AbstractKlebsiella pneumoniae is a Gram-negative, multidrug-resistant (MDR) bacteria, causing nosocomial pneumonia, urinary tract infections (UTIs) and sepsis. Klebsiella’s subversion of the host immune response is critical for infection. Therefore, Klebsiella employs a range of novel mechanisms, including the targeting of host post-translational modifications (PTMs), to attenuate inflammation. The Bengoechea laboratory has previously demonstrated that Klebsiella triggers a type I interferon (IFN) response in vivo and here it is shown Klebsiella targets ISGylation, a type I IFN-dependent PTM, in vitro. ISGylation is analogous to ubiquitylation and involves the reversible conjugation of the ubiquitin-like modifier (UBL), IFN-Stimulated Gene 15 (ISG15) to target substrates. Whilst a crucial component of the host anti-viral response, the function of ISG15 and its associated PTM in the context of bacterial infection remains poorly understood.
Using an in vitro infection model, I show that K. pneumoniae targets ISGylation and additional host PTMs to attenuate the innate immune response to infection. These results uncovered that Klebsiella inhibits IFN-β induced ISGylation and levels of unconjugated ISG15 levels in infected epithelial cells (A549 cell line). Klebsiella was found to reduce isg15 transcription by blocking the phosphorylation of ISG15’s transcription factor IRF3. Remarkably, Klebsiella targets two PTMs to achieve this, namely phosphorylation and ubiquitylation. First, by elevating expression of the ubiquitously expressed phosphatase PP2Ac, Klebsiella effectively reduces phosphorylation of IRF3.
Furthermore, Klebsiella inhibits K48-linked polyubiquitylation to prevent PP2Ac degradation. It was also determined that Klebsiella reduces the PTM NEDDylation, via increased expression of the deNEDDylases CSN5 and SENP8. This prevents activation of the E3 ubiquitin ligase component Cullin1 to reduce ubiquitin-mediated PP2Ac degradation.
Reduced ISGylation was achieved by increased expression of the deISGylase USP18 during infection. Remarkably, as was the case for PP2Ac, Klebsiella’s inhibition of ubiquitin-dependent proteolysis accounted for the elevated levels of USP18. Furthermore, elevated USP18 was also dependent on reduced Cullin1 NEDDylation. These findings indicate that Klebsiella increases USP18 expression to limit activation of NF-kB-governed host antibacterial responses.
It was determined that Klebsiella targets EGFR signalling to affect levels of the proteins involved in ISGylation and NEDDylation. Activation of an EGFR-PI3KAKT-ERK axis accounted for elevated USP18, CSN5, SENP8 and PP2Ac levels. Additionally, the bacterial factor activating this pathway was identified to be capsule polysaccharide (CPS).
Furthermore, Klebsiella induces secretion of ISG15 from cells during initial infection. This extracellular ISG15 functions as a pro-inflammatory cytokine and, in conjunction with IL-12, is sufficient to trigger IFN-γ release by peripheral blood mononuclear cells (PBMCs). Remarkably, Klebsiella subsequently blocks this inflammatory loop, completely abrogating extracellular ISG15 secretion by three hours post-infection.
Taken together, these results demonstrate that K. pneumoniae manipulates several host PTMs and extracellular cytokine release to enhance infection. Such findings add to the current understanding of the complex infection biology that Klebsiella engages in.
|Date of Award||Jul 2020|
|Supervisor||Jose Bengoechea (Supervisor), Paul Moynagh (Supervisor) & Mark Robinson (Supervisor)|