Molecular insights into the fine-tuning of pH-dependent ArsR-mediated regulation of the SabA adhesin in Helicobacter pylori.

Adaptation to variations in pH is crucial for the ability of Helicobacter pylori to persist in the human stomach. The acid responsive two-component system ArsRS, constitutes the global regulon that responds to acidic conditions, but molecular details of how transcription is affected by the ArsR response regulator remains poorly understood. Using a combination of DNA-binding studies, in vitro transcription assays, and H. pylori mutants, we demonstrate that phosphorylated ArsR (ArsR-P) forms an active protein complex that binds DNA with high specificity in order to affect transcription. Our data showed that DNA topology is key for DNA binding. We found that AT-rich DNA sequences direct ArsR-P to specific sites and that DNA-bending proteins are important for the effect of ArsR-P on transcription regulation. The repression of sabA transcription is mediated by ArsR-P with the support of Hup and is affected by simple sequence repeats located upstream of the sabA promoter. Here stochastic events clearly contribute to the fine-tuning of pH-dependent gene regulation. Our results reveal important molecular aspects for how ArsR-P acts to repress transcription in response to acidic conditions. Such transcriptional control likely mediates shifts in bacterial positioning in the gastric mucus layer.

Campylobacter jejuni Surface-Bound Protease HtrA, but Not the Secreted Protease nor Protease in Shed Membrane Vesicles, Disrupts Epithelial Cell-to-Cell Junctions.

Fundamental functions of the intestinal epithelium include the digestion of food, absorption of nutrients, and its ability to act as the first barrier against intruding microbes. Campylobacter jejuni is a major zoonotic pathogen accounting for a substantial portion of bacterial foodborne illnesses. The germ colonizes the intestines of birds and is mainly transmitted to humans through the consumption of contaminated poultry meat. In the human gastrointestinal tract, the bacterium triggers campylobacteriosis that can progress to serious secondary disorders, including reactive arthritis, inflammatory bowel disease and Guillain-Barré syndrome. We recently discovered that C. jejuni serine protease HtrA disrupts intestinal epithelial barrier functions via cleavage of the tight and adherens junction components occludin, claudin-8 and E-cadherin. However, it is unknown whether epithelial damage is mediated by the secreted soluble enzyme, by HtrA contained in shed outer-membrane vesicles (OMVs) or by another mechanism that has yet to be identified. In the present study, we investigated whether soluble recombinant HtrA and/or purified OMVs induce junctional damage to polarized intestinal epithelial cells compared to live C. jejuni bacteria. By using electron and confocal immunofluorescence microscopy, we show that HtrA-expressing C. jejuni bacteria trigger efficient junctional cell damage, but not soluble purified HtrA or HtrA-containing OMVs, not even at high concentrations far exceeding physiological levels. Instead, we found that only bacteria with active protein biosynthesis effectively cleave junctional proteins, which is followed by paracellular transmigration of C. jejuni through the epithelial cell layer. These findings shed new light on the pathogenic activities of HtrA and virulence strategies of C. jejuni.

Phosphatidic acid-mediated binding and mammalian cell internalization of the Vibrio cholerae cytotoxin MakA.

Vibrio cholerae is a noninvasive intestinal pathogen extensively studied as the causative agent of the human disease cholera. Our recent work identified MakA as a potent virulence factor of V. cholerae in both Caenorhabditis elegans and zebrafish, prompting us to investigate the potential contribution of MakA to pathogenesis also in mammalian hosts. In this study, we demonstrate that the MakA protein could induce autophagy and cytotoxicity of target cells. In addition, we observed that phosphatidic acid (PA)-mediated MakA-binding to the host cell plasma membranes promoted macropinocytosis resulting in the formation of an endomembrane-rich aggregate and vacuolation in intoxicated cells that lead to induction of autophagy and dysfunction of intracellular organelles. Moreover, we functionally characterized the molecular basis of the MakA interaction with PA and identified that the N-terminal domain of MakA is required for its binding to PA and thereby for cell toxicity. Furthermore, we observed that the ΔmakA mutant outcompeted the wild-type V. cholerae strain A1552 in the adult mouse infection model. Based on the findings revealing mechanistic insights into the dynamic process of MakA-induced autophagy and cytotoxicity we discuss the potential role played by the MakA protein during late stages of cholera infection as an anti-colonization factor.

Helicobacter pylori Outer Membrane Vesicles Protect the Pathogen From Reactive Oxygen Species of the Respiratory Burst.

Outer membrane vesicles (OMVs) play an important role in the persistence of Helicobacter pylori infection. Helicobacter OMVs carry a plethora of virulence factors, including catalase (KatA), an antioxidant enzyme that counteracts the host respiratory burst. We found KatA to be enriched and surface-associated in OMVs compared to bacterial cells. This conferred OMV-dependent KatA activity resulting in neutralization of H2O2 and NaClO, and rescue of surrounding bacteria from oxidative damage. The antioxidant activity of OMVs was abolished by deletion of KatA. In conclusion, enrichment of antioxidative KatA in OMVs is highly important for efficient immune evasion.

BabA dependent binding of Helicobacter pylori to human gastric mucins cause aggregation that inhibits proliferation and is regulated via ArsS.

Mucins in the gastric mucus layer carry a range of glycan structures, which vary between individuals, can have antimicrobial effect or act as ligands for Helicobacter pylori. Mucins from various individuals and disease states modulate H. pylori proliferation and adhesin gene expression differently. Here we investigate the relationship between adhesin mediated binding, aggregation, proliferation and adhesin gene expression using human gastric mucins and synthetic adhesin ligand conjugates. By combining measurements of optical density, bacterial metabolic activity and live/dead stains, we could distinguish bacterial aggregation from viability changes, enabling elucidation of mechanisms behind the anti-prolific effects that mucins can have. Binding of H. pylori to Leb-glycoconjugates inhibited the proliferation of the bacteria in a BabA dependent manner, similarly to the effect of mucins carrying Leb. Furthermore, deletion of arsS lead to a decrease in binding to Leb-glycoconjugates and Leb-decorated mucins, accompanied by decreased aggregation and absence of anti-prolific effect of mucins and Leb-glycoconjugates. Inhibition of proliferation caused by adhesin dependent binding to mucins, and the subsequent aggregation suggests a new role of mucins in the host defense against H. pylori. This aggregating trait of mucins may be useful to incorporate into the design of adhesin inhibitors and other disease intervention molecules.

Structural Insights into Polymorphic ABO Glycan Binding by Helicobacter pylori.

The Helicobacter pylori adhesin BabA binds mucosal ABO/Le(b) blood group (bg) carbohydrates. BabA facilitates bacterial attachment to gastric surfaces, increasing strain virulence and forming a recognized risk factor for peptic ulcers and gastric cancer. High sequence variation causes BabA functional diversity, but the underlying structural-molecular determinants are unknown. We generated X-ray structures of representative BabA isoforms that reveal a polymorphic, three-pronged Le(b) binding site. Two diversity loops, DL1 and DL2, provide adaptive control to binding affinity, notably ABO versus O bg preference. H. pylori strains can switch bg preference with single DL1 amino acid substitutions, and can coexpress functionally divergent BabA isoforms. The anchor point for receptor binding is the embrace of an ABO fucose residue by a disulfide-clasped loop, which is inactivated by reduction. Treatment with the redox-active pharmaceutic N-acetylcysteine lowers gastric mucosal neutrophil infiltration in H. pylori-infected Le(b)-expressing mice, providing perspectives on possible H. pylori eradication therapies.

Helicobacter pylori vesicles carrying CagA localize in the vicinity of cell-cell contacts and induce histone H1 binding to ATP in epithelial cells.

Helicobacter pylori produces outer membrane vesicles (OMV), delivering bacterial substances including the oncogenic cytotoxin-associated CagA protein to their surroundings. We investigated the effects of H. pylori OMV carrying CagA (OMV-CagA) on cell junctions and ATP-binding proteome of epithelial monolayers, using proteomics, mass spectrometry and imaging. OMV-CagA localized in close vicinity of ZO-1 tight junction protein and induced histone H1 binding to ATP. We suggest the expression of novel events in the interactions between H. pylori OMV and epithelia, which may have an influence on host gene transcription and lead to different outcomes of an infection and development of cancer.

A repetitive DNA element regulates expression of the Helicobacter pylori sialic acid binding adhesin by a rheostat-like mechanism.

During persistent infection, optimal expression of bacterial factors is required to match the ever-changing host environment. The gastric pathogen Helicobacter pylori has a large set of simple sequence repeats (SSR), which constitute contingency loci. Through a slipped strand mispairing mechanism, the SSRs generate heterogeneous populations that facilitate adaptation. Here, we present a model that explains, in molecular terms, how an intergenically located T-tract, via slipped strand mispairing, operates with a rheostat-like function, to fine-tune activity of the promoter that drives expression of the sialic acid binding adhesin, SabA. Using T-tract variants, in an isogenic strain background, we show that the length of the T-tract generates multiphasic output from the sabA promoter. Consequently, this alters the H. pylori binding to sialyl-Lewis x receptors on gastric mucosa. Fragment length analysis of post-infection isolated clones shows that the T-tract length is a highly variable feature in H. pylori. This mirrors the host-pathogen interplay, where the bacterium generates a set of clones from which the best-fit phenotypes are selected in the host. In silico and functional in vitro analyzes revealed that the length of the T-tract affects the local DNA structure and thereby binding of the RNA polymerase, through shifting of the axial alignment between the core promoter and UP-like elements. We identified additional genes in H. pylori, with T- or A-tracts positioned similar to that of sabA, and show that variations in the tract length likewise acted as rheostats to modulate cognate promoter output. Thus, we propose that this generally applicable mechanism, mediated by promoter-proximal SSRs, provides an alternative mechanism for transcriptional regulation in bacteria, such as H. pylori, which possesses a limited repertoire of classical trans-acting regulatory factors.

Uptake of Helicobacter pylori vesicles is facilitated by clathrin-dependent and clathrin-independent endocytic pathways.

UNLABELLED: Bacteria shed a diverse set of outer membrane vesicles that function as transport vehicles to deliver effector molecules and virulence factors to host cells. Helicobacter pylori is a gastric pathogen that infects half of the world's population, and in some individuals the infection progresses into peptic ulcer disease or gastric cancer. Here we report that intact vesicles from H. pylori are internalized by clathrin-dependent endocytosis and further dynamin-dependent processes, as well as in a cholesterol-sensitive manner. We analyzed the uptake of H. pylori vesicles by gastric epithelial cells using a method that we refer to as quantification of internalized substances (qIS). The qIS assay is based on a near-infrared dye with a cleavable linker that enables the specific quantification of internalized substances after exposure to reducing conditions. Both chemical inhibition and RNA interference in combination with the qIS assay showed that H. pylori vesicles enter gastric epithelial cells via both clathrin-mediated endocytosis and additional endocytic processes that are dependent on dynamin. Confocal microscopy revealed that H. pylori vesicles colocalized with clathrin and dynamin II and with markers of subsequent endosomal and lysosomal trafficking. Interestingly, however, knockdown of components required for caveolae had no significant effect on internalization and knockdown of components required for clathrin-independent carrier (CLIC) endocytosis increased internalization of H. pylori vesicles. Furthermore, uptake of vesicles by both clathrin-dependent and -independent pathways was sensitive to depletion, but not sequestering, of cholesterol in the host cell membrane suggesting that membrane fluidity influences the efficiency of H. pylori vesicle uptake. IMPORTANCE: Bacterial vesicles act as long-distance tools to deliver toxins and effector molecules to host cells. Vesicles can cause a variety of host cell responses via cell surface-induced cell signaling or internalization. Vesicles of diverse bacterial species enter host cells via different endocytic pathways or via membrane fusion. With the combination of a fluorescence-based quantification assay that quantifies internalized vesicles in a large number of cells and either chemical inhibition or RNA interference, we show that clathrin-mediated endocytosis is the major pathway for uptake of Helicobacter pylori vesicles and that lipid microdomains of the host cell membrane affect uptake of vesicles via clathrin-independent pathways. Our results provide important insights about membrane fluidity and its important role in the complex process that directs the H. pylori vesicle to a specific endocytic pathway. Understanding the mechanisms that operate in vesicle-host interactions is important to fully recognize the impact of vesicles in pathogenesis.

The complement regulator CD46 is bactericidal to Helicobacter pylori and blocks urease activity.

BACKGROUND & AIMS: CD46 is a C3b/C4b binding complement regulator and a receptor for several human pathogens. We examined the interaction between CD46 and Helicobacter pylori (a bacterium that colonizes the human gastric mucosa and causes gastritis), peptic ulcers, and cancer. METHODS: Using gastric epithelial cells, we analyzed a set of H pylori strains and mutants for their ability to interact with CD46 and/or influence CD46 expression. Bacterial interaction with full-length CD46 and small CD46 peptides was evaluated by flow cytometry, fluorescence microscopy, enzyme-linked immunosorbent assay, and bacterial survival analyses. RESULTS: H pylori infection caused shedding of CD46 into the extracellular environment. A soluble form of CD46 bound to H pylori and inhibited growth, in a dose- and time-dependent manner, by interacting with urease and alkyl hydroperoxide reductase, which are essential bacterial pathogenicity-associated factors. Binding of CD46 or CD46-derived synthetic peptides blocked the urease activity and ability of bacteria to survive in acidic environments. Oral administration of one CD46 peptide eradicated H pylori from infected mice. CONCLUSIONS: CD46 is an antimicrobial agent that can eradicate H pylori. CD46 peptides might be developed to treat H pylori infection.

Effects of blood group antigen-binding adhesin expression during Helicobacter pylori infection of Mongolian gerbils.

Helicobacter pylori outer membrane proteins, such as the blood group antigen-binding adhesin (BabA), are associated with severe pathological outcomes. However, the in vivo role of BabA during long-term infection is not clear. In this study, Mongolian gerbils were infected with H. pylori and necropsied continuously during 18 months. Bacterial clones were recovered and analyzed for BabA expression, Leb-binding activity, and adhesion to gastric mucosa. BabA expression was completely absent by 6 months post-infection. Loss of BabA expression was attributable to nucleotide changes within the babA gene that resulted in a truncated BabA. In response to the infection, changes in the epithelial glycosylation pattern were observed that were similar to responses observed in humans and monkeys. Furthermore, infections with BabA-expressing and BabA-nonexpressing H. pylori showed no differences in colonization, but infection with the BabA-expressing strain exhibited histological changes and increased inflammatory cell infiltration. This suggests that BabA expression contributes to severe mucosal injury.

Biochemical and functional characterization of Helicobacter pylori vesicles.

Helicobacter pylori can cause peptic ulcer disease and/or gastric cancer. Adhesion of bacteria to the stomach mucosa is an important contributor to the vigour of infection and resulting virulence. H. pylori adheres primarily via binding of BabA adhesins to ABO/Lewis b (Leb) blood group antigens and the binding of SabA adhesins to sialyl-Lewis x/a (sLex/a) antigens. Similar to most Gram-negative bacteria, H. pylori continuously buds off vesicles and vesicles derived from pathogenic bacteria often include virulence-associated factors. Here we biochemically characterized highly purified H. pylori vesicles. Major protein and phospholipid components associated with the vesicles were identified with mass spectroscopy and nuclear magnetic resonance. A subset of virulence factors present was confirmed by immunoblots. Additional functional and biochemical analysis focused on the vesicle BabA and SabA adhesins and their respective interactions to human gastric epithelium. Vesicles exhibit heterogeneity in their protein composition, which were specifically studied in respect to the BabA adhesin. We also demonstrate that the oncoprotein, CagA, is associated with the surface of H. pylori vesicles. Thus, we have explored mechanisms for intimate H. pylori vesicle-host interactions and found that the vesicles carry effector-promoting properties that are important to disease development.

Semiconstant-time P,H-COSY NMR: analysis of complex mixtures of phospholipids originating from Helicobacter pylori.

Lipids play a central role in numerous biological events, ranging from normal physiological processes to host-pathogen interactions. The proposed semiconstant-time (31)P,(1)H-COSY NMR experiment provides identification of known and structural characterization of unknown phospholipids in complex membrane extracts with high sensitivity, based on the combination of their (1)H and (31)P chemical shifts and coupling patterns. Furthermore, the spectra allow quantification of phospholipid composition. Analysis of the phospholipid composition of Helicobacter pylori, the causative agent of peptic ulcer disease, showed the presence of uncommon phospholipids. This novel NMR approach allows the study of changes in membrane composition in response to biological stimuli and opens up the possibility of identifying soluble phosphorus species in a number of research fields.

Helicobacter pylori BabA expression, gastric mucosal injury, and clinical outcome.

BACKGROUND & AIMS: The blood grou. METHODS: We compared the ability of published PCR-based methods to assess BabA status with BabA immunoblotting and Lewis b (Le(b)) binding activity assays. We also used immunoblotting to examine the relationship between clinical presentation and levels of BabA expression. RESULTS: Immunoblotting and Le(b) binding assays for 80 strains revealed 3 levels of BabA expression: BabA high producers (BabA-H) with Le(b) binding activity, BabA low producers (BabA-L) without Le(b) binding activity, and BabA-negative. BabA-negative strains lacked the babA gene. PCR methods to determine BabA status yielded poor results. babA1 sequences were never detected. BabA expression was examined in 250 strains from Western countries and 270 strains from East Asia. The results failed to confirm any relationship between triple-positive status (cagA-positive/vacA s1/BabA-H) and clinical outcome. BabA-negative strains typically were cagA-negative/vacA s2 and were associated with gastritis. BabA-L strains showed a higher level of mucosal injury and were associated more frequently with duodenal ulcer and gastric cancer than the other groups. CONCLUSIONS: Information gained from currently used PCR-based methods must be interpreted with caution. Le(b) binding activity does not accurately reflect the severity of mucosal damage or the clinical outcome. Quantitation of BabA expression revealed that Le(b)-nonbinding BabA-L strains are associated with higher levels of mucosal injury and clinical outcome.

Helicobacter pylori adhesion to carbohydrates.

Adherence of bacterial pathogens to host tissues contributes to colonization and virulence and typically involves specific interactions between bacterial proteins called adhesins and cognate oligosaccharide (glycan) or protein motifs in the host that are used as receptors. A given pathogen may have multiple adhesins, each specific for a different set of receptors and, potentially, with different roles in infection and disease. This chapter provides strategies for identifying and analyzing host glycan receptors and the bacterial adhesins that exploit them as receptors, with particular reference to adherence of the gastric pathogen Helicobacter pylori.

SabA is the H. pylori hemagglutinin and is polymorphic in binding to sialylated glycans.

Adherence of Helicobacter pylori to inflamed gastric mucosa is dependent on the sialic acid-binding adhesin (SabA) and cognate sialylated/fucosylated glycans on the host cell surface. By in situ hybridization, H. pylori bacteria were observed in close association with erythrocytes in capillaries and post-capillary venules of the lamina propria of gastric mucosa in both infected humans and Rhesus monkeys. In vivo adherence of H. pylori to erythrocytes may require molecular mechanisms similar to the sialic acid-dependent in vitro agglutination of erythrocytes (i.e., sialic acid-dependent hemagglutination). In this context, the SabA adhesin was identified as the sialic acid-dependent hemagglutinin based on sialidase-sensitive hemagglutination, binding assays with sialylated glycoconjugates, and analysis of a series of isogenic sabA deletion mutants. The topographic presentation of binding sites for SabA on the erythrocyte membrane was mapped to gangliosides with extended core chains. However, receptor mapping revealed that the NeuAcalpha2-3Gal-disaccharide constitutes the minimal sialylated binding epitope required for SabA binding. Furthermore, clinical isolates demonstrated polymorphism in sialyl binding and complementation analysis of sabA mutants demonstrated that polymorphism in sialyl binding is an inherent property of the SabA protein itself. Gastric inflammation is associated with periodic changes in the composition of mucosal sialylation patterns. We suggest that dynamic adaptation in sialyl-binding properties during persistent infection specializes H. pylori both for individual variation in mucosal glycosylation and tropism for local areas of inflamed and/or dysplastic tissue.

Immunoproteomics of Actinobacillus actinomycetemcomitans outer-membrane proteins reveal a highly immunoreactive peptidoglycan-associated lipoprotein.

In a search for novel bioactive cell surface structures of periodontal pathogens, it was found that sera from two patients with Actinobacillus actinomycetemcomitans-associated infections reacted strongly at 17 kDa on immunoblots of A. actinomycetemcomitans outer-membrane protein (OMP) preparations. The 17 kDa antigen was also recognized by anti-CsgA (Escherichia coli curli major subunit) antibody. The 17 kDa A. actinomycetemcomitans protein was identified as peptidoglycan-associated lipoprotein (PAL; AaPAL) by two-dimensional immunoblotting and subsequent sequence analysis by mass spectrometry and bioinformatics tools. AaPAL was an OMP and a lipoprotein, and it had an OmpA-like domain. In a group of middle-aged subjects (n = 26), serum reactivity to AaPAL was associated with the presence of periodontitis but not with the oral detection of A. actinomycetemcomitans. Both human sera and rabbit antisera against three different types of antigens, the gel-purified AaPAL, A. actinomycetemcomitans whole-cell antigens, and CsgA, recognized putative PALs of oral haemophili in addition to AaPAL. The results demonstrated that the novel AaPAL is a conserved bacterial lipoprotein. It is expressed in vivo and is strongly immunoreactive. The antigenic cross-reactivity found between AaPAL and oral haemophili may enhance local and systemic immuno-inflammatory reactions in periodontitis.

Metastability of Helicobacter pylori bab adhesin genes and dynamics in Lewis b antigen binding.

Heterogeneity among Helicobacter pylori strains in gastric epithelial adherence is postulated to contribute to pathogen fitness in the physiologically diverse human population. H. pylori adherence to ABO and Lewis b (Leb) blood group antigens in the human stomach is mediated by the blood group antigen-binding adhesin BabA. Approximately 70% of Swedish and U.S. H. pylori clinical isolates exhibit Leb binding, but here we show that the babA gene is present in each of 10 Leb-nonbinding strains. Fluorescence microscopy identified occasional bacterial cells with a Leb-binding phenotype in populations of Leb-nonbinding strains. Thus, nonbinding seemed to be a metastable phenotype. To model metastable transition into the virulence-associated Leb-binding mode, Leb-binding clones were isolated from nonadherent strains by panning with Leb-magnetic beads and characterized. Strain 17875 has two babA genes, babA1 (silent) and babA2 (expressed). We found that a babA2-cam derivative of strain 17875 regained Leb binding by recombination of the formerly silent babA1 gene into the expressed and partially homologous babB locus. The chimeric BabB/A adhesin binds Leb with an affinity similar to that of wild-type BabA adhesin, but its expression level was lower and was subject to phase variation through slipped-strand mispairing. Equivalent results were obtained with strain NCTC11638. We propose that adhesin metastability and heterogeneity contributes to bacterial fitness and results in some clones having potential for periodic activation and deactivation of virulence appropriate for intensity of the host response to infection.

Functional adaptation of BabA, the H. pylori ABO blood group antigen binding adhesin.

Adherence by Helicobacter pylori increases the risk of gastric disease. Here, we report that more than 95% of strains that bind fucosylated blood group antigen bind A, B, and O antigens (generalists), whereas 60% of adherent South American Amerindian strains bind blood group O antigens best (specialists). This specialization coincides with the unique predominance of blood group O in these Amerindians. Strains differed about 1500-fold in binding affinities, and diversifying selection was evident in babA sequences. We propose that cycles of selection for increased and decreased bacterial adherence contribute to babA diversity and that these cycles have led to gradual replacement of generalist binding by specialist binding in blood group O-dominant human populations.

Helicobacter pylori SabA adhesin in persistent infection and chronic inflammation.

Helicobacter pylori adherence in the human gastric mucosa involves specific bacterial adhesins and cognate host receptors. Here, we identify sialyl-dimeric-Lewis x glycosphingolipid as a receptor for H. pylori and show that H. pylori infection induced formation of sialyl-Lewis x antigens in gastric epithelium in humans and in a Rhesus monkey. The corresponding sialic acid-binding adhesin (SabA) was isolated with the "retagging" method, and the underlying sabA gene (JHP662/HP0725) was identified. The ability of many H. pylori strains to adhere to sialylated glycoconjugates expressed during chronic inflammation might thus contribute to virulence and the extraordinary chronicity of H. pylori infection.