Characterisation of the L. pneumophila effectors LtpK and LtpM

Abstract

Legionella pneumophila is an intracellular pathogen and the causative agent of a pneumonia called Legionnaire’s disease or legionellosis. L. pneumophila translocates ~ 300 so called effector proteins into the host cells via a type IVB secretion system to enable its survival and replication. The role of most of these effectors is still unknown, however several have been characterised to modulate vesicle trafficking or phosphoinositol composition of the Legionella containing vacuole (LCV). Here we characterised two effector proteins: LtpK, which is conserved amongst clinical and environmental isolates, and LtpM, which is only found in a subset of L. pneumophila strains. LtpK was found to have a diffuse cytoplasmic localisation upon transfection, during infection it also locates diffusely in the cytoplasm but also shows nuclear localisation. Different potential cellular interaction partners for LtpK were identified in a Yeast-2-Hybrid screen, namely ASXL1, ACBD3 and VBP1. The role of these interactions requires further investigation. It should be noted, however, that it was not possible to confirm these interactions in a pBio-pulldown. LtpM on the other hand shows a vesicular cellular localisation upon transfection and appears on the membrane of the Legionella containing vacuole (LCV) and vesicular structures upon infection. These vesicular structures were identified as early endosomes by staining for the early endosomal marker EEA1. Furthermore, binding of LtpM to the phosphoinositide (PI) phosphatidylinositol-3-phosphate (PI3P) was characterized. LtpM binds to PI3P via its C-terminal domain (LtpM455-639), which is 63% identical to the PI3P binding domain of the previously characterised L. pneumophila effector LtpD. Moreover, LtpM shares secondary structure homology in its N-terminus (LtpM1-145) to a glycosyltransferase. It also harbours a DxN motif, which is as DxD or DxN motif characteristic of glycosyltransferases. It could be shown that LtpM has indeed mono-o-glycosyltransferase activity. LtpM inhibits growth of yeast after transformation and this growth inhibition depends on the enzymatic activity of the glycosyltransferase domain. As the enzymatic activity of LtpM could be resolved, however, a cellular target for LtpM could not be identified. More research is necessary to find the target of LtpM that gets glycosylated and to therefore fully understand the role of LtpM for the pathogenicity of L. pneumophila.