Systematic Identification of Host Cell Regulators of Legionella pneumophila Pathogenesis Using a Genome-wide CRISPR Screen

Abstract

During infection, Legionella pneumophila translocates over 300 effector proteins into the host cytosol, allowing the pathogen to establish an endoplasmic reticulum (ER)-like Legionella-containing vacuole (LCV) that supports bacterial replication. Here, we perform a genome-wide CRISPR-Cas9 screen and secondary targeted screens in U937 human monocyte/macrophage-like cells to systematically identify host factors that regulate killing by L. pneumophila. The screens reveal known host factors hijacked by L. pneumophila, as well as genes spanning diverse trafficking and signaling pathways previously not linked to L. pneumophila pathogenesis. We further characterize C1orf43 and KIAA1109 as regulators of phagocytosis and show that RAB10 and its chaperone RABIF are required for optimal L. pneumophila replication and ER recruitment to the LCV. Finally, we show that Rab10 protein is recruited to the LCV and ubiquitinated by the effectors SidC/SdcA. Collectively, our results provide a wealth of previously undescribed insights into L. pneumophila pathogenesis and mammalian cell function.

Keywords: C1ORF43; CRISPR screen; Legionella pneumophila; RAB10; host-pathogen interaction; intracellular bacteria; pathogen; phagocytosis.

Figure 1.. CRISPR-Cas9 knockout screens for modifiers of L. pneumophila pathogenesis.

(A) U937 cell killing by L. pneumophila. U937 cells were infected with the indicated MOI of WT or ΔdotA L. pneumophila and live cells were counted 24 hours after infection using flow cytometry and compared between strains (n = 2 technical replicates, two-tailed Student’s t test, **P<0.01; error bars, s.d. (B) Schematic of screen. Cas9-expressing U937 cells were infected with a genome-wide lentiviral sgRNA library. Half of the population was treated with 5 rounds of L. pneumophila infection while the other half was maintained in log-phase growth before quantification by deep sequencing. (C) Volcano plot of confidence score vs. effect size for all genes. Blue dots indicate genes whose knockout protect host cells from L. pneumophila infection and red dots indicate genes whose knockout sensitize host cells to L. pneumophila infection using a 10% FDR cutoff. Genome-wide screen results are in Table S1. (D) Adjusted P values for select non-redundant enriched gene-ontology (GO) terms using GOrilla on a ranked list of genome-wide screen hits. (E) Schematic of genes included in batch retest library. Genes in batch retest library are listed in Table S2. (F) Correlation of signed confidence scores between genome-wide and batch retest screens. See also Figure S1.

Figure 2.. Summary of modifiers of L. pneumophila pathogenesis from CRISPR screens

(A) Schematic of selected hits in cellular compartments or processes. Genes are color-coded by effect size. Previously known host factors hijacked by L. pneumophila are outlined in orange. Complete batch retest screen results are in Table S3. (B) Validation of selected hits using individual sgRNAs in a competitive killing assay. Knockout or negative control cells (GFP+) were cocultured with negative control cells (mCherry+) in equal numbers and either infected with L. pneumophila at MOI = 100 or left uninfected. The percentage of GFP+ cells was measured by Incucyte and the % GFP change at 48 h was compared between L. pneumophila-infected and uninfected populations (n = 3 technical replicates, two-tailed Student’s t test, ns = not significant, *P<0.05, ***P<0.001; error bars, s.d.). Data shown are representative of 2 independent experiments. See also Figure S2.

Figure 3.. C1orf43 and KIAA1109 are regulators of phagocytosis

(A) Effect of gene knockouts on phagocytosis. Differentiated U937 knockout cells (GFP+) were treated with pHrodo Red-labeled ΔdotA L. pneumophila at MOI = 10. Phagocytic index was measured as total mCherry fluorescence intensity at 6 h normalized to the number of GFP+ cells. Knockout cells were compared to three negative controls with the most stringent significance value reported (n = 4 technical replicates, one-way ANOVA, Dunnett’s multiple comparison test, ns = not significant, *P<0.05, **P<0.01, ***P<0.001; error bars, s.d.). Data shown are representative of 3 independent experiments. (B) Timecourse measurements of pHrodo Red-labeled ΔdotA L. pneumophila phagocytosis for C1orf43 clonal knockout cells. Negative control cells treated with 5 μg/mL Cytochalasin D were used as a non-phagocytic control. Data represent mean ± s.d. of 4 technical replicates and are representative of 3 independent experiments. (C) Phagocytosis of various prey particles in C1orf43 clonal knockout cells. U937 C1orf43 knockout clones or negative control cells were treated with pHrodo Red-labeled ΔdotA L. pneumophila, E. coli, S. aureus, zymosan, and 1 μm amino magnetic polystyrene beads and phagocytic index was measured at 6 h. Negative control cells treated with 5 μg/mL Cytochalasin D were used as a non-phagocytic control. Cytochalasin D-treated and C1orf43 knockout cells were compared to WT cells (n = 3 independent experiments, two-tailed Student’s t test, **P<0.01, ***P<0.001; error bars, s.d. (D) Phagocytosis of L. pneumophila in Raw 264.7 mouse macrophages. Raw 264.7 cells were treated with pHrodo Red-labeled ΔdotA L. pneumophila and phagocytic index was measured at 6 h. Data represent mean ± s.d. of 4 individual knockout clones for each gene, with each clone used to perform the experiment in technical quadruplicates. Knockout cells were compared to negative control cells (n = 4 individual knockout clones, two-tailed Student’s t test, **P<0.01 (E) Overexpression of C1orf43 rescues C1orf43 knockout phenotype in a competitive killing assay. GFP+ U937 C1orf43 clonal knockout cells or negative control cells were lentivirally infected with constructs encoding C-terminal 3xFLAG-tagged full-length C1orf43 or the first 41 amino acids of C1orf43 (C1orf43_1–41), or a negative control (GFP). These cells were cocultured with mCherry+ negative control cells in equal numbers and infected with L. pneumophila at MOI = 100 or left uninfected. The percentage of GFP+ cells was measured by Incucyte and the % GFP change at 24 h was calculated. L. pneumophila-infected cells were compared to uninfected cells (n = 3 technical replicates, two-tailed Student’s t test with Holm-Sidak multiple comparisons correction, ns = not significant, ***P<0.001; error bars, s.d.). Data shown are representative of 2 independent experiments. Cartoon shows C1orf43 domain structure and C1orf43_1–41 truncation mutant. TM indicates the single-pass transmembrane domain in C1orf43. See also Figure S3.

Figure 4.. RABIF regulates L. pneumophila replication and ER recruitment to the LCV by stabilizing Rab10 expression

(A) Effect of gene knockouts on L. pneumophila burden. Differentiated U937 knockout cells were infected with mCherry-expressing L. pneumophila at MOI = 1. Total mCherry fluorescence intensity per well was measured by Incucyte at 36 h as a combined metric of L. pneumophila uptake and intracellular replication. Knockout cells were compared to three negative controls with the most stringent significance value reported (n = 3 technical replicates, one-way ANOVA, Dunnett’s multiple comparison test, *P<0.05, **P<0.01, ***P<0.001; error bars, s.d.). Data shown are representative of 2 independent experiments. (B) L. pneumophila intracellular replication analysis in RABIF and RAB10 clonal knockout cells. Differentiated U937 clonal knockout cells were infected with mCherry-expressing L. pneumophila at MOI = 1. Total mCherry fluorescence intensity was measured by Incucyte every 2 h for 36 h and compared between knockout cells and negative control cells. Data represent mean ± s.d. of 3 technical replicates and are representative of 2 independent experiments. (C) Results from GFP-RABIF affinity purification followed by mass spectrometry (APMS). U937 cells stably expressing GFP-RABIF or GFP (negative control) were lysed and co-immunoprecipitated using anti-GFP beads. Each data point represents a protein identified by mass spectrometry. The x-axis shows log₂ fold enrichment of the protein in the GFP-RABIF pull-down vs. GFP pull-down using the geometric mean of three experimental replicates. The y-axis shows the confidence score (SAINT score). Tabular AP-MS results are in Table S4. (D) Immunoblot analysis of RABIF, Rab10, and Rab1a levels in U937 RABIF and RAB10 knockout cells or negative control cells. (E) HeLa FcγRII RABIF and RAB10 clonal knockout cells or negative control cells were infected with WT or ΔdotA L. pneumophila (L.p.) at MOI = 1 for 4 h, fixed, and immunostained for L. pneumophila (red) and RTN4 (green). Nuclei were stained with Hoechst dye (blue). Scale bar, 10 μm. (F) Quantification of the percentage of individual intracellular L. pneumophila co-localizing with RTN4. Knockout cells were compared to negative control cells for infection with WT L. pneumophila (n = 3 technical replicates with at least 50 LCVs analyzed per replicate, two-tailed Student’s t test, *P<0.05, **P<0.01, ***P<0.001; error bars, s.d. See also Figures S4 and S5.

Figure 5.. Rab10 is recruited to the LCV and ubiquitinated by SidC/SdcA during L. pneumophila infection

(A) HeLa FcγRII cells stably expressing GFP-Rab10 were infected with WT, ΔdotA, or ΔsidC-sdcA complemented with empty vector, or plasmid-encoded SidC or SdcA L. pneumophila at MOI = 3 for 1 h, fixed, and immunostained for L. pneumophila (red). Nuclei were stained with Hoechst dye (blue). Scale bars, 10 μm. (B) Quantification of the percentage of intracellular L. pneumophila co-localizing with GFP-Rab10 for the indicated L. pneumophila strains. Mutant L. pneumophila strains were compared to WT L. pneumophila (n = 3 technical replicates with at least 50 LCVs analyzed per replicate, two-tailed Student’s t test, ns = not significant, ***P<0.001; error bars, s.d. (C and D) HEK293 FcγRII cells were infected with WT or ΔdotA L. pneumophila at MOI = 100 for 1 h or 8 h and Rab10 ubiquitination (C) and protein abundance (D) were analyzed by mass spectrometry (n = 3 biological replicates). Plots show log₂ fold change vs. uninfected control (x-axis) vs. P value (y-axis). Dotted line represents p-value cutoff of 0.05. (E) Immunoblot analysis of Rab10 ubiquitination. HEK293 FcγRII cells stably expressing 3xFLAG-Rab10 were either untransfected (lanes 1–5) or transfected with FLAG-tagged full-length SdcA or SdcA lacking residues 222–315 (lanes 6 and 7); 21 h after transfection, cells were either uninfected or infected with WT, ΔdotA, ΔsidC-sdcA, or ΔsidC-sdcA complemented with plasmid-encoded SdcA L. pneumophila. Cells were lysed 1 h post-infection and probed with anti-FLAG antibody. (F) Schematic and 3D structure showing Rab10 residues ubiquitinated by L. pneumophila during infection. Green, Mg²⁺; Teal, GTP; Magenta, ubiquitinated residues. See also Figure S5.