Thesis Format
Monograph
Degree
Doctor of Philosophy
Program
Microbiology and Immunology
Supervisor
Creuzenet, Carole
Abstract
Helicobacter pylori (HP) colonizes half the world population. It causes chronic gastric inflammation that often leads to ulcers and cancers. Considering rising antibiotic resistance and patient noncompliance, the search for an alternative treatment is essential. HcpE, a member of the Helicobacter cysteine-rich proteins (Hcps) family, contains the largest number of cysteine residues and is secreted extracellularly. Its structure is made of Sel-Like Repeats (SLR) motifs–stabilized by disulfide bonds–which are important in protein-protein interactions and signal transduction. DsbK is key for HcpE’s disulfide bond formation and its secretion. Our aim was to identify the immunostimulatory effects of HcpE and DsbK, determine HcpE’s secretion/translocation via the Type-IV-Secretion-System-(T4SS), identify candidate host interacting partners and the subcellular localization of HcpE within host cells. AGS (adenocarcinoma gastric) cells were exposed to either live bacteria, bacterial culture supernatants, outer membrane vesicles (OMVs), supernatants devoid of OMVs of wild-type and knock-out mutants of DsbK and HcpE in two HP background strains or purified recombinant HcpE (rHcpE), followed by IL-8 secretion assessment. The relative expression levels of the various Hcps were assessed to comprehend their contribution in mutants’ IL-8 response. A disrupted T4SS was used to assess HcpE’s secretion/translocation. To identify host potential interacting partners, BioID (proximity-dependent biotin identification) and Immunoprecipitation (affinity purification) were implemented, and candidates were identified by mass spectrometry. Finally, the subcellular localization of HcpE in host cells was assessed by immunofluorescence microscopy-(IFM). We show a significant DsbK and -HcpE-dependent proinflammatory response by increased IL-8 levels in response to live HP-strain, but not their bacterial secretions. HcpE’s proimmunostimulatory-inducing effect was established in a dose-dependent manner. The T4SS does not secrete HcpE extracellularly. Furthermore, BioID and Immunoprecipitation identified several potential partner proteins, involved in signal transduction, peroxisomal pathways, and cytoskeletal functions. Finally, IFM did not detect intracellular HcpE within the assessed timepoints. Future studies will validate selected candidate interacting partners and will assess different timepoints and techniques for HcpE’s localization. The iii identification of interacting proteins will provide a better understanding of HcpE’s mechanism in inducing inflammation and provide targets for inhibition that could be applied as novel therapeutics for HP infections.
Summary for Lay Audience
Helicobacter pylori (HP) is a bacterium thatinfects 50% of the world’s population. HP chronic infections can cause ulcers and cancers. HP colonization increases the risk of gastric cancer ~10 -times. Although its occurrence is decreasing, the prognosis is poor. Because of HP’s high incidence and the severity of the disease, HP significantly burdens the health care system. Effectively treating HP infections is key to preventing these serious diseases, but current treatments are cumbersome, poorly tolerated by patients, and are jeopardized by antibiotic resistance. Thus, understanding the inflammatory effect and secretion method of HcpE-an HP virulence factor-would allow us to design new anti-virulence agents as alternative treatments. New therapeutics can be designed to inhibit the interaction between HcpE and its gastric cell targets. These inhibitors can eliminate the inflammatory effects of HcpE; and when used alone or in combination with existing treatments, they could significantly improve outcomes of anti-HP therapies. Here, two proteins produced by HP were evaluated for their inflammatory capacities on gastric cells. Their production activates the patient’s immune system and produces inflammatory molecules that are supposed to help eliminate the bacterium. Despite the inflammatory response activation, the body does not clear the bacterium. As the immune system persists in trying to eliminate the bacterium, it constantly produces inflammatory compounds which damage the gastric tissue and increase the risks for ulcers or cancer. We found that these proteins stimulate inflammation in the gastric cells when live bacteria were used, while other bacterial secretions didn’t have an effect. To better understand their function, we studied their effects in two different strains of HP. Preventing the secretion or activity of these proteins should help the immune system regain control and clear the infection. Therefore, we determined a particular secretion system was not implicated in the secretion/translocation or inflammatory response of our secreted protein. Also, we identified several candidates that HcpE may interact with to perturb the normal function of gastric cells. Finally, to visualize HcpE’s localization in the gastric cell, we used fluorescence imaging, which did not detect signals from inside the cell, rather only on the outside. These findings are important as they expand the knowledge on infection and inflammation of HP.
Recommended Citation
Salloum, Noor, "Assessing Helicobacter pylori's HcpE and DsbK's Effects on Immunostimulation in Host Gastric Cells" (2024). Electronic Thesis and Dissertation Repository. 10314.
https://ir.lib.uwo.ca/etd/10314
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