Characterisation of the Legionella pneumophila effector RidL

Finsel, Ivo (2014): Characterisation of the Legionella pneumophila effector RidL. Dissertation, LMU München: Medizinische Fakultät

Abstract

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.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 6. Mai 2014
• 1. Bericht­erstatter:in: Hilbi, Hubert