Assessing Helicobacter pylori's HcpE and DsbK's Effects on Immunostimulation in Host Gastric Cells
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.