Oral Presentation 14th Lorne Infection and Immunity 2024

Targeted degradation of host messenger RNA by a bacterial effector modulates macrophage metabolism (#17)

Kitty McCaffrey 1 , Hari Sivaraman 1 , Joshua von Ameln 1 , Raissa Wibawa 1 , Garrett Ng 1 , Natalio Garbi 2 , Ian R van Driel 3 , Wilson Wong 1 , Elizabeth L Hartland 1
  1. Hudson Institute, Clayton, VICTORIA, Australia
  2. University of Bonn, Bonn, Germany
  3. University of Melbourne, Melbourne, VICTORIA, Australia

Macrophages are the first line of defence against bacterial infection and, upon activation, undergo a shift to glycolytic metabolism that is essential for the antimicrobial and pro-inflammatory functions of these innate immune cells. Macrophages are also the primary target of many intracellular bacterial pathogens that inject virulence effector proteins into the macrophage intracellular environment to alter cellular responses, thereby enabling the survival and replication of the pathogen. In this study, we observed that infection with the intracellular bacterial pathogen Legionella pneumophila depleted host-cellular messenger RNA (mRNA) encoding metabolic enzymes, including the rate-limiting glycolytic enzyme GAPDH. By screening a library of L. pneumophila deletion mutant strains, we identified the secreted Dot/Icm effector LegC4 as essential for the degradation of host mRNA and the suppression macrophage glycolysis during infection. Using cross-linking immunoprecipitation (CLIP), we demonstrated that LegC4 directly interacted with GAPDH mRNA during infection. Electrophoretic mobility shift assays (EMSA) also showed that LegC4 bound directly to GAPDH mRNA in vitro in the absence of other bacterial or host factors. Analysis of RNA bound to LegC4 showed enrichment for guanine (G)-rich RNA recognition motifs that occurred within the GAPDH transcript and within all mRNA-degradation targets, suggesting that these motifs conferred LegC4-mRNA specificity. Finally, cryo-EM structural analysis of an inactive mutant of LegC4 (LegC4H60A) in complex with RNA revealed a non-canonical RNA-binding domain (RBD) and the molecular basis for the putative ribonuclease activity of LegC4. This work is the first description of a secreted bacterial effector that binds to eukaryotic host mRNA and has revealed a novel mechanism by which functionally related mRNAs may be post-transcriptionally regulated to alter cellular metabolism during infection.