Legionella pneumophila and the Host Endoplasmic Reticulum: How a Single Bacterial Protein Catalyzes a Novel Multi-Step Ubiquitination Pathway to Manipulate Tubular ER
Kotewicz, Kristin.
2018
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Abstract: Intracellular pathogens manipulate host organelles to support
replication within cells. In the case of Legionella pneumophila, the bacterium translocates
proteins that establish an endoplasmic reticulum (ER)-associated replication compartment.
Using high-resolution electron microscopy and fluorescence microscopy we show that the
bacterial Sde proteins target host reticulon 4 (Rtn4) t... read moreo control tubular ER dynamics,
resulting in tubule rearrangements as well as structural alterations to Rtn4 proximal to
the Legionella pneumophila containing vacuole (LCV). We utilized both in vitro recombinant
protein systems and L .pneumophila infection models to characterize the Sde proteins we
were able to identified the molecular mechanism promoting these ER rearrangements. Sde
ubiquitin (Ub) ligation to Rtn4, which occurs almost immediately after bacterial uptake, is
dependent on two sequential enzymatic activities expressed from a single Sde polypeptide:
an ADP-ribosyltransferase (ART) and a nucleotidase/phosphohydrolase (NP). The
ADP-ribosylated moiety of ubiquitin is a substrate for the nucleotidase/phosphohydrolase,
resulting in either transfer of ubiquitin to Rtn4, or phosphoribosylation of ubiquitin in
the absence of a ubiquitination target. These enzymatic activities collaborate to promote
ubiquitin ligation through a phosphodiester bond, rather than the classic isopeptide
ubiquitin linkage and specifically target Rtn4 and a subset of Rabs, including Rab33b and
Rab1[1]. These enzymatic activities are essential for intracellular replication and ER
association in amoeba, but not macrophages. We show Sde targeting of LCV associated polyUb
conjugates with ADP-ribosylate or phosphoribosylate modifications protect these products
from host disassembly and are required for intracellular replication in amoeba. We also
identified a third enzymatic domain in Sde proteins, a K63-polyubiquitin specific
deubiquitinase (DUB) that limits LCV polyUB within the first 30 minutes of infection that
fuels Sde Ub ligase function by generation of a free monoubiquitin substrate pool, while
also preventing lysosomal targeting ubiquitin signals (K63) from accumulating on the
Legionella vacuole membrane. This work demonstrates that Sde mediated ubiquitination
transforms peripheral ER tubules associated with the LCV through a novel molecular
mechanism, where a single bacterial protein catalyzes a multistep biochemical pathway to
control ubiquitination via both ubiquitin conjugation and deconjugation.
Thesis (Ph.D.)--Tufts University, 2018.
Submitted to the Dept. of Molecular Microbiology.
Advisor: Ralph Isberg.
Committee: Ekaterina Heldwein, John Leong, and Bree Aldrige.
Keywords: Microbiology, Cellular biology, and Molecular biology.read less - ID:
- 8623j883g
- Component ID:
- tufts:25430
- To Cite:
- DCA Citation Guide EndNote
