The Legionella effector WipB is a translocated Ser/Thr phosphatase that targets the host lysosomal nutrient sensing machinery

Legionella pneumophila infects human alveolar macrophages and is responsible for Legionnaire’s disease, a severe form of pneumonia. L. pneumophila encodes more than 300 putative effectors, which are translocated into the host cell via the Dot/Icm type IV secretion system. These effectors highjack the host’s cellular processes to allow bacterial intracellular growth and replication. Here we adopted a multidisciplinary approach to investigate WipB, a Dot/Icm effector of unknown function. The crystal structure of the N-terminal domain at 1.7 Å resolution comprising residues 25 to 344 revealed that WipB harbours a Ser/Thr phosphatase domain related to the eukaryotic phospho-protein phosphatase (PPP) family. The C-terminal domain (residues 365–524) is sufficient to pilot the effector to acidified LAMP1-positive lysosomal compartments, where WipB interacts with the v-ATPase and the associated LAMTOR1 phosphoprotein, key components of the lysosomal nutrient sensing (LYNUS) apparatus that controls the mammalian target of rapamycin (mTORC1) kinase complex at the lysosomal surface. We propose that WipB is a lysosome-targeted phosphatase that modulates cellular nutrient sensing and the control of energy metabolism during Legionella infection.


Supplementary Figure 1.
Structure-based sequence alignment of the WipB 25-344 with WipA (PDB entry code 5N6X), PP2B (PDB entry code 5C1V), PP2A (PDB entry code 2IAE) and PP1 (PDB entry code 5INB). Similar structures search was performed using the Dali server 1 , selected top-scores aligned with PDBeFold 2 and the resulting alignment was rendered with ESPript 3 . The WipB secondary structure elements are shown above the aligned sequences. Conserved phosphatase motifs are labeled DXH, GDxxDR, and NHE, and color-coded orange, green and blue, respectively.

P S H R L T A K E V F D N D G K P R V D I L K A H L M K E G R L E E S V A L R I I T E G A S I L R Q E K N L L D I D
. . . . . . . . . L N I D S I I Q R L L E V R G S K P G K N V Q L Q E N E I R G L C L K S R E I F L S Q P I L L E L E A 3 3 4 3 5 3 6 3 7 3       and Asp331 (396 in WipB) are involved in catalysis while Arg369 of WipA was hypothesized to be involved in pTyr recognition. In WipB, the equivalent residue of Arg369 is Arg310, which is conformationally restrained away from the active site and therefore cannot assume a role in substrate recognition. We hypothesize that this is the reason why WipB is inactive against pTyr-containing peptides (see main text).

Supplementary Figure 4: Localisation of GFP-WipB in cultured cells
HeLa cells expressing GFP-WipB (green) were fixed and stained with an anti-giantin or anti-calreticulin antibody (red), prior to fixation with Mitotracker (red), or loaded with transferrin (magenta). Scale bars, 10µm.

Figure 3d
The membrane was cut in 3 horizontal strips around the molecular weight of the protein of interest (v-ATPase A, 68kDa; v-ATPase B, 57kDa; LAMTOR1, 17kDa). Each strip was incubated with the corresponding primary antibody, then the secondary HRP-conjugated antibody, and finally the ECL reagents. The 3 strips were then re-assembled carefully before exposure.

Figure 3e
The membrane was cut in 5 horizontal strips around the molecular weight of the protein of interest (v-ATPase A, 68kDa; v-ATPase B, 57kDa; LAMTOR1, 17kDa; GFP 86kDa and 30kDa). Each strip was incubated with the corresponding primary antibody, then the secondary HRP-conjugated antibody, and finally the ECL reagents. The 5 strips were then re-assembled carefully before exposure.

Figure S3a
The membrane was cut in 2 horizontal strips around the molecular weight of the protein of interest (GFP, from 20kDa to top, Sec61 from 20kDa to bottom). Each strip was incubated with the corresponding primary antibody, then the secondary HRPconjugated antibody, and finally the ECL reagents. The 2 strips were then reassembled carefully before exposure.

Figure S5b
The membrane was cut in 2 horizontal strips around the molecular weight of the protein of interest (GFP, from 20kDa to top, Sec61 from 20kDa to bottom). Each strip was incubated with the corresponding primary antibody, then the secondary HRPconjugated antibody, and finally the ECL reagents. The 2 strips were then reassembled carefully before exposure.