference genes. Then, the evolutionary tree was drawn based on multi-omics, and the differences of virulence factors (VF) and gene function were analyzed

Results: In GC stains, the 1544-1640 coding genes, with a total length of 1,549,790-1,605,249 bp, were predicted. In GI stains, the 1552-1668 coding genes, with a total length of 1,552,426-1,665,981 bp, were identified. In addition, the average length of coding genes in GC and GI strain, was approximately 1594 (90.91%) and 1589 genes (90.81%), respectively. We found that the VFs predicted by the two cohort strains had high consistency, but their cagA status was significantly different. Additionally, the clustering results indicated that there were significant differences in core Single Nucleotide Polymorphism (SNP) between GC and GI strains, but no significant differences in homologous proteins and gene island prediction between the two strains. Subsequently, the results of pan-genomic and Average Nucleotide Identity (ANI) analyses suggested that GC, GI and other reference H. pylori strains had high homology consistency. Furthermore, the gene function annotation results suggested that the H. pylori strains of GC and GI also had high similarity in gene function, and their specific gene functions were mainly concentrated in the process of metabolism, transcription and repair.

Conclusions: GC and GI patient-derived H. pylori have some differences in VF and SNP, but they also have high homologous consistency at other level of the genome in Chinese population.

gastric cancer

gastritis

Helicobacter pylori

genome

Helicobacter pylori (H. pylori) is a highly successful gram-negative bacterium that chronically infects the human gastric mucosa, infecting an estimated 4.3 billion people worldwide in 2022 [1]. H. pylori infection is closely associated with gastritis (GI) and gastric cancer (GC), of which about 1–3% cases will develop gastric cancer [2, 3]. The prevalence of H. pylori infection is higher in developing countries than in developed countries [4]. At present, China is one of the countries with the highest H. pylori infection rate in the world, and with an infection rate of more than 50% [5]. Meanwhile, according to GLOBOCAN 2020, the age-standardized rate (ASR) of GC prevalence in China is as high as 15.8% [6]. However, there is minimal knowledge about the origin of H. pylori genomes and pathogenicity differences in patients with GI and GC in this region, which limits the application of molecular diagnostics, such as screening for early GC, prevention of precancerous lesions, and vaccine drug development.

Previously, several studies applied the first-generation sequencing method to initially explore and compare the genetic differences of H. pylori from GC and benign gastric diseases. In 2009, by comparing H. pylori from GC and benign gastric ulcers, it has been first found that highly differentiated alleles and strain specific genes may be useful biomarkers for analyzing the geographic distribution of H. pylori and identifying strains capable of inducing malignant or precancerous gastric lesions [7]. Subsequently, similar studies focused more on the differences in specific genes and virulence factors of the two strains, which could be used as biomarkers of GC risk, such as VacA and CagA genes [8, 9]. However, due to the limitation of sequencing technology, lack of reports on the specific genomic differences of H. pylori strains in GC and GI. In this study, we isolated and cultured H. pylori strains from patients with GC and GI (GC: n = 6, GI: n = 6), and applied whole genome sequencing methods to deeply explore and compare the possible differences in the genomics and pathogenicity of the two cohort strains in the Chinese population.

H. pylori isolates

Twelve strains of H. pylori were isolated from tissue samples of patients undergoing gastric surgery (open surgery and endoscopic resection) at Fudan University Shanghai Cancer Center (FUSCC) (Supplemental Table S1). Based on the diagnosis, the isolates were classified into GC (n = 6) and GI (n = 6) subgroup. With the consent of all patients, at least 2 pieces of tissue were taken from the margin of gastric benign lesions or cancer tissue for bacterial culture and pathological examination. Two experienced clinical pathologists performed pathological checks on gastric tissue specimens. This study was approved by the FUSCC review board in accordance with Chinese bioethical regulations, with written informed consent from all subjects (Ethics Approval Number: FUSCC-D-2022-144). In addition, genome sequences of 20 H. pylori reference strains were downloaded from GenBank, and the detailed information is provided in Supplemental Table S2.

Genomic DNA extraction and sequencing

The DNA of H. pylori isolates were extracted using a bacterial genomic DNA extraction kit (Beyotime Biotechnology Co., Ltd., China) according to the manufacturer’s instructions [10]. DNA content and purity were determined using a qubit fluorometer and a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientifc, Carlsbad, USA). DNA content and purity were determined using a qubit fluorometer and a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientifc, Carlsbad, USA). Whole-genome sequence (WGS) of H. pylori strains were sequenced at the Shanghai PersonalBio Technology Company (SPTC, Shanghai) using the Illumina NovaSeq 6000 platform.

Data preparation and genome assembly

Using fastp (version 0.20.0, https://github.com/OpenGene/fastp) software for filtering the raw sequencing data (The sequencing junctions and primers in Reads were removed; Constant quality value ≤ 5 bases were removed; Reads containing 5% of N-containing bases were deleted. Remove repeated contamination), and then obtain clean reads [11]. Next, the data was assembled using the default parameters of the Unicycler (v0.4.8) software and the genome sequence was subsequently obtained [12].

Genomic structural and functional annotation

Structural annotation of the genome sequence was performed using prokka(v 1.12) software. After obtaining the gene set of sequenced strains, the genes are compared and annotated through multiple databases to find the gene function and related description information. The amino acid sequences of each strain gene were compared with each database to obtain the corresponding functional annotation information and selected three databases for general functional analysis: Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG), and Gene Ontology (GO). Additionally, Pathogen virulence factor database (VFDB) was used for pathogenicity.

Comparative genomic analysis

OrthoFinder (v2.3.5) software was used to compare the predicted protein sequences of 32 strains of H. pylori and identify homologous gene clusters.

Then, the basic situation of core and non-core genes was analyzed, and the differences within species were studied from the point of view of specific gene sequences [13]. The single-copy homologous genes identified by OrthoFinder were aligned using MAFFT (v 7.427). Meanwhile, snippy (v4.6.0) and gubbins (v2.4.1) were used to identify the core SNPs, and then we applied FastTree (2.1.10) and iTols for construct the phylogenetic tree.

Average Nucleotide Identity (ANI) is an index that compares the relationship between two genomes at the nucleotide level. It can calculate the similarity or evolutionary distance between different genomes for species classification and kinship comparison. Using FastANI version 1.31 software, ANI analysis was performed on 32 sorted strains in pairings, and sorted into matrix form, and then visualized. Pan-genome is a general term for all genes of a species, reflecting all genetic information at the gene level of the species [14].

Statistical analyses

Statistical analyses were performed using SPSS software version 19.0 for windows. Differences between the groups were compared using Chi-square or Pearson Chi-square tests, Fisher’s exact method, or Kruskal-Wallis analysis according to the data type and the number of comparisons. Statistical significance were considered clinically significant at a p value < 0.05. All P-values were two-sided. When the results of Pearson Chi-square tests showed a statistical difference, the intra-group pairwise comparison was adjusted using the Bonferroni formula.

Clinical characteristics of patient information and strains isolation

Between 1 March 2021 and 31 October 2022, thirty-nine GC and GI patients were hospitalized in our department for surgery and twenty patients tested with H pylori positive. Next, H pylori isolate from each patient (from ten single colony isolates stored for each patient) was selected and sub-cultured. Finally, twelve H pylori isolates were selected through 3-step randomization procedure and were analyzed with WGS (Fig. 1). Of these patients, six underwent gastrectomy for GC and six underwent Endoscopic submucosal dissection (ESD) for GI, respectively. The average age of the patients was 46.2 years; 37–69 years range. Seventy-five percent (8/12) of the patients were male (Table S1).

The whole genome of H. pylori strains were sequenced and analyzed using Illumina NovaSeq sequencing platforms in this study. Table 1 presents the general characteristics for each of the 12 genome sequences. There were 24–43 contigs with a high (310×-1040×) genome coverage. The mean genome size was 1.57 Mb and the mean G + C content was 38.8%. Each genome contains between 1544 and 1688 annotated CDS with an average ~ 91% of the genome is used for the coding region.

Table 1

Genome statistics of the whole genome sequences of the 12 *H pylori* isolates
Strain ID	Disease	Genome coverage	No. of contigs	Genome size	No. of CDS	Coding percentage	G + C percentage	rRNA	tRNA
GI-01	GI	830	35	1540791	1552	90.89	38.91	2	36
GI-02	GI	1040	24	1538881	1553	91.19	38.89	2	36
GI-03	GI	310	43	1665981	1688	90.14	38.61	2	36
GI-04	GI	1020	36	1563078	1578	90.92	38.80	2	36
GI-05	GI	800	36	1555888	1598	90.57	38.73	2	36
GI-06	GI	610	36	1552426	1565	91.17	38.86	2	36
GC-01	GC	580	27	1591503	1607	91.09	38.72	2	36
GC-02	GC	760	30	1549790	1544	91.16	38.85	2	36
GC-03	GC	960	40	1560160	1590	90.63	38.88	2	36
GC-04	GC	490	40	1573950	1598	90.90	38.88	2	36
GC-05	GC	1000	28	1597182	1583	90.80	38.75	2	36
GC-06	GC	800	33	1605249	1640	90.87	38.70	2	36
CDS, Coding DNA Sequence. GI, Gastritis. GC, Gastric Cancer.

Circle diagram of the chromosome of H. pylori isolates strain

The genome sequence of H. pylori in GC strains was composed of a circular chromosome with an average total length of 1,579,639 bp and G + C average ratio of 38.8% (Table 1 and Fig. 2A). In six GC stains of H. pylori, the 1544–1640 coding genes, with a total length of 1,549,790-1,605,249 bp, were predicted in the genome of H. pylori GC strains, which accounted for 90.63%-91.16% of the total genome. In GI stains of H. pylori (Fig. 2B), the 1552–1668 coding genes, with a total length of 1,552,426-1,665,981 bp, were predicted in the genome of six H. pylori GI strains, which accounted for 90.14%-91.19% of the total genome. In addition, the average length of coding genes in GC and GI strain, was approximately 1594 (90.91%) and 1589 genes (90.81%), respectively.

Based on the VFDB (Virulence Factors of Pathogenic Bacteria) database, these virulence genes were divided into 7 VF (virulence factor) classes including acid resistance, adherence, immune evasion, immune modulator, motility and so forth. In detail, each VF class contained several kinds of VFs and the related genes as shown in Table 2. We compared the VFs of H. pylori derived from GC and GI, and found that the VFs predicted by the two cohort strains had a high consistency, but there were also obvious differences. For example, cagA, a well-known virulence factor related gene, was found in GC strains compared to those derived from GI.

Table 2

Comparison of virulence factor prediction of H. pylori from GC and GI.
Class	Virulence factors	Related gene-GC	Related gene-GI
Acid resistance	Urease	UreA/E/F/G/H/I	UreA/B/E/F/G/H/I
Adherence	AlpB (hopB)	alpB/hopB	-
	H. pylori adhesin A	hpaA	hpaA
	HopZ	hopZ	hopZ
	HorB	horB	horB
	Sialic acid binding adhesins	sabA/hopP	-
	Adherence-associated lipoprotein AlpA (hopC)	alpA/hopC	-
Immune evasion	Lipopolysaccharide Lewis antigens	futB/C	futC
Immune modulator	Neutrophil-activating protein (HP-NAP)	napA	napA
Motility	Flagella	flaA/B/G, flagA/B/C/D/E1/E2/G2/H/I/K, flhA/B1/B2/F, fliA/D/E/F/G/H/I/L/M/N/P/Q/R/Y,pflA	flaA/B/G, flagA/B/C/D/E1/E2/G1/G2/H/I/K/L, flhA/B1/B2, fliA/E/F/G/H/I/L/M/N/P/Q/R/S/Y, motA/B, pflA
Secretion system	Cag PAI type IV secretion system	Cag1/3/4/5/C/D/E/F/G/H/I/L/M/N/P/Q/S/T/U/V/W/X/Z, virB11	Cag1/3/4/5/C/D/E/F/G/H/I/L/M/N/P/Q/S/T/U/V/W/X/Z, virB11
Secretion system	T4SS effectors cytotoxin-associated gene A	-	cagA
Toxin	Vacuolating cytotoxin	vacA	vacA
GC, Gastric Cancer. GI, Gastritis.

Phylogenetic tree of the 12 H. pylori isolates and the reference genomes

Our H. pylori isolates genomes were assessed for their phylogenetic relationships using core genome SNPs with 20 public reference genomes (Table S1). The core genome consisted of were separated into four distinct population according to the area within reference genomes. 30.0% (6/20) of the isolates belonged to Asia, 30.0% (6/20) of the isolates to America, 30.0% (6/20) of the isolates to Europe, and 10.0% (2/20) of the isolates to Australia.

The phylogenomics of our H. pylori strains were analyzed according to the flow diagram in Figure S1. Pangenome analysis using orthoFinder identified 2492 genes among 32 H. pylori strains, which included 1054 conserved genes (42.4%) present in all (100%) of all strains (Supplementary Table S3). We used snippy to identify SNPs of all species using CP003904.1_Helicobacter_pylori_26695 (1,667,892 bp) as a reference sequence. A total of 206,091 core SNPs were identified. Gubbins were then used to reassemble 188,039 SNPs, and finally Fasttree was used to construct core SNP evolutionary tree. Additionally, ModelFinder selected the best fit model of substitution according to the relationship of grouping and cross-validation errors to build the phylogenetic tree (Figure S2). The phylogeny of 20 strains is shown in Fig. 3 and Figure S3 and represented four H. pylori populations: HP-Asia (Japan-1, Japan-2, India-1, India-2, Bangladesh-1, and Bangladesh-2), HP-America (United states-1, United states-2, Mexico-1, Mexico-2, Brazil-1, and Brazil-2), HP-Europe (France-1, France-2, Belgium-1, Belgium-2, Spain-1, and Spain-2), and HP-Oceania (Australia-1, and Australia-2). According to the clustering with reference strains, our strains (GC and GI strains) were assigned to HP-Asia population, especially with high homology with Japanese strains (Fig. 3A). Subsequently, we also mapped the evolutionary tree of strains based on homologous proteins, and the results also suggested the homology of our strains with Asian strains, especially Japanese strains (Fig. 3B). At the same time, the clustering results indicated that there were significant differences in core SNP between GC and GI strains, but no significant differences in homologous proteins between the two cohort strains.

Synteny in genome organization of the 12 H. pylori isolates and the reference strains

Next, to illustrate the utility of IslandCompare, we performed an analysis for prediction, comparison and exploratory visualization of gene islands of GC, GI, and the reference of H. pylori strains. The gene-island prediction visualization features an interactive linear display covering each genome (Fig. 4). Gene islands are uniformly colored according to their sequence clusters, with the highlighted colors highlighting similar gene islands across all genomes. Through the results of gene island prediction, the location of each gene island, the relationship between each other and the possible evolution sequence of all strains in the cluster were visualized. The prediction results of H. pylori strains for GC and GI based on gene islands showed that the locations of gene islands for 91.7% (11/12) strains were concentrated in 1-1.4Mb and were mainly divided into two parts. Four of the strains (GC-05, GC-06, GI-02, and GI-05) were clustered in the upper half of the region, adjacent to GI-03 and India-1. Five other strains (GC-01, GC-02, GI-01, GI-04, and GI-06) were clustered in the lower half of the region, adjacent to other strains of Asian origin (Japan-2, India-2, Bangladesh-1, and Bangladesh-1) and GC-03, GC-04.

Genome homology and difference of H. pylori in GC and GI patients

We used FastANI software to perform pair-to-pair ANI analysis on 32 sorted H. pylori strains, and visualized them. In Fig. 5A, the results of ANI analysis suggested that the GC and GI strains studied in our study had high homology similarity with strains of Asian origin (Japan-2, India-1, India-2, Bangladesh-1, and Bangladesh-2). In this study, H. pylori strains of GC and GI shared 1103 genes, 160 genes specific to GC and 144 genes specific to GI (Fig. 5B).

The pan-genomic analysis of GC, GI, and reference strains showed that all strains had a large number of common genetic components (1215 core genes shared), while GC and GI strains had fewer specific genes, ranging from 2–8 (Figure S4A). GO functional annotation found that the specific genes of GC and GI were mainly concentrated in cellular anatomical entity, binding, cellular process, metabolic process (Fig. 4C and Figure S4B).

At the same time, COG analysis found that the specific gene functions of GC and GI strains were mainly concentrated in metabolism, translation, biogenesis, replication, recombination, and repair (Figure S4C-D). In addition, KEGG analysis results in Fig. 4D indicated that the top four functions of specific genes of GC strains were nucleotide metabolism, amino acid metabolism, metabolism of cofactors and vitamins. The top functions of the specific genes of GC strain were metabolism of cofactors and vitamins, translation, amino acid metabolism, replication and repair (Figure S4E).

H. pylori has a complex and long-term coexistence relationship with humans, and is closely related to the occurrence of GI and GC [15, 16]. Whether the pathogenicity of H. pylori has population, source, gene specificity, and whether there is a specific highly pathogenic strain has been a research hotspot in the field [17, 18]. Hence, it is critical to identify the key factors of strain pathogenicity and to understand the regional highly pathogenic strains. Here, we sequenced 12 H pylori isolates of derived from GC or GI patients and performed whole genome–based comparative analysis to investigate and understand the genetic structure and genetic differences between GC and GI strains.

East Asia has always been a high incidence area of GI and GC, and H. pylori is highly correlated with the occurrence of GI and GC [19, 20]. In this study, 12 H. pylori strains from GI and GC patients in China were selected as research objects, and it is representative to explore the pathogenicity and genetic differences of the two cohort strains. Meanwhile, we selected strains representative of other regions of the world from public databases as references [15]. Therefore, the results of our study can reflect the homology and difference between the strains of GI and GC, and with other reference strains to a certain extent.

In the present study, we used next-generation sequencing technology to sequence the whole genome of H. pylori strains that were isolated from GI and GC patients in Shanghai (China). The genome sequence of GC strain H. pylori consists of a ring chromosome with an average total length of 1,579,639 bp and average coding genes sequence (CDS) of 1594. Simultaneously, the average genome size and number of CDS in GI strains was lower than that of GC group (size: 1,569,508 bp, CDS: 1589). Previous studies have reported that the whole genome sequence size of Helicobacter pylori is about 1.5–1.9 Mb, and the coding genes are about 1600 [21, 22]. Additionally, VFs of H. pylori from GC and GI, although with high consistency. However, cagA gene sequence was found in GC strains compared with GI strains, which may indicate higher virulence and aggressiveness of GC strains. H. pylori strains with cagA-positive are associated with acute GI, peptic ulcer and GC [23]. First reported in 1995, infection with cagA-positive strains increases the risk of stomach cancer by at least an order of magnitude more than with cagA-negative strains [24].

In this study, the results of SNP evolutionary tree first displayed that the 12 strains from GC and GI patients in china, were not uniform in their H. pylori lineage. The intersections of GC and GI H. pylori lineages sequenced in this study are consistent with the co-evolution of H. pylori lineages in Japan, especially strains from GI patients. In addition, phylogenetic tree based on homologous proteins and visualization of gene island prediction did not distinguish H. pylori strains from GC and GI. The observation of two distinct lineages has been also reported previously. Previous genome-wide association studies of strains of hp-East Asia by Japanese scholars have shown that differences in SNPs between strains of GC and duodenal ulcer can be detected, and potential pathogenic mechanisms such as charge changes in ligand-binding pockets, changes in subunit interactions, and pattern switching DNA methylation have been proposed [25]. Yamaoka et al found that virulence genes in the genomes of CHC155 (GC) and VN1291 (Duodenal ulcer) strains are a key and risk factor for H. pylori pathogenicity [26]. Another study based on comparing GC and duodenal ulcer H. pylori infection suggests that vacA genotype status will help to identify patients at high risk for GC development [8]. The comparison results of strains VF described above in this study also found that the cagA status of H. pylori strains in GC and GI was different, which may be an important genomic feature of the two cohort strains.

Subsequently, the results of pan-genomic and ANI analyses suggested that GC, GI and other reference H. pylori strains had high homology consistency. This is similar to previous research results. H. pylori lineages are related to geographical regions and can be divided into 7 lineages: HP-Africa1, HP-Africa2, HP-Sahul, HP-Europe, HP-Asia2, HP-Amerind and HP-East Asia [15]. But the expectation is that these coexisting lineages will converge over time due to the plasticity of strains at the genomic level [16]. In addition to genome homology, the gene function annotation results of COG, GO and KEGG further suggested that the H. pylori strains of GC and GI also had high similarity in gene function, and their specific gene functions were mainly concentrated in the process of metabolism, transcription and repair. Previously, the gene annotation for other regions strains were similar to our results [10, 27].

There are certain limitations to our study. First of all, due to the limited sample size in the study design and the regional nature of the samples, the obtained research results need to be further confirmed by large sample size and multi-center studies. Secondly, the reference genome in the study only selected a few representative strains from each continent, which may also have a certain selection bias. Thirdly, the differences between VF and SNP in strains of GC and GI have not been further explored, nor have relevant basic experiments been designed for verification, and more detailed answers need to be provided in future studies.

In conclusion, GC and GI patient-derived H. pylori have some differences in VF and SNP, but they also have high homologous consistency at other level of the genome in Chinese population.

VacA Vacuolating cytotoxin A

CagA Cytotoxin-associated gene A

Acknowledgments: We thank Dr. He Teng for statistical advising and review of the manuscript.

Ethics approval and consent to participate: The study was reviewed and approved by Ethics Committee of Fudan University Shanghai Cancer Center Review Board [Approval No. FUSCC-D-2022-144]. All study participants or their legal guardian provided informed written consent about personal and medical data collection prior to study enrolment.

Consent for publication: Not applicable.

Availability of data and materials: The original data related to 16S rRNA sequencing in this study were uploaded in the attachment

Competing interests: The authors declare no conflicts of interest for this article.

Funding: This work was supported by the Natural Science Foundation of China under grants 81972213.

Authors' contributions: Xu DZ and Kong PF designed the research study; Xu DZ, Kong PF, Yan YH, Duan YT, Fang YT, Dou Y and Xu YH performed the research; Kong PF, Yan YH, and Fang YT analyzed the data and wrote the manuscript; Xu DZ made the final review and revision of the manuscript; all authors have read and approve the final manuscript.

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No competing interests reported.

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A comparative whole genome comparison analysis of Helicobacter pylori from gastric cancer and gastritis in China setting

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Version 1

Abstract

Figures

Background

Methods

H. pylori isolates

Genomic DNA extraction and sequencing

Data preparation and genome assembly

Genomic structural and functional annotation

Comparative genomic analysis

Statistical analyses

Results

Clinical characteristics of patient information and strains isolation

Circle diagram of the chromosome of H. pylori isolates strain

Phylogenetic tree of the 12 H. pylori isolates and the reference genomes

Synteny in genome organization of the 12 H. pylori isolates and the reference strains

Genome homology and difference of H. pylori in GC and GI patients

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1