Helicobacter pylori evolution and phenotypic diversification in a changing host

Helicobacter pylori colonizes the stomachs of more than 50% of the world's population, making it one of the most successful of all human pathogens. One striking characteristic of H. pylori biology is its remarkable allelic diversity and genetic variability. Not only does almost every infected person harbour their own individual H. pylori strain, but strains can undergo genetic alteration in vivo, driven by an elevated mutation rate and frequent intraspecific recombination. This genetic variability, which affects both housekeeping and virulence genes, has long been thought to contribute to host adaptation, and several recently published studies support this concept. We review the available knowledge relating to the genetic variation of H. pylori, with special emphasis on the changes that occur during chronic colonization, and argue that H. pylori uses mutation and recombination processes to adapt to its individual host by modifying molecules that interact with the host. Finally, we put forward the hypothesis that the lack of opportunity for intraspecies recombination as a result of the decreasing prevalence of H. pylori could accelerate its disappearance from Western populations.     

Genomic changes during chronic Helicobacter pylori infection

The gastric pathogen Helicobacter pylori shows tremendous genetic variability within human populations, both in gene content and at the sequence level. We investigated how this variability arises by comparing the genome content of 21 closely related pairs of isolates taken from the same patient at different time points. The comparisons were performed by hybridization with whole-genome DNA microarrays. All loci where microarrays indicated a genomic change were sequenced to confirm the events. The number of genomic changes was compared to the number of homologous replacement events without loss or gain of genes that we had previously determined by multilocus sequence analysis and mathematical modeling based on the sequence data. Our analysis showed that the great majority of genetic changes were due to homologous recombination, with 1/650 events leading to a net gain or loss of genes. These results suggest that adaptation of H. pylori to the host individual may principally occur through sequence changes rather than loss or gain of genes.     

Isogenic variation of Helicobacter pylori strain resulting in heteroresistant antibacterial phenotypes in a single host in vivo

BACKGROUND: Antibiotic-susceptible and -resistant Helicobacter pylori can be present simultaneously in the same host. The aim of this study was to evaluate the genomic diversity of H. pylori strains resulting in heteroresistant antibacterial phenotypes. MATERIALS AND METHODS: Twenty-one pairs of H. pylori strains isolated from the antrum and body displaying heteroresistant antibacterial phenotypes were included. We compared the genotypes of paired-isolates by random arbitrarily primed polymerase chain reaction (PCR), flagella gene PCR-based restriction fragment length polymorphism, and flaA gene sequencing. In metronidazole-heteroresistant isolates, the sequence variation of rdxA and frxA genes was analyzed using phylogenetic analysis. RESULTS: The DNA fingerprinting patterns of the paired isolates revealed that 12 pairs (57.1%) were identical, whereas one pair (3.8%) was different. The remaining eight pairs (38.1%) of isolates showed minor heterogenecity in fingerprinting patterns. In flaA gene sequencing, these identical and similar isolates showed close sequence similarity between the antrum and body, whereas different isolate showed 31 points of different nucleotide sequences. Phylogenetic analysis of the metronidazole-heteroresistant pairs showed consistent genetic relatedness of each paired isolates despite the sequence variation of the rdxA or frxA genes in five pairs (71.4%). CONCLUSIONS: These results suggest that continuing genomic diversities in the same strain may play an important role in modulating the antibiotic-heteroresistant H. pylori in vivo.     

Analysis of host responses of guinea pigs during Helicobacter pylori infection

Host responses of guinea pigs infected with Helicobacter pylori were investigated. Passaged H. pylori colonised the stomach for up to 13 weeks after infection, but after 1 month the number of bacteria fell sharply. Specific antibodies, predominantly of the IgG2 subtype, were present from week 3 onwards. Antibodies to urease A and flagella were abundant. Severe inflammation of the gastric mucosa and damage to the stomach epithelium was seen. Infiltrates of mononuclear cells and eosinophils were found near the parietal glands. As infection progressed, inflammation and tissue damage became more localised and more variable between individual animals. These parameters can be used as markers for colonisation of the stomach by H. pylori.     

No association between Helicobacter pylori genotypes and antibiotic resistance phenotypes within families

Background. Triple therapy combining a proton pump inhibitor with two antibiotics, e.g. clarythromycin (CLR), metronidazole (MTZ) or amoxicillin (AMX), represents the standard in Helicobacter pylori eradication regimens. Resistance to antimicrobial agents, particularly MTZ (up to 56% in Western countries) and CLR (up to 15% in southern Europe), is frequently observed and may be associated with treatment failure [ 1 ]. Recently, several studies indicated that individual H. pylori colonies from a single anatomic site may not always yield identical genotypes, or the identical patterns of susceptibility to antibiotics [ 2 - 5 ]. Representative for every single patient we analyzed 27 H. pylori antrum isolates for susceptibility to antimicrobial agents in order to test whether identical H. pylori genotypes exhibit a similar pattern of susceptibility to antibiotics. Methods. PCR, RELP, PFGE, antibiotic susceptibility testing. Results. H. pylori genotype and antibiotic susceptibility pattern in families do not segregrate. Conclusion. Molecular typing of H. pylori from family members does not predict antibiotic susceptibility pattern.     

Effect of host species on recG phenotypes in Helicobacter pylori and Escherichia coli

Recombination is a fundamental mechanism for the generation of genetic variation. Helicobacter pylori strains have different frequencies of intragenomic recombination, arising from deletions and duplications between DNA repeat sequences, as well as intergenomic recombination, facilitated by their natural competence. We identified a gene, hp1523, that influences recombination frequencies in this highly diverse bacterium and demonstrate its importance in maintaining genomic integrity by limiting recombination events. HP1523 shows homology to RecG, an ATP-dependent helicase that in Escherichia coli allows repair of damaged replication forks to proceed without recourse to potentially mutagenic recombination. Cross-species studies done show that hp1523 can complement E. coli recG mutants in trans to the same extent as E. coli recG can, indicating that hp1523 has recG function. The E. coli recG gene only partially complements the hp1523 mutation in H. pylori. Unlike other recG homologs, hp1523 is not involved in DNA repair in H. pylori, although it has the ability to repair DNA when expressed in E. coli. Therefore, host context appears critical in defining the function of recG. The fact that in E. coli recG phenotypes are not constant in other species indicates the diverse roles for conserved recombination genes in prokaryotic evolution.     

Circulating T-cell response to Helicobacter pylori infection in chronic gastritis

Background. Helicobacter pylori elicits a specific humoral and cellular immune response. There is increasing evidence that the type of T-cell response contributes to clinical outcome in H. pylori infection.
Materials and Methods. The host response to H. pylori infection in 34 subjects with chronic gastritis was examined in terms of T-cell proliferation and cytokine production in whole-blood cultures stimulated or unstimulated with H. pylori acid-glycine extract antigens (AGE).
Results. The proliferative response in whole-blood cultures was similar for both H. pylori –positive and –negative subjects stimulated with H. pylori AGE. While an increase in interferon-γ (IFN-γ) production was observed from both H. pylori –positive and –negative subjects with gastritis, significantly higher levels of IFN-γ were detected in the former when stimulated with H. pylori AGE. In contrast, interleukin 4 (IL-4) was undetectable regardless of antigen stimulation. However, if an in situ IL-4 antibody capture assay was used, antigen-independent production of IL-4 was detected, but there was no difference between H. pylori –positive and –negative subjects with gastritis. After eradication of H. pylori , antigen-induced production of IL-4 was increased, with no decrease in the levels of secretion of IFN-γ. IL-4 production was dependent on CD4+ T cells, as addition of anti-CD4 but not anti-CD8 mouse monoclonal antibody or matched IgG isotype to the whole-blood culture inhibited the production of IL-4.
Conclusion. The results suggest that a shift toward a balanced Th1-Th2 response due to an increase in antigen-induced IL-4 production from CD4+ T cells follows eradication. We suggest that the downregulation of mucosal inflammation consequent on reduction in antigen levels or removal of downregulation after eradication of H. pylori contributes to this shift in cytokine balance.     

Helicobacter pylori infection in children and adults: a single pathogen but a different pathology

Background. The aims of this retrospective study were to ascertain in large series of children and adults: the relationship of the infecting strain to gastric mucosal lesions; and the relationship of the infecting strain to its duodenal localization. Materials and Methods. We studied 307 and 604 consecutive children and adults. In gastric mucosal samples H. pylori was cultured, genotyped and histologically assessed, while inflammation, activity and intestinal metaplasia were graded. In a subset of 171 patients H. pylori ureaseA (ureA) and cagA genes were amplified (PCR) using mucosal biopsies from the duodenum. Results. H. pylori infection was diagnosed in 40 children and 308 adults. cagA was identified in 50% and 65.5% of infected children and adults. Antral activity was associated with the density of infecting bacteria (p < .001) and with cagA (p < .01). Intestinal metaplasia was correlated with cagA (p < .001). The ureA gene was found in 56 duodenal samples from 82 H. pylori positive patients. Duodenal H. pylori ureA was significantly more frequent in patients with duodenal diseases than in those without (p < .01), cagA positive strains being mainly involved in the infection of this anatomical area (p < .01). Conclusions. A severe H. pylori‐associated gastritis is more prevalent when the density of infecting bacteria is high and when cagA positive strains cause the infection. The most virulent cagA positive H. pylori colonizes not only the gastric, but also the duodenal mucosa, which can be directly damaged by the bacteria itself or by its products.     

Helicobacter pylori infection in patients with Hepatitis C Virus positive chronic liver diseases

BACKGROUND AND GOALS: One-third of patients with liver cirrhosis suffers from acute peptic ulcer, a disease strongly correlated with Helicobacter pylori (H. pylori) infection. We report the seroprevalence of antibodies to H. pylori in 179 patients with Hepatitis C Virus (HCV)-related chronic active hepatitis and cirrhosis. MATERIALS AND METHODS: Among patients, 135 (86 males and 49 females, mean age 51.2 +/- 13.28, range 27-77 years) had chronic active hepatitis (CAH) and 44 cirrhosis (28 males and 16 females, mean age 62.4 +/- 9.2, range 37-77 years). Serum antibodies to H. pylori were tested using a commercial enzyme immunosorbent assay. The control population consisted of 619 consecutive blood donors (523 males, 96 females, mean age 47 +/- 5.3 years, range 18-65). RESULTS: The overall prevalence of antibodies to H. pylori was 73.1% (131/179) among patients and 47% (291/619) among blood donors (p<0.0001; OR 3.08 [95%CI, 2.10-4.51]). 70.5% (24/34) of patients aged less than 40 years were seropositive for H. pylori versus 34.2% (90/263) of controls (p<0.0001; OR 4.61[95%CI, 2.0-10.85]). Among cirrhosis patients, the prevalence of antibodies to H. pylori was 79.5% (35/44) versus 47% (291/619) of controls (p<0.0001; OR 4.38 [95%CI, 1.98-9.98]). Overall seroprevalence among CAH patients was 71.1% (96/135) versus 47% (291/619) of blood donors (p<0.0001; OR 2.77 [95%CI, 1.82-4.24]). CONCLUSIONS: The high seroprevalence of antibodies to H. pylori in patients with HCV-positive liver diseases explains the elevated incidence of peptic ulcer, and warrants studies on the pathogenic role in human liver diseases of Helicobacter spp which is known to cause chronic hepatitis and hepatocellular carcinoma in mice.     

Interactions between Helicobacter pylori infection and host metabolic homeostasis: A comprehensive review

The microbiota actively and extensively participates in the regulation of human metabolism, playing a crucial role in the development of metabolic diseases. Helicobacter pylori (H. pylori), when colonizing gastric epithelial cells, not only induces local tissue inflammation or malignant transformation but also leads to systemic and partial changes in host metabolism. These shifts can be mediated through direct contact, toxic components, or indirect immune responses. Consequently, they influence various molecular metabolic events that impact nutritional status and iron absorption in the host. Unraveling the intricate and diverse molecular interaction links between H. pylori and human metabolism modulation is essential for understanding pathogenesis mechanisms and developing targeted treatments for related diseases. However, significant challenges persist in comprehensively understanding the complex association networks among H. pylori itself, the infected host's status, the host microbiome, and the immune response. Previous metabolomics research has indicated that H. pylori infection and eradication may selectively shape the metabolite and microbial profiles of gastric lesions. Yet, it remains largely unknown how these diverse metabolic pathways, including isovaleric acid, cholesterol, fatty acids, and phospholipids, specifically modulate gastric carcinogenesis or affect the host's serum metabolism, consequently leading to the development of metabolic-associated diseases. The direct contribution of H. pylori to metabolisms still lacks conclusive evidence. In this review, we summarize recent advances in clinical evidence highlighting associations between chronic H. pylori infection and metabolic diseases, as well as its potential molecular regulatory patterns.     

Emergence of recombinant strains of Helicobacter pylori during human infection

Genetic recombination can be important evolutionarily in speeding the adaptation of organisms to new environments and in purging deleterious mutations. Here, we describe polymerase chain reaction (PCR), hybridization and DNA sequence-based evidence of six such exchanges between two strains of Helicobacter pylori during natural mixed infection of a patient in Lithuania. One parent strain contained the 37 kb long, virulence-associated cag pathogenicity island (PAI), and the other strain lacked this PAI. Most H. pylori from the patient had descended from the cag ⁺ parent, but had become cag ⁻ during infection. This had resulted from transfer of DNA containing the 'empty site' allele from the cag ⁻ strain and homologous recombination, not from excision of the cag PAI without DNA transfer. Other cases of recombination involved genes for an outer membrane protein ( omp 5 and omp 29; also called HP0227 and HP1342) and a putative phosphoenolpyruvate synthase ( ppsA  ; HP0121). Replacement of a short patch of DNA sequence (36–124 bp) was also seen. As the chance of forming any given recombinant is small, the abundance of recombinants in this patient suggests selection for particular recombinant genotypes during years of chronic infection. We suggest that genetic exchange among unrelated H. pylori strains, as documented here, is important because of the diversity of this gastric pathogen and its human hosts. Certain H. pylori recombinants may grow better in a given host than either parent. The vigour of growth, in turn, could impact on the severity of disease that infection can elicit.     

Helicobacter pylori cholesteryl glucosides interfere with host membrane phase and affect type IV secretion system function during infection in AGS cells

Helicobacter pylori infection is an aetiological cause of gastric disorders worldwide. H. pylori has been shown to assimilate and convert host cholesterol into cholesteryl glucosides (CGs) by cholesterol-α-glucosyltransferase encoded by capJ. Here, we show that CapJ-deficient (ΔcapJ) H. pylori resulted in greatly reduced type IV secretion system (TFSS)-associated activities, including the hummingbird phenotype of AGS cells, IL-8 production, CagA translocation/phosphorylation and CagA-mediated signalling events. Complementation of the ΔcapJ mutation with wild type cagJ or by adding CGs-containing lysates or exogenous fluorophore-tagged CGs reversed the mutant phenotypes. We also show that the wild-type but not ΔcapJ H. pylori recruited raft-associated components to sites of bacterial attachment. Fluorescence recovery after photobleaching (FRAP) analysis of AGS cells treated with fluorescence-tagged cholesterol/CGs revealed that there was a higher proportion of CGs associated with immobile fractions. CGs-associated membranes were also more resistant to a cold detergent extraction. Thus, we propose that CGs synthesized by H. pylori around host-pathogen contact sites partition in detergent-resistant membranes (DRMs), alters lateral-phase segregation in membrane and reorganizes membrane architecture. These processes together promote the formation of a functional TFSS and H. pylori infection.     

Organ-specific autoantibodies in children with Helicobacter pylori infection

BACKGROUND: We compared the prevalence of organ-specific autoantibodies in a group of Helicobacter pylori infected children and a group of uninfected children and investigated the relationship between the presence of relevant autoantibodies and the status of the target organs. PATIENTS AND METHODS: One hundred and twenty-four children with dyspepsia (54 boys, 70 girls; mean age 10.5 years; range 4-19) underwent gastroscopy: 56 had H. pylori infection (31 girls, 25 boys), while 68 (37 girls and 31 boys), were H. pylori-negative. All sera were tested for the presence of: parietal cell autoantibodies (PCA), intrinsic factor autoantibodies (IFA), microsomial autoantibodies, thyroglobulin autoantibodies, islet cell autoantibodies, glutamic acid decarboxylase autoantibodies, adrenal cortex autoantibodies, steroid-producing cell autoantibodies; gastrin, pepsinogen A, pepsinogen C and anti-H. pylori antibodies. The histological features and the ureA and cagA genes were also considered. RESULTS: The frequency of organ-specific autoantibodies was higher in patients with H. pylori infection than in uninfected patients (chi2-test p < .0001). Specifically gastric autoantibodies were significantly higher: seven of the 56 H. pylori-positive children were PCA-positive and one was IFA-positive (chi2-test p = .0004). The presence of autoantibodies was not associated with any clinical or biohumoral signs of disease. CONCLUSIONS: Our study detected a relationship between H. pylori infection in childhood and the presence of organ-specific autoantibodies unassociated with any clinical or biohumoral signs of disease. Helicobacter pylori infection in childhood could trigger the onset of clinical autoimmune gastritis, and/or other clinical autoimmune diseases.     

Host use dynamics in a heterogeneous fitness landscape generates oscillations in host range and diversification

Colonization of novel hosts is thought to play an important role in parasite diversification, yet little consensus has been achieved about the macroevolutionary consequences of changes in host use. Here, we offer a mechanistic basis for the origins of parasite diversity by simulating lineages evolved in silico. We describe an individual‐based model in which (i) parasites undergo sexual reproduction limited by genetic proximity, (ii) hosts are uniformly distributed along a one‐dimensional resource gradient, and (iii) host use is determined by the interaction between the phenotype of the parasite and a heterogeneous fitness landscape. We found two main effects of host use on the evolution of a parasite lineage. First, the colonization of a novel host allowed parasites to explore new areas of the resource space, increasing phenotypic and genotypic variation. Second, hosts produced heterogeneity in the parasite fitness landscape, which led to reproductive isolation and therefore, speciation. As a validation of the model, we analyzed empirical data from Nymphalidae butterflies and their host plants. We then assessed the number of hosts used by parasite lineages and the diversity of resources they encompass. In both simulated and empirical systems, host diversity emerged as the main predictor of parasite species richness.     

Pathophysiological functions of the CagA oncoprotein during infection by Helicobacter pylori

Infection with Helicobacter pylori cagA-positive strains plays an essential role in the development of gastric carcinoma. This review summarizes the pathophysiological functions of the cagA gene product, CagA, particularly focusing on the molecular mechanisms underlying CagA translocation into the host cells as well as CagA-mediated deregulation of host cell signaling.     

Epithelial cell kinetics of the gastric mucosa during Helicobacter pylori infection

Helicobacter pylori is an important pathogen in major gastroduodenal diseases, including inflammation with ulceration and gastric malignancies. Alterations in H. pylori associated cell turnover in gastric epithelial cells are examined in relation to inflammatory activity, bacteria load and cytokines which may improve knowledge concerning the outcome of gastric diseases caused by H. pylori. Antral biopsies from 42 dyspeptic patients including 27 H. pylori-positive and 15 H. pylori-negative patients were tested for apoptotic activity by the TUNEL assay, and immuno-histochemically for p53 and the proliferative marker Ki-67. H. pylori infection, bacteria load and inflammatory activity were associated with increased cell turnover as judged by enhanced activities of TUNEL, p53 and Ki-67. Only p53 was significantly correlated to IFN-gamma, IL-8 and IL-10. The H. pylori-positive state was furthermore accompanied by varying degrees of altered distribution pattern of the markers studied, with occasional presence of apoptosis in the deeper pit zones, upward extension of Ki-67 and to a lesser degree of p53. Given a similar pattern of change in proliferation and apoptosis in some neoplastic lesions in other parts of the gastrointestinal tract, such studies in cell turnover may provide insights valuable in the investigations of potential precursors of gastric malignancies.     

Evaluation of therapeutic efficacy of adjuvant Helicobacter pylori whole cell sonicate in mice with chronic H. pylori infection

Successful prophylactic administration of Helicobacter pylori whole cell sonicate (WCS) plus complete Freund's adjuvant (CFA) or aluminum hydroxide (ALM) against subsequent H. pylori infection was reported recently. Here we tested the effect of WCS plus TiterMax Gold (TMX) or ALM in mice with chronic H. pylori infection. Mice with chronic (18 weeks) H. pylori infection were injected intraperitoneally with H. pylori (Sydney strain) WCS plus ALM or TMX once weekly for three times. The number of colonizing H. pylori in the stomach, IgG1 and IgG2a levels, and local inflammatory status were determined after therapeutic immunization. H. pylori specific IgG1, but not IgG2a, was significantly induced in mice immunized with H. pylori WCS plus TMX or ALM. Immunization did not result in reduction of bacterial count or recruiting inflammatory cells to the stomach. Adjuvant H. pylori WCS resulted in induction of CD4+ Th2 cell-mediated immunity although it did not reduce bacterial density in mice with chronic H. pylori infection. Our results implied that CD4+ Th1 cell-mediated immunity, rather than Th2 cell dominant immunity, might play a role in reducing the number of bacteria in chronic H. pylori infection.