Helicobacter suis infection alters glycosylation and decreases the pathogen growth inhibiting effect and binding avidity of gastric mucins

Helicobacter suis is the most prevalent non-Helicobacter pylori Helicobacter species in the human stomach and is associated with chronic gastritis, peptic ulcer disease, and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. H. suis colonizes the gastric mucosa of 60-95% of pigs at slaughter age, and is associated with chronic gastritis, decreased weight gain, and ulcers. Here, we show that experimental H. suis infection changes the mucin composition and glycosylation, decreasing the amount of H. suis-binding glycan structures in the pig gastric mucus niche. Similarly, the H. suis-binding ability of mucins from H. pylori-infected humans is lower than that of noninfected individuals. Furthermore, the H. suis growth-inhibiting effect of mucins from both noninfected humans and pigs is replaced by a growth-enhancing effect by mucins from infected individuals/pigs. Thus, Helicobacter spp. infections impair the mucus barrier by decreasing the H. suis-binding ability of the mucins and by decreasing the antiprolific activity that mucins can have on H. suis. Inhibition of these mucus-based defenses creates a more stable and inhabitable niche for H. suis. This is likely of importance for long-term colonization and outcome of infection, and reversing these impairments may have therapeutic benefits

Glycan dependent Helicobacter spp. and Streptococcus oralis binding to mucins in the gastric and oral mucosal niches

Helicobacter pylori infects the stomach of half of the world’s population, while Helicobacter suis colonizes pigs and is the most common non-H. pylori Helicobacter species that also infects human stomach. Infection with Helicobacter spp. is associated with chronic gastritis, peptic ulcer disease, and gastric cancer. Streptococcus oralis colonizes human oral cavity and can cause infective endocarditis (IE). First barrier pathogens encounter is the mucus layer constituted by highly glycosylated glycoproteins, the mucins. Mucin glycans provide an extensive surface of interaction for bacteria. Here, we show the interactions of Helicobacter spp. and S. oralis with glycans in the gastric and oral mucosal niche. In paper I, the glycans from H. pylori infected and non-infected human stomachs were characterized by mass spectrometry. An enormous diversity of glycosylation exists in the human stomach. Infection with Helicobacter spp. is associated with large inter- and intra-individual diversity. The differences in glycosylation between mucins from infected and non-infected individuals are reflected by differences in binding of H. pylori to the mucins. In paper II, the binding of different H. pylori strains J99, P12, 26695 and G27 was analyzed. We show that these strains differ in their binding preferences and that mucins from infected or non-infected human stomachs affect the adhesion of different strains differently. Further, we show that infection, rather than inflammation, determines these effects. In paper III, we showed that experimental H. suis infection alters the composition of mucins and their glycosylation in a manner that reduces the amount of H. suis binding glycan structures, decreases H. suis binding ability, and changes mucin phenotype towards more Helicobacter spp. growth promoting. Thus, Helicobacter spp. infections impair the mucus barrier to create a stable niche in the stomach. In paper IV, the carbohydrate binding of IE isolates of S. oralis subspecies was investigated. Mucins were isolated from the saliva from blood group A and B positive individuals. We show that S. oralis adhesion occurs to salivary mucins and the binding differs between strains. S. oralis binding differs between mucins and individuals. Further, we show that S. oralis subsp. oralis binding to oral mucins is mediated by a cell wall anchored surface protein(s) and Leb, SLex and LNT like glycans present on the mucins. We demonstrate that mucin glycans are highly diverse and differ between individuals and with infection status. The glycan repertoire governs the ability of the mucins to bind to pathogens. Helicobacter spp. infection increases the diversity of glycosylation in the host and changes the host mucin composition. Understanding the adhesion mechanisms of H. pylori, H. suis and S. oralis could help develop preventive strategies against these pathogens

The Importance of Exosite Interactions for Substrate Cleavage by Human Thrombin

Thrombin is a serine protease of the chymotrypsin family that acts both as a procoagulant and as an anticoagulant by cleaving either factor VIII, factor V and fibrinogen or protein C, respectively. Numerous previous studies have shown that electropositive regions at a distance from the active site, so called exosites, are of major importance for the cleavage by human thrombin. Upstream of all the known major cleavage sites for thrombin in factor VIII, factor V and fibrinogen are clusters of negatively charged amino acids. To study the importance of these sites for the interaction with the exosites and thereby the cleavage by thrombin, we have developed a new type of recombinant substrate. We have compared the cleavage rate of the minimal cleavage site, involving only 8-9 amino acids (typically the P4-P4' positions) surrounding the cleavage site, with the substrates also containing the negatively charged regions upstream of the cleavage sites. The results showed that addition of these regions enhanced the cleavage rate by more than fifty fold. However, the enhancement was highly dependent on the sequence of the actual cleavage site. A minimal site that showed poor activity by itself could be cleaved as efficiently as an optimal cleavage site when presented together with these negatively charged regions. Whereas sites conforming closely to the optimal site were only minimally enhanced by the addition of these regions. The possibility to mimic this interaction for the sites in factor V and factor VIII by recombinant substrates, which do not have the same folding as the full size target, indicates that the enhancement was primarily dependent on a relatively simple electrostatic interaction. However, the situation was very different for fibrinogen and protein C where other factors than only charge is of major importance

Extended Cleavage Specificity of Human Neutrophil Elastase, Human Proteinase 3, and Their Distant Ortholog Clawed Frog PR3-Three Elastases With Similar Primary but Different Extended Specificities and Stability

Serine proteases are major granule constituents of several of the human hematopoietic cell lineages. Four proteolytically active such proteases have been identified in human neutrophils: cathepsin G (hCG), N-elastase (hNE), proteinase 3 (hPR-3), and neutrophil serine protease 4 (hNSP-4). Here we present the extended cleavage specificity of two of the most potent and most abundant of these enzymes, hNE and hPR-3. Their extended specificities were determined by phage display and by the analysis of a panel of chromogenic and recombinant substrates. hNE is an elastase with a relatively broad specificity showing a preference for regions containing several aliphatic amino acids. The protease shows self-cleaving activity, which results in the loss of activity during storage even at +4 degrees C. Here we also present the extended cleavage specificity of hPR-3. Compared with hNE, it shows considerably lower proteolytic activity. However, it is very stable, shows no self-cleaving activity and is actually more active in the presence of SDS, possibly by enhancing the accessibility of the target substrate. This enables specific analysis of hPR-3 activity even in the presence of all the other neutrophil enzymes with addition of 1% SDS. Neutrophils are the most abundant white blood cell in humans and one of the key players in our innate immune defense. The neutrophil serine proteases are very important for the function of the neutrophils and therefore also interesting from an evolutionary perspective. In order to study the origin and functional conservation of these neutrophil proteases we have identified and cloned an amphibian ortholog, Xenopus PR-3 (xPR-3). This enzyme was found to have a specificity very similar to hPR-3 but did not show the high stability in the presence of SDS. The presence of an elastase in Xenopus closely related to hPR-3 indicates a relatively early appearance of these enzymes during vertebrate evolution

Analyses of the importance of Phe8, Gly12 and three negatively charged residues in the N-terminal region of fibrinogen α chain for the cleavage by thrombin.

<p>The name and sequence of the substrates are indicated above the gel pictures. The time of cleavage (in minutes) is also indicated above their corresponding lanes on the gel. The mutations are marked in green in the sequences above each gel. Panels B and D shows the results from a scanning of the individual gels with corresponding percentages for a more easy evaluation of the result.</p

Schematic 3-D models of human thrombin showing the position of the N-terminal region of fibrinogen α chain.

<p>Panel A shows a space-filling model with the alpha chain peptide in purple. Panel B shows the interaction between thrombin (ribbon structure in beige) and the N-terminal region of fibrinogen α chain (ball and stick structure in purple) in detail. The same orientation as panel A is shown with the catalytic residues His57, Asp102 and Ser195 together with the S1 pocket residues Asp 189, Gly216 and 226 in green. Thrombin structure from PDB, code 1DM4 run using UCSF Chimera v1.8 and annotated in Adobe Illustrator CS5.</p

The importance of exosite interactions for the cleavage efficiency of cleavage sites in fibrinogen α chain (panel A), β chain (panel C) and protein C (panel E).

<p>The name and sequence of the substrates are indicated above the gel pictures. The time of cleavage (in minutes) is also indicated above their corresponding lanes on the gel. Panels B, D and F shows the results from a scanning of the individual gels with corresponding percentages for a more easy evaluation of the result.</p

The importance of exosite interactions for the cleavage efficiency of cleavage sites in FVIII.

<p>The name and sequence of the substrates are indicated above the gel pictures. The time of cleavage (in minutes) is also indicated above their corresponding lanes on the gel. Panels A-C shows the results for the individual cleavage sites in FVIII, R372, R740 and R1689, respectively. Panels D, E and F shows the results from a scanning of the individual gels with corresponding percentages for a more easy evaluation of the result.</p

Schematic 3-D models of human thrombin.

<p>Panel A shows a schematic 3D structure of human thrombin with the charge density illustrated with blue for basic or positively charged regions and red for negatively charged or acidic regions. The color intensity reflects the charge intensity. Panel B shows a schematic 3D structure of human thrombin where some of the positively charged amino acid positions studied by site directed mutagenesis in exosites I and II are marked in blue. The active site serine is shown in green and the histidine and aspartic acids are hidden under an extending loop and therefore not visible in this angle of the molecule. Two extended surface loops, the 60 loop and the γ-loop are shown in yellow and orange, respectively. Thrombin structure from PDB, code 1DM4 run using UCSF Chimera v1.8 and annotated in Adobe Illustrator CS5.</p