Legionella pneumophila regulates host cell motility by targeting Phldb2 with a 14-3-3ζ-dependent protease effector
- Department of Respiratory Medicine, Center for Pathogen Biology and Infectious Diseases, Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, State Key Laboratory for Zoonotic Diseases, The First Hospital of Jilin University, China
- Department of Microbiology, School of Basic Medical Sciences, Peking University Health Science Center, China
- Department of Biological Sciences, Purdue University, United States
- School of Life Science, Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, China
Figures
Figure 1 with 2 supplements
Lem8 is a cysteine protease-like Dot/Icm effector toxic to yeast.
(A) Alignment of Lem8 with several known cysteine proteases obtained by PSI-BLAST analysis. The strictly conserved catalytic residues are marked in red. Shown cysteine proteases are HopN1 and …
Figure 1—figure supplement 1
Sequence alignment of Lem8 with four bacterial cysteine protease effectors.
The strictly conserved residues were shown in dark purple background and the Cys-His-Asp motif are marked with white letters in a red background. HopN1 and AvrpphB are from P. syringae. YopT and …
Figure 1—figure supplement 2
Lem8 is dispensable for intracellular growth of L. pneumophila.
(A) Subcellular distribution of Lem8. Two hours after infected with the indicated bacterial strains, bone marrow-derived macrophages (BMDMs) were immunostained using anti-Legionella antibody to …
Figure 2 with 1 supplement
The interactions between Lem8 and 14-3-3ζ.
(A) Interactions between Lem8 and 14-3-3ζ detected by yeast two-hybrid assay. Yeast strains harboring the indicated constructs were streaked on Leu− and Trp− medium to select for plasmids (left) or …
Figure 2—figure supplement 1
Phosphorylation of Lem8 is not required for 14-3-3ζ binding.
Lysates of HEK293T cells expressing indicated HA-tagged proteins were subjected to immunoprecipitation with agarose beads coated with the HA antibody. The precipitates, as well as His6-Lem8 purified …
Figure 3 with 1 supplement
14-3-3ζ induces Lem8 to undergo self-cleavage.
(A) Self-processing of Lem8 requires 14-3-3ζ. His6-Lem8 or His6-Lem8C280S was incubated with His6-14-3-3ζ for 2 hr, proteins resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis …
Figure 3—figure supplement 1
Identification of the self-cleavage sites of Lem8.
(A) Determination of the self-cleavage site of Lem8 by mass spectrometry. A diagram of the sequence containing the recognition site with the two diagnostic peptides used to determine the cleavage …
Figure 4 with 1 supplement
Lem8 cleaves Phldb2 in a manner that requires 14-3-3ζ.
(A) Multiple alignments of the self-cleavage site of Lem8 with potential targets in human cells identified by bioinformatic analysis. Identical residues are highlighted in red. (B) Lem8 reduces the …
Figure 4—figure supplement 1
Verification of Lem8-mediated cleavage of candidate proteins and its cleavage of phldb2 at multiple sites.
(A) Cleavage of substrate candidates by Lem8. Flag- or HA-tagged Rasgrp2, Pak6, Exoc8, Ankrd13B, Chkb, Ppp6R1, Kiaa1033, Gnal, and Gpr61 expressed in HEK293T cells each was immunoprecipitated with …
Figure 5
A coiled coil motif in Lem8 is important for its interactions with 14-3-3ζ.
(A) Lem8 harbors a putative coil motif. A predicted coiled coil motif located in the amino end of Lem8 (top panel). The sequence ranges from the 45th residue to the 73rd residue with a coiled coil …
Figure 6
Autoprocessed Lem8 retains the cysteine protease activity.
(A) The autoprocessed form of Lem8 cleaves Phldb2 in cells. HA-Phldb2-Flag was coexpressed in HEK293T cells with Lem8 or the indicated truncation mutants including the self-processed form, Lem8∆C52. …
Figure 7 with 1 supplement
Lem8 contributes to cell migration inhibition by L. pneumophila.
(A) Establishment of cell lines stably expressing Lem8 or its enzymatically inactive mutant. HEK293T cells were transduced with lentiviral particles harboring the indicated plasmid at an …
Figure 7—figure supplement 1
Overexpression of Phldb2 suppressed the inhibitory effects of Lem8 on cell migration.
(A) Overexpression of Phldb2 in 293T cells stably expressing Lem8. A HEK293T-derived cell line stably expressing Lem8 was transfected with empty vector or HA-Phldb2 for 18 hr, and the cell lysates …
Author response image 1
Author response image 2
Additional files
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/73220/elife-73220-transrepform1-v2.docx
-
Supplementary file 1
Potential Lem8 interacting proteins identified by yeast two-hybrid screenings.
- https://cdn.elifesciences.org/articles/73220/elife-73220-supp1-v2.docx
-
Supplementary file 2
Bacterial strains, plasmids and primers used in the study.
- https://cdn.elifesciences.org/articles/73220/elife-73220-supp2-v2.docx
-
Source data 1
Source data for figures and figure supplements.
- https://cdn.elifesciences.org/articles/73220/elife-73220-data1-v2.zip
