Exploration Of The Association Of Polymorphisms In Helicobacter Pylori CagA, VacA And Outer Membrane Protein Homa, Homb With The H.pylori Related Disease

Background and objective:The outcome of H.pylori infection is diverse, which could be asymptomatic, orincrease the incidence of chronic gastritis, peptic ulcer, gastric cancer and gastricmucosa-associated lymphoid tissue (MALT) lymphoma. The result of H.pyloriinfection is mainly influenced by three factors which included bacteria, host andenvironment. Furthermore，the bacteria play the major role in the pathopoiesis ofH.pylori infection, and polymorphism of virulence gene play a significant role in thevirulence of the bacteria. Among them, the cytotoxin associated protein gene of H.pylori (cagA) and vacuolating cytotoxin A gene (vacA) are the two of the mostimportant virulence genes. Additionally, previous studies had found that H.pyloriouter membrane protein HomB also play a key role in the pathopoiesis of H.pyloriinfection. In China, most H.pylori strains have vacA and cagA genes, and it is veryhard to simply use their expression states to determine their virulence and therelationship with diseases. Recent studies indicated that polymorphisms in signalregion, intermediate region of open reading frame and intermediate region of vacAgene, and CagA EPIYA motif polymorphism were closed H.pylori related diseaseoutcomes. Other researches indicated that HomA and HomB participate in thepathogenesis of H.pylori related disease, but the results were inconsistent, which maydue to their gene polymorphisms or the geographical differences. Herein,we isolate170H. pylori clinical strains from different H. pylori related disease and the analysedthe gene expression of cagA, vacA, homA and homB. Moreover, the genotyping ofCagA EPIYA motif and vacA, and full-length sequencing of homB were alsoperformed to analyze these three genes and their relationship between the genepolymorphisms with H. pylori related disease.Methods:1.DNA manipulation of H.pylori HomA and HomB genes.(1)170clinical H.pylori strains were resuscitated and cultured, then genomicDNAs were extracted. (2) The genomic DNAs were diluted.(3) Designing primers according to different middle region of homA and homBwas conducted to detect homA and homB states.(4) PCR amplification of homB (161bp) and homA (128bp) to obtain homA andhomB positive strains.2.Analysis of H.pylori homB sequence characteristics, CagA (EPIYA) andtypes of VacA (s/i/m)(1) Designing multi-pairs specific full-length sequencing primers for homB,cagA and vacA.(2) Amplification of target genes was performed, and the products weresequenced in two directions.(3) Using bioinformatics software, such as Primer5.0, MEGA, AlignX andrelated database to analyze and align DNA sequences and amino acid sequences, andthen analysis of association between hom B and two virulence genes’ polymorphismswith clinical disease and pathology.3. Statistical analysisEnumeration data were quantifed by chi-square test or Fisher’s exact test. A pvalue of less than0.05was accepted as statistically signifcant. Data were analyzedusing SPSS statistical software package version17.0.Results:1. Analysis of characteristics of cagA carboxyl-terminal variable regionamino acid sequence (EPIYA):For170clinical H.pylori strains,100%of them have cagA gene. CagA3’endvariable region full-length sequences were obtained by sequencing, and the aminoacid sequences suggested that the C-terminal variable region was starting with5or6poly-aspartic acid NNNNNN, ending with sequence LSKVG. There were0-4EPIYAmotifs in them, and4strains contained4EPIYA motifs, including two strains ofgastric cancer, and2strains containing2EPIYA motifs were all chronic gastritisstrains.161strains containing3EPIYA motifs and3strains without EPIYA motifswere no significant correlation with diseases. All H.pylori isolates can be divided into3sequence types, including AB type (2EPIYA motifs), ABD type (3EPIYA motifs) and AABD type (4EPIYA motifs), all of which are oriental type(TIDD). In this study,all strains were identified as TIDD.We further analyzed EPIYA motif polymorphisms and found2strains withEPIYA-A mutation were from chronic gastritis.2/9strains with EPIYA-B mutationswere from gastric cancer, and7/9were from duodenal ulcer. These resultsdemonstrated that the EPIYA-B mutated strains had stronger virulence2. VacA genotyping results:The vacA gene of170H.pylori strains were amplified by PCR method. ExceptNO.152strain which did not express i1and i2, the phenotype of other169strains iss1a/m2/i1.Herein, s1phenotype is virulent, and s2type is not virulent and virulencein m1type is stronger than m2type. Moreover, i1type is virulent and virulence of i2type is weak or absent, all of above results suggest that vacA genotypes of H.pyloristrains from Jiangxi region are often conservative, and most are moderately strongvirulent strains. To some extent, different diseases caused by H.pylori may be due toplurality virulence factors.3. H.pylori homA and B gene expression（1） The distribution of homAand homB in clinical disease①170H.pylori strains were isolated from clinical patients, of them28fromgastric cancer,19from gastric ulcer,75from duodenal ulcer, and48from gastritispatients.②HomA single positive rate was14.7%(25/170), and its positive rates inH.pylori from gastric cancer, gastric ulcer, duodenal ulcer and gastritis samples were22.2%(6/27),20.0%(4/20),13.3%(10/75),10.4%(5/48), respectively. There was nosignificant difference within each group (p>0.05).③HomB single positive rate was70%(119/170), and its positive rates inH.pylori from gastric ulcer, duodenal ulcer and gastritis patients were74.1％（20/27）,75.0％（15/20）,68.0%(51/75),68.8%(33/48), respectively. There was no significantdifference within each group (p>0.05).④HomA and homB double-positive rate was15.3%(26/170), which were3.7％（1/27）,5.0％（1/20）,18.7%(14/75),20.8%(10/48) in strains isolated fromgastric cancer, gastric ulcer, duodenal ulcer and gastritis samples. Further analysis showed that there was a statistical difference between gastric cancer group andgastritis group (p=0.044), while no significant difference existed within other groups.（2） The distribution of homAand homB in clinical pathology①170H.pylori strains were isolated from the clinical samples, of which90strains (duodenal ulcers were not performed pathological examination) had clinicaland pathological test results. Of them,42strains were from chronic superficialgastritis,21from intestinal metaplasia and dysplasia, and the rest27were fromgastric cancer.②HomA single positive rate was14.4%(13/90), and its positive rates were9.5%(4/42),14.3%(3/21),22.2%(6/27) in isolates from chronic superficial gastritis,intestinal metaplasia and dysplasia, gastric cancer patients. There was no significantdifference within each group (p>0.05).③HomB single positive rate was68.9%(62/90), and its positive rates were64.3%(27/42),71.4%(15/21),74.1%(20/27) in isolates from chronic superficialgastritis, intestinal metaplasia and dysplasia, gastric cancer samples. There was nosignificant difference within each group (p>0.05).④HomA and homB double-positive rate was16.7%(15/90), which were26.2%(11/42),14.3%(3/21),3.7%(1/27) in H.pyloris from chronic superficialgastritis, intestinal metaplasia and dysplasia, gastric cancer. Base on the results ofstatistical analysis, there was a siginificant difference in three pathology groups (p=0.047), and also between chronic superficial gastritis group and gastric cancer group(p=0.016), while no significant difference existed within other groups.1. The sequencing of homB gene and alignment analysis resultsAfter optimizing PCR and sequencing conditions for three times,59full-lengthsequences were obtained ultimately from145homB gene positive strains. Amongthem, the sequencing success rate of gastric cancer (9.5%) was significantly lowerthan the other three groups (50.0%~66.7%)(p<0.05).To our knowledge, the mainreason for failure of sequencing was that the senior structure and repeats may causecombination or heterozygous when sequencing, and the low sequencing success rateof homB from gastric cancer strains indicated that they are more complex senior structure or multiple repeats in gartric cancer, which may be one of reasons whystrains isolated from gastric cancer have stronger virulence.After analysis these sequences by using MEGA5.0and AlignX softwares,alignment with standard sequences showed that variety in entire homB sequences wasvery complex, and polymorphism in100-1600gene locus was more abundant.However, the relatively small changes were existed in latter gene sequence, but entiresequence was less conservative. Moreover, these59homB gene sequences showed noapparent diseases aggregation, which may be due to the failure of sequencing causedby complex structure and abundant polymorphism in gene sequence.Alignment and cluster analysis were conducted between homB gene sequencesamplificated from strains in Jiangxi region and sequences from14standard strainsby MEGA software, and we found the mutation are widely existed in strains fromdifferent regions. There was a siginificant difference between eastern and westernnational standard strains, and also display a siginificant difference in strains fromJiangxi region and standard strains of east Asian countries (Japan and Korea).Meanwhile, there was not obvious regional aggregation between strains in Jiangxiregion and standard strains in Japan or Korea, furthermore, distribution of standardstrains from western countries was more discrete.Conclusion:1. CagA gene positive rate in Jiangxi region was100%which was significantlyhigher than other regions, and all the cagA gene types are East Asian type.2. H.pylori pathogenicity enhanced with the number of CagA EPIYA motifs.And the virulence of strains with EPIYA-B mutation was stronger than strains withEPIYA-A mutantion and non-mutantion.3. VacA genes of H.pylori in Jiangxi region were all expressed as s1a/m2/i1typeexcept only one strain which didn’t have i-type expression, and virulence of isolatesfrom different diseases has no significant difference.4. HomA gene positive rate of H.pylori from Jiangxi region was lower thanhomB gene, and homB positive rate was much higher than that of Western countries.5. HomA and homB single positive rate was no significant difference withindifferent diseases, but homA and homB double positive rate in gastric cancer strains was significantly lower than that in chronic gastritis strains. Furthermore, sequencingfailure proportion of homB from gastric cancer strains was significantly higher thanother three kinds of diseases, suggesting homB gene of gastric cancer strains mayoccupied complex senior structure and multi-repetitive sequences.