Investigating the functions and interactions of Salmonella effector SspH2 with intracellular innate immune receptors

• Author / Creator

  Delyea, Cole J.

• As part of its pathogenesis, Salmonella enterica serovar typhimurium delivers effector proteins into host cells. One such effector is SspH2, a member of the novel E3 ubiquitin ligase (NEL) family, which has been reported to interact with, and enhance, NOD1 pro-inflammatory signaling. It is still unknown as to how SspH2 is specifically mediating increased NLR activity. I transfected host NLRs and bacterial SspH2 in mammalian epithelial cell culture and monitored various aspects of SspH2 interactions in host cells. I demonstrate that SspH2 interacts with two out of three different regions on NOD1, namely its central Nucleotide binding domain, and its C-terminal Leucine rich repeat domain by using co-immunoprecipitations (co-IP). Using the same methods, I also determined that SspH2 also interacts with NOD2, a closely related NLR to NOD1. This interaction induces super-activation of NOD2, resulting in increased IL-8 secretion that was measured by ELISA. Furthermore, I show that catalytically active SspH2 significantly increases NLR-mediated cytokine secretion via the NF-κB pathway. With the help of Shu Luo from the Julien lab, I immunoprecipitated NOD1 in the presence of SspH2 and utilized liquid chromatography with tandem mass spectrometry (LC-MS/MS) to uncover specific sites of ubiquitination on NOD1. I then semi-quantitatively analyzed the peptide fragment intensities of LC-MS/MS and identified lysines of statistical importance. Based off of these identified lysines, I mutated them to arginine residues, such that they could no longer be ubiquitinated. These NOD1 variants retained their interaction with SspH2 and showed similar basal activity compared to wildtype NOD1 through analysis by co-IP. Interestingly by utilizing IL-8 ELISAs, I identified 4 key lysine residues in NOD1 that are specifically required for enhanced cytokine secretion in the presence of SspH2. Here, I provide evidence for post-translational modification of NOD1 by ubiquitin, and uncover a unique mechanism of targeted ubiquitination to enhance the activation of an archetypal NLR. To my knowledge this is the first demonstration that NLRs are specifically ubiquitinated by bacterial effectors.

• Subjects / Keywords

  • Innate Immune system
  • Natural immunity
  • Salmonella typhimurium
  • NOD-like receptors

• Graduation date

  Fall 2021

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-7800-qp50

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.