Discipline: Biological Sciences
Lauren Kunselman - University of South Carolina
Co-Author(s): Kristin Kotewicz, Tufts University, Boston, MA; Mengyun Zhang, Tufts University, Boston, MA; Ralph Isberg, Tufts University, Boston, MA
Legionella pneumophila is an intravacuolar bacterial pathogen that can translocate over 300 proteins into a host cell through its main virulence factor, the type four secretion system (T4SS). The Sde family proteins are important virulence factors for L. pneumophila intracellular replication in amoebal hosts. A single Sde protein can conjugate ubiquitin (Ub) to host proteins such as Reticulon 4 through a novel mechanism independent of host Ub machinery using the concerted activity of an ADP-ribosyltransferase (ART) and nucleotidase. This mechanism and Ub linkages have not been previously observed in nature. Sde proteins also ubiquitinate specific host translation initiation factors and phosphoribosylate polyubiquitin chains in extracts or using pure proteins. Yet despite both Sde-mediated mono-ubiquitination and host-promoted polyubiquitination at the L. pneumophila containing vacuole (LCV), autophagy does not proceed in infected host cells. Sde modifications of polyubiquitin chains prevents p62, an autophagic receptor, from binding, perhaps providing an answer. We hypothesize that phosphoribosylation of Ub blocks p62 from initiating autophagy of the LCV, and ubiquitination of initiation factors leads to translation inhibition.
To investigate autophagy suppression, A/J macrophages were infected with Sde family knockouts or a wild type (WT) strain and fixed 1-hour post-infection. The host nucleus, p62, and L. pneumophila were stained with antibodies and analyzed via immunofluorescence microscopy. With mutant strains, there were high levels of p62 colocalization with the bacterial compartment compared to the WT. The scarcity of p62 staining in the WT indicates that autophagy is blocked, while the presence of p62 in the knockouts is consistent with autophagic recognition.
To test translation inhibition, a HeLa cell lysate translation assay was performed in the presence of purified SdeC, and GFP was measured by fluorometry. There was a significant reduction in translation in the presence of SdeC (p < 0.01). The results indicate that the Sde family can interfere with host autophagy and translation. This research will promote a better understanding of the biological pathways that L. pneumophila manipulates and could lead to the development of novel therapeutics. References: Isberg, R.R., O'Connor, T.J., & Heidtman, M. 2009. The Legionella pneumophila replication vacuole: making a cosy niche inside host cells. Nature Reviews Microbiology, 7(1):13-24. Kotewicz, K.M., Ramabhadran, V., Sjoblom, N., Vogel, J.P., Haenssler, E., Zhang, M., Behringer, J., Scheck, R.A., & Isberg, R.R. 2017. A Single Legionella Effector Catalyzes a Multistep Ubiquitination Pathway to Rearrange Tubular Endoplasmic Reticulum for Replication. Cell Host Microbe, 21(2):169-181.
Funder Acknowledgement(s): This study was supported by a grant from NSF REU awarded to Lauren Kunselman, University of South Carolina, Columbia, SC.
Faculty Advisor: Ralph Isberg, firstname.lastname@example.org
Role: I harvested A/J macrophages, infected the macrophages with the different pathogen strains, fixed the macrophages, performed the antibody staining, photographed the macrophages under the microscope, and analyzed the images for p62 staining. I also performed statistical analyses on the data acquired from the images. In addition, I performed the HeLa cell lysate translation assay and measured GFP using fluorometry. I performed statistical analyses on this data, as well.