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) ... read moreto 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
