The Role of Human Plasminogen in the Virulence of Helicobacter pylori

Abstract

Helicobacter pylori is an important pathogen inhabiting the human stomach. H. pylori binds plasminogen, a 90 kDa glycoprotein secreted by liver cells, and utilizes it for enhancing virulence. Surface receptors on the bacteria recognize plasminogen that is then activated into plasmin by host factors. The active plasmin can degrade a range of host proteins including proteins of the extracellular matrix (ECM), the complement system and IgG. This in turn enhances the virulence of the pathogen and decreases complement-mediated killing. In this study, we investigated the interaction between plasminogen and the different H. pylori plasminogen binding proteins. Previously, seven different plasminogen binding genes were identified in our lab and cloned into pET26b for expression in E. coli BL21 (DE3). Another important goal was to create deletion mutants of the seven candidate genes in H. pylori to perform functional studies on bacterium-plasminogen binding. We identified Hpg 1, 5 and 6 and PgbA as proteins with highest binding affinity to plasminogen by ELISA and biolayer interferometry, whereas Hpg 2, 3 and 4 bound plasminogen with low affinity. Two different H. pylori strains were incubated with plasminogen, which was activated by urokinase Plasminogen Activator (uPA) and the degradation of the complement molecules C3b and C5 as well as IgG was studied. The assays revealed that the plasmin bound by H. pylori still degraded all three tested substrates in a time-dependent manner. We were successful in making mutants for Hpg1, 6 and PgbA plasminogen binding genes. Taken together, this study provides new insight into the role of plasminogen-binding proteins in the virulence of H. pylori.

Popular Abstract

Helicobacter pylori employs host proteins to cause infection

Plasminogen is a glycoprotein, which is produced and secreted by liver cells in the form of zymogen as a 810 amino acid (90kDa) polypeptide chain. It is converted into the serine protease plasmin by host factors. A large number of pathogens have been reported to bind plasminogen that can subsequently be activated to plasmin. Active plasmin can degrade host ECM proteins , some proteins of the complement system and, finally, IgG. Binding plasmin thus enhances the invasive capacity of pathogens and dampens complementmediated killing of bacteria.

H. pylori has previously been reported to bind human plasminogen. Two H. pylori plasminogen binding proteins (PgbA & PgbB) have been characterized previously and they bind in a lysine-dependant manner. Several other plasminogen-binding proteins of H. pylori have been identified in our laboratory and were successfully cloned into the pET26b expression vector and expressed in E. coli BL21 (DE3) as Hpg 1 to Hpg 7 and Pgb A. Protein-protein interactions were studied by using Bio-layer Interferometry and ELISA. Proteins Hpg 1, 5, 6 and Pgb A showed high binding affinity to immobilized plasminogen in the ELISA assay. Bio layer Interferometry uses an optical biosensor to determine the protein and protein interaction by steady state kinetics. KD is the equilibrium dissociation constant that determines the strength of an interaction between two protein molecules. Hpg 1, Hpg 5, Hpg 6 and Pgb A have very low KD values. A lower KD value corresponds to a higher binding affinity and vice versa. Hpg 2, 3 and Hpg 4 have relatively higher KD values and thus have lower binding affinity. This result is in agreement with our ELISA results.

Plasminogen is cleaved into active plasmin by the urokinase plasminogen activators (uPA) or tissue plasminogen activators. Degradation of complement molecules and IgG antibodies by active plasmin were investigated in our study. It was observed that the complements C3b and C5 were degraded by plasmin in a time- dependant manner. The degradation of alpha and beta chains of complement molecules increases as the incubation time of the bacteria bound to activated plasminogen increased. IgG degradation in samples containing bacteria, plasminogen, uPA and IgG, was measured through absorbance at 450 nm. Lower absorbance values correspond to a higher degradation of IgG. It was evident from the assay that those samples incubated with activated plasmin had lower absorbance values when compared to the controls that had only plasminogen activators or only plasminogen. The function of plasminogen binding genes could be assessed by gene knock out experiments and by studying the binding affinity of mutants to plasminogen. We used the gene replacement method, “allelic exchange mutagenesis” to produce recombinant genes with kanamycin cassette. We were successful in producing mutant gene constructs for Hpg 1, Hpg 6 and Pgb A. These gene fragments were electroporated into H. pylori clinical strains. It would be interesting to investigate the interactions of these mutant strains with plasminogen to study how Helicobacter pylori employs plasminogen binding in evading the innate immune system. In conclusion, we have characterized the plasminogen-binding receptors of Helicobacter pylori that evades the innate immunity by binding plasminogen. In conclusion, characterisation of these bacterial surface receptors that bound plasminogen showed that Helicobacter pylori can degrade complement components and human IgG.

Supervisors: Dr. Birendra Singh, Dr. Corinna Richter, Dr. Kristian Riesbeck.
Master´s Degree Project 45 credits in Molecular Biology, 2016
Department of Biology, Lund University
Department of Translational medicine, Lund University.

• author: Babu Sait, Mohammed Rizwan
• supervisor: Corinna Richter; Birendra Singh
• organization: Degree Projects in Molecular Biology
• course: MOBN01 20161
• year: 2016
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 8891473
• date added to LUP: 2016-09-12 13:43:36
• date last changed: 2016-09-12 13:43:36