Helicobacter pylori is a gastric pathogen that colonizes approximately 50% of the world's population. Infection with H. pylori causes chronic inflammation and significantly increases the risk of developing duodenal and gastric ulcer disease and gastric cancer. Infection with H. pylori is the strongest known risk factor for gastric cancer, which is the second leading cause of cancer-related deaths worldwide. Once H. pylori colonizes the gastric environment, it persists for the lifetime of the host, suggesting that the host immune response is ineffective in clearing this bacterium. In this review, we discuss the host immune response and examine other host factors that increase the pathogenic potential of this bacterium, including host polymorphisms, alterations to the apical-junctional complex, and the effects of environmental factors. In addition to host effects and responses, H. pylori strains are genetically diverse. We discuss the main virulence determinants in H. pylori strains and the correlation between these and the diverse clinical outcomes following H. pylori infection. Since H. pylori inhibits the gastric epithelium of half of the world, it is crucial that we continue to gain understanding of host and microbial factors that increase the risk of developing more severe clinical outcomes.

Helicobacter pylori and Gastric Cancer: Factors That Modulate Disease Risk

A high-molecular-mass (120- to 128-kDa) Helicobacter pylori antigen has been associated with peptic ulcer disease. We created a bank of 40,000 random chromosomal fragments of H. pylori 84-183 by using lambda ZapII. Screening of this bank in Escherichia coli XL1-Blue with absorbed serum from an H. pylori-infected person permitted the isolation and purification of a clone with a 3.5-kb insert. Subcloning of this insert (pMC3) permitted the expression of a recombinant H. pylori protein that had a mass of approximately 96 kDa and that was recognized by the human serum. Sera that were obtained from H. pylori-infected persons and that recognized the native 120- to 128-kDa H. pylori antigen recognized the recombinant 96-kDa pMC3 protein to a significantly greater extent than did sera that did not recognize the native H. pylori antigen. All 19 H. pylori isolates producing the 120- to 128-kDa antigen hybridized with pMC3; none of 13 nonproducers did so (P < 0.001). Because all 15 isolates producing the vacuolating cytotoxin hybridized with pMC3, we called the gene cagA (cytotoxin-associated gene). Sequence analysis of pMC3 identified an open reading frame of 859 amino acids, without a termination codon. Parallel screening of a lambda gt11 library with human serum revealed positive plaques with identical 0.6-kb inserts and sequences matching the sequence of the downstream region of pMC3. To clone the full-length gene, we used the 0.6-kb fragment as a probe and isolated a clone with a 2.7-kb insert from the lambda ZapII genomic library. Nucleotide sequencing of this insert (pYB 2) revealed a 785-bp sequence that overlapped the downstream region of pMC3. Translation of the complete nucleotide sequence of cagA revealed an open reading frame of 1,181 amino acids yielding a protein of 131,517 daltons. There was no significant homology with any previously reported protein sequence. These findings indicate the cloning and characterization of a high-molecular-mass H. pylori antigen potentially associated with virulence and with cytotoxin production.

https://iai.asm.org/content/iai/61/5/1799.full.pdf

Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: evidence of linkage to cytotoxin production.

Since Helicobacter pylori was first cultivated from human gastric biopsy specimens in 1982, it has become apparent that many related species can often be found colonizing the mucosal surfaces of humans and other animals. These other Helicobacter species can be broadly grouped according to whether they colonize the gastric or enterohepatic niche. Gastric Helicobacter species are widely distributed in mammalian hosts and are often nearly universally prevalent. In many cases they cause an inflammatory response resembling that seen with H. pylori in humans. Although usually not pathogenic in their natural host, these organisms serve as models of human disease. Enterohepatic Helicobacter species are an equally diverse group of organisms that have been identified in the intestinal tract and the liver of humans, other mammals, and birds. In many cases they have been linked with inflammation or malignant transformation in immunocompetent hosts and with more severe clinical disease in immunocompromised humans and animals. The purpose of this review is to describe these other Helicobacter species, characterize their role in the pathogenesis of gastrointestinal and enterohepatic disease, and discuss their implications for our understanding of H. pylori infection in humans.

https://cmr.asm.org/content/cmr/14/1/59.full.pdf

Emergence of Diverse Helicobacter Species in the Pathogenesis of Gastric and Enterohepatic Diseases

The two complete genomic sequences of Helicobacter pylori J99 and 26695 were used to compare the paralogous families (related genes within one genome, likely to have related function) of genes predicted to encode outer membrane proteins which were present in each strain. We identified five paralogous gene families ranging in size from 3 to 33 members; two of these families contained members specific for either H. pylori J99 or H. pylori 26695. Most orthologous protein pairs (equivalent genes between two genomes, same function) shared considerable identity between the two strains. The unusual set of outer membrane proteins and the specialized outer membrane may be a reflection of the adaptation of H. pylori to the unique gastric environment where it is found. One subfamily of proteins, which contains both channel-forming and adhesin molecules, is extremely highly related at the sequence level and has likely arisen due to ancestral gene duplication. In addition, the largest paralogous family contained two essentially identical pairs of genes in both strains. The presence and genomic organization of these two pairs of duplicated genes were analyzed in a panel of independent H. pylori isolates. While one pair was present in every strain examined, one allele of the other pair appeared partially deleted in several isolates. Helicobacter pylori is a gram-negative bacterial pathogen, and almost 50% of the world’s population, approaching 100% in some countries, is infected (43). Infection with H. pylori has been associated with chronic gastritis and other severe gastroduodenal diseases such as peptic and gastric ulcers, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma (14, 29, 35). Several molecular techniques suggest that independent H. pylori isolates exhibit extensive genetic diversity (2, 3, 5, 8, 24, 25, 31, 42, 58‐60) which has been predicted to be important in pathogenesis, possibly relating to the wide variation in patient symptomology. Comparison of two completely sequenced H. pylori isolates, 26695 and J99, showed considerable allelic diversity at the nucleotide level between the gene coding sequences (4). Further, the comparison demonstrated that the chromosomes of these two strains were organized differently in a limited number of discrete regions but the overall gene order was more similar than would have been expected (4). The gram-negative bacterial outer membrane is an asymmetric bilayer with phospholipids in the inner monolayer and the bulky glycolipid lipopolysaccharide (LPS) in the outer monolayer. Outer membranes constitute a semipermeable, size-dependent permeability barrier representing an effective barrier to hydrolytic enzymes, detergents, dyes, and hydrophobic antimicrobials. Channel-forming proteins, termed porins, also determine the permeability properties of the outer membrane. Porins contain transmembrane diffusion channels that allow small hydrophilic molecules, nutrients, and even small antibiotics to passively diffuse across the outer membrane. Most bacterial species possess only a modest number of different porins that constitute the most abundant species in the outer membrane. Many porins are nonselective and limit substrate diffusion mainly by size, whereas others have been shown to possess a high degree of selectivity for specific substrates (12, 40, 57). The primary amino acid sequences of porins from different bacterial species generally exhibit little sequence similarity, although all are characterized by a series of amphipathic amino acid sequence motifs (alternating hydrophilic and hydrophobic residues) that form the antiparallel b-sheet structures of the membrane-spanning core region (the b barrel). These b strands are connected on the periplasmic side by short amino acid loops and on the external side of the porin by longer loops. The external loops can then fold back into the core of the b

https://iai.asm.org/content/iai/68/7/4155.full.pdf

Comparative Genomics of Helicobacter pylori: Analysis of the Outer Membrane Protein Families

Pathogenic bacteria frequently express surface proteins with affinity for components of the mammalian extracellular matrix, i.e. collagens, laminin, fibronectin or proteoglycans. This review summarizes our current knowledge on the mechanisms of bacterial adherence to extracellular matrices and on the biological significance of these interactions. The best-characterized bacterial proteins active in these interactions are the mycobacterial fibronectin-binding proteins, the fibronectin- and the collagen-binding proteins of staphylococci and streptococci, specific enterobacterial fimbrial types, as well as the polymeric surface proteins YadA of yersinias and the A-protein of Aeromonas. Some of these bacterial proteins are highly specific for an extracellular matrix protein, some are multifunctional and express binding activities towards a number of target proteins. The interactions can be based on a protein-protein or on a protein-carbohydrate interaction, or on a bridging mechanism mediated by a bivalent soluble target protein. Many of the interactions have also been demonstrated on tissue sections or in vivo, and adherence to the extracellular matrix has been shown to promote bacterial colonization of damaged tissues.

Bacterial proteins binding to the mammalian extracellular matrix

Summary

Flagellar hooks were purified from Helicobacter pylori and Helicobacter mustelae. The 70 × 16nm H. pylori hook was composed of FIgE subunits of 78kDa, while the 72 × 16nm H. mustelae hook was composed of 87kDa subunits. N-terminal sequence was obtained for the FIgH proteins of both species, and for an internal H. mustelae FlgE peptide. Degenerate oligonucleotide primers allowed amplification of a 1.2 kb fragment from the H. mustelae chromosome, which carried part of the flgE gene. The corresponding H. pylori gene was cloned by immunoscreening of a genomic library constructed in λZAP Express, The translated H. pylori flgE sequence indicated a protein with limited homology with the hook proteins from Salmonella typhimurium and Treponema phagedenis. Mutants of H. pylori and H. mustelae defective in hook production generated by allele replacement were non-motile and devoid of flagellar filaments but produced both flagellin subunits, which were localized in the soluble fraction of the cell. The level of flagellin production was unchanged in the mutants, indicating that the regulation of flagellin expression in Helicobacter differs from that in the Enterobacteriaceae.

Non‐motile mutants of Helicobacter pylori and Helicobacter mustelae defective in flagellar hook production

Antisera raised in rabbits to whole cells of Helicobacter pylori recognized as a major antigen a protein with an apparent molecular weight of 20,000. The antigen was purified by differential solubilization with N-octyl-beta-D-glucopyranoside, urea, and sodium dodecyl sulfate followed by molecular sieving. The mass of the protein, Lpp20, was 18,283 Da as determined by mass spectrometry. The lpp20 gene encoding this protein was cloned in Escherichia coli by using the vector lambda EMBL3, and plasmid subclones expressed the full-length protein from the native H. pylori promoter. lpp20 was mapped to the same 358-kb NruI fragment as flaB. DNA sequence analysis showed that the gene was 525 bp long and encoded a 175-amino-acid protein with a molecular weight of 19,094 containing a 21-residue typical lipoprotein signal peptide and consensus prolipoprotein processing site. The mass of the deduced 154-residue mature protein was 16,865 Da. Growth of E. coli cells expressing the cloned H. pylori lpp20 gene in the presence of [3H]palmitic acid resulted in radiolabelled Lpp20 while treatment of the E. coli cells with globomycin caused accumulation of unprocessed Lpp20, consistent with Lpp20 being a lipoprotein. Lpp20 cofractionated with the cytoplasmic membrane fraction, although a proportion of the protein was also found in the outer membrane. A mutant generated by mutant-allele exchange displayed normal viability, showing that Lpp20 belonged to the nonessential class of lipoproteins.

Molecular characterization of a conserved 20-kilodalton membrane-associated lipoprotein antigen of Helicobacter pylori.

The surface of the fish pathogen Aeromonas salmonicida is covered by a paracrystalline array (the A-layer) which is a virulence factor for the organism. Quantification of the ability of A. salmonicida cells to bind collagen types I and IV in a 125I-radiolabelled liquid-phase assay showed that A-layer-positive cells bound high levels of collagen type IV, but significantly lower levels of collagen type I. Collagen type IV binding was confirmed using non-radiolabelled enzyme-linked immunosorbent assays. 125I-Collagen type IV binding was rapid, specific, saturable, high affinity, and essentially irreversible by unlabelled collagen type IV. The A-layer was responsible for collagen type IV binding because binding was inactivated by selective removal of the A-layer at pH 2.2, and neither isogenic A-layer-deficient A. salmonicida mutants nor strains of Aeromonas hydrophila possessing a morphologically similar paracrystalline array bound this basement membrane protein.

High‐affinity binding of the basement membrane protein collagen type IV to the crystalline virulence surface protein array of Aeromonas salmonicida

A protein ofMr26,000 whichwaspresent inlarge quantities inextracts ofcells ofHelicobacter pyloni was purified tohomogeneity byammoniumsulfate precipitation followed bygelfiltration andreversed-phase chromatography oranion-exchange chromatography. Theprotein appeared tobeassociated withthesoluble fraction ofthecell, andantibodies raised against theprotein werereactive withwhole-cell lysates ofavariety ofH.pylori strains inasimple immunodot blotassay. Thisreaction wasspecies specific. Protein sequence determination oftheaminoterminus andinternal cyanogen bromide fragments andaminoacidcomposition analysis wereperformed. Anoligonucleotide derived fromthese datawasusedtoclone afragment encoding mostofthecoding sequence. Expression inEscherichia coli wasdependent onvector promoters. TheDNA sequence ofthefragment wasdetermined. DNA probes derived fromthecloned fragment hybridized to genomic DNAofall H.pylon strains tested, butnottoDNAsofHelicobacter mustelae, Wolinella succinogenes, various Campylobacter species, andapanel ofgram-negative enteric bacteria. Theapparent uniqueness ofthis protein maybeexploited forthedevelopment ofspecies-specific diagnostics forthis gastric pathogen. Helicobacter (Campylobacter) pylori (11)isacurved or spiral gram-negative microaerophilic bacterium whichwas first isolated fromahumangastric biopsy specimen in1983 (46). Since this first isolation, ithasbecomeapparent that thisorganism maybeoneofthemostcommonbacterial pathogens ofhumans. Epidemiological evidence hasshown thatH.pylori colonizes theuppergastrointestinal tract of morethanoneintwoindividuals during their lifespan; in manyofthesepersons, theorganism isassociated with disease ofthegastrointestinal tract (12, 13,22).Indeed, H. pylori appears tobecausally associated withactive and chronic gastritis aswellaswithpeptic andduodenal ulcers (4,5,7,12,22)andmayalso beassociated withcarcinoma of thestomach (17, 21). Additional evidence forthepathogenic activity ofH.pylori hasbeenprovided bystudies with gnotobiotic andbarrier-born pigs(18, 20)andstudies with twohumanvolunteers (29, 35). Because oftheclinical importance ofthis pathogen and thelarge number oflaboratory identifications being routinely undertaken around theworld onadaily basis, there isaneed forthedevelopment ofrapid diagnostic testprotocols. Liberation of14Cfromlabeled ureabythepotent bacterial urease isreliable fordetection ofanactive H.pylori infection(30,33)buthasdisadvantages incost,time,and exposure toundesirable radiation. Cellextracts orpurified cell-surface components havebeenevaluated asantigens in thedevelopment ofserological tests forinfection (9,36). DNA-based approaches haveincluded restriction endonuclease analysis fortyping (28, 42)andthedevelopment of specific probes (6,34). We haveidentified a protein that appears tobeunique toH.pylori. We haveisolated this protein andcharacterized itbiochemically. Inthis paper we

Magdalena Kostrzynska

Papers

Non‐motile mutants of Helicobacter pylori and Helicobacter mustelae defective in flagellar hook production

Molecular characterization of a conserved 20-kilodalton membrane-associated lipoprotein antigen of Helicobacter pylori.

High‐affinity binding of the basement membrane protein collagen type IV to the crystalline virulence surface protein array of Aeromonas salmonicida

Isolation andBiochemical andMolecular Analyses ofa Species-Specific Protein Antigen fromtheGastric Pathogen Helicobacter pylori