Characterisation of the Legionella pneumophila effector RidL

The Gram-negative bacterium Legionella pneumophila naturally
parasitises environmental amoebae, but is also able to infect human
alveolar macrophages in a mechanistically similar manner. This can
result in the mild "Pontiac fever", a flu-like illness, or a
potentially lethal pneumonia termed Legionnaires' disease". Crucial
for establishing an intracellular replication niche is the Icm/Dot
type IV secretion system (T4SS), which translocates approximately
300 different "effector" proteins into the host cell. These
substrates enhance uptake efficiency into phagocytes and direct
formation of a replication-permissive compartment, called the
Legionella-containing vacuole (LCV), and ultimately the egress of
the bacteria. Some of the effectors interfere with small GTPases,
phosphoinositide metabolism or the ubiquitination machinery, and
modulate host cell signalling and vesicle trafficking. We developed
a method to isolate intact LCVs by using immuno-magnetic separation
with an LCV-specific antibody followed by density gradient
centrifugation. Proteomic analysis of the purified phagosomes
together with findings of previous studies showed, that the
vacuoles harbour markers of the endosomal network, associate with
mitochondria, early secretory vesicles and the endoplasmic
reticulum, but avoid fusion with lysosomes. Our investigations of
the novel L. pneumophila effector RidL revealed that the LCV also
communicates with the retrograde vesicle trafficking pathway of
infected cells. This pathway recycles amongst others acid-hydrolase
receptors, such as the cation-independent mannose 6-phosphate
receptor (CIMPR), from the tubular endosomal network back to the
trans-Golgi. This transport requires the multiprotein "retromer"
complex, which consists of two major subunits: the heterotrimeric
cargo-selective subcomplex comprising the proteins Vps26, Vps29 and
Vps35 and the membrane-deforming heterodimeric subcomplex composed
of any combination of the phosphoinositide (PI)-binding sorting
nexins SNX1 or SNX2 plus SNX5 or SNX6. Pull-down experiments with
lysates of RAW 264.7 macrophages or D. discoideum amoebae revealed
Vps26, Vps29 and Vps35 to be retained by the then uncharacterised
protein RidL, which represented an intriguing, novel effector
interaction. Like most T4SS substrate mutants, L. pneumophila
lacking ridL showed no phenotype for growth in liquid AYE medium
and uptake into phagocytes compared to wild-type bacteria. However,
intracellular replication was strongly impaired for the mutant
strain in several host cell lines. RidL is preferentially expressed
in the late post-exponential growth phase and translocated in an
T4SS-dependent manner at early time-points of the infection,
suggesting a role shortly after the uptake of the bacteria. The
effector exhibited a bipolar localisation on the LCV membrane, but
upon overexpression the protein covered the entire vacuole.
Interestingly, RidL bound the lipid phosphatidylinositol
3-phosphate (PtdIns(3)P), a known eukaryotic endosomal membrane
anchor, and also specifically bound to the retromer subunit Vps29.
Although the protein had no effect on the acquisition of Vps26,
Vps29 and Vps35, the percentage of LCVs positive for the retrograde
cargo receptors CIMPR or sortilin was reduced in presence of RidL,
suggesting interference with the retrograde transport pathway.
Furthermore, significantly less SNX1- and SNX2-positive LCVs were
detected in cells infected with wild-type L. pneumophila compared
to the ridL mutant strain. Moreover, RidL competed with SNX1 for
binding at PtdIns(3)P-positive membranes. To directly examine the
influence of RidL on retrograde trafficking, the retromer-dependent
transport of cholera and Shiga toxin inside cells was analysed in
macrophages infected with wild-type or ridL L. pneumophila, and in
HeLa cells ectopically producing RidL, respectively. In both cases,
the trafficking was inhibited by RidL, and for cholera toxin the
transport was arrested at the endosomal stage. In line with these
findings, siRNA knockdown experiments revealed that a functional
retrograde pathway restricted intracellular growth of L.
pneumophila. Taken together, we postulate that RidL (Retromer
interactor decorating LCVs) inhibits retrograde trafficking at
endosomes by binding to the retromer subunit Vps26 and/or by
competition with sorting nexins, thus promoting intracellular
replication of L. pneumophila. Collectively, the results obtained
in this thesis shed light on the host factor composition of LCVs
and provide mechanistic insights into a novel L. pneumophila
effector protein.