Judith Lind

Suche


B10 Molecular function and signal transduction of the serine protease HtrA, a novel secreted effector protein of bacterial pathogens

Principal investigator
Steffen Backert

Mentor
Anja Lührmann

PhD exam: 02.12.2015

Interaction of enteric pathogens with the polarized epithelium: Role of bacterial and host factors for colonization, transmigration and disease development

Helicobacter pylori is a Gram-negative, flagellated bacterium colonizing the human stomach. It is capable to withstand the harsh conditions in this highly acidic environment and to colonize and persist there for lifetime. Approximately half of the world population is infected with the bacterium where it results in chronic gastric inflammation for the majority of people. However, in about 10-20% of patients it can lead to more severe gastric or duodenal pathologies such as ulcer formation or even adenocarcinoma. It was thus classified as a type I carcinogen by the WHO in 1994.
The ability of the bacterium to circumvent and prevent the clearance or elimination by the human immune system remains an open question. In general, upon infection the human mucus surface layer is naturally protected by the so called innate immune system that builds a first line of defense upon pathogen invasion. The immune system is able to recognize different microbial components, so called pathogen-associated molecular patterns (PAMPs), for example flagellin, lipopolysaccharide or peptidoglycan. These PAMPs are sensed and recognized by different pattern recognition receptors (PRRs). The best studied family of these PRRs is the Toll-like receptor (TLR) family. However, the role of TLRs in H.pylori's colonization and persistence in the human stomachs is highly uncertain.
Although H. pylori infection is leading to an activation and expression of various proinflammatory chemokines (e.g. IL-8, NF-κB) due to TLR activation, how and which bacterial factors trigger this immune response is not clear. The aim of this project is therefore to clarify which bacterial factors are capable to activate and stimulate TLR2 and TLR4. The classical role of TLRs in being activated by lipoprotein (TLR2), LPS (TLR4) is somewhat perturbed and confusing regarding current H. pylori literature. Thus, we are screening transposon-based mutant libraries of H. pylori to identify and characterize TLR2 and TLR4 dependent bacterial factors and downstream signaling pathways using reporter cell lines.
Another important part of the project investigates the variable recognition of EPIYA (Glu-Pro-Ile-Tyr-Ala) motifs in the H. pylori< CagA protein by the use of commercially available antibodies. Helicobacter strains that are associated with higher virulence are known to express a type-IV secretion system (T4SS) able to inject the effector protein CagA into the host cells. Once translocated, CagA is tyrosine-phosphorylated at EPIYA sequence motifs by members of the host c-Src and c-Abl kinases. Phosphorylated CagA is then able to exploit several downstream signaling pathways by binding to various SH-2 domain containing host proteins. In general, detection of tyrosine-phosphorylated CagA is taken as a measure for CagA injection and based on the use of pan-phosphotyrosine-specific antibodies. These antibodies were originally designed to detect a large range of phosphorylated tyrosine residues in mammalian proteins.
The question which bacterial tyrosine residues in the EPIYAs can be detected with these antibodies has so far not been examined. Thus, phospho- and non-phospho peptides of CagA EPIYA motifs are synthesized and used for dotblot analyses. The recognition pattern of the different antibodies is then also screened during infection of gastric epithelial cells with worldwide H. pylori strains carrying different CagA EPIYA motifs by Western blotting.

Figure: Toll-like receptors and pathogen recognition

 

Publications

Lind J, Backert S, Pfleiderer K, Berg DE, Yamaoka Y, Sticht H, Tegtmeyer N. 2014. Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of Western-type Helicobacter pylori strains. PLoS One. 9(5): e96488.

Koch KN, Hartung ML, Urban S, Kyburz A, Bahlmann AS, Lind J, et al. 2015. Helicobacter urease triggers TLR2 signaling to license NLRP3 inflammasome activation, promote regulatory T-cell response and protect against allergic asthma. J Clin Invest. pii: 79337.

Boehm M, Lind J, Backert S, Tegtmeyer N. 2015. Campylobacter jejuni serine protease HtrA plays an important role in heat tolerance, oxygen resistance, host cell adhesion, invasion, and transmigration. Eur J Microbiol Immunol (Bp). 5(1): 68-80.

Pachathundikandi SK, Lind J, Tegtmeyer N, El-Omar EM, Backert S. 2015. Interplay of the Gastric Pathogen Helicobacter pylori with Toll-like receptors. Biomed Res Int. 2015: 192420.

Tegtmeyer N, Lind J, Schmid B, Backert S. 2014. Helicobacter pylori CagL Y58/E59 mutations turns-off type IV secretion dependent delivery of CagA into host cells. PLoS One. 9(6): e97782.

Tenguria S, Ansari SA, Khan N, Ranjan A, Devi S, Tegtmeyer N, Lind J, Backert S, Ahmed N. 2014. Helicobacter pylori cell translocating kinase (CtkA/JHP0940) is pro-apoptotic in mouse macrophages and acts as auto-phosphorylating tyrosine kinase. Int J Med Microbiol. 304(8): 1066-76.

 

Presentations

July 2014 6th Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
 “Systematic phosphorylation analysis of the injected H. pylori type IV secretion effector protein cagA”
Talk
     
July 2013 5th Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Interaction of Helicobacter pylori with toll-like receptors
Talk