Aggravating genetic interactions allow a solution to redundancy in a bacterial pathogen

Abstract

Interactions between hosts and pathogens are complex, so understanding the events that govern these interactions requires the analysis of molecular mechanisms operating in both organisms. Many pathogens use multiple strategies to target a single event in the disease process, confounding the identification of the important determinants of virulence. We developed a genetic screening strategy called insertional mutagenesis and depletion (iMAD) that combines bacterial mutagenesis and RNA interference, to systematically dissect the interplay between a pathogen and its host. We used this technique to resolve the network of proteins secreted by the bacterium Legionella pneumophila to promote intracellular growth, a critical determinant of pathogenicity of this organism. This strategy is broadly applicable, allowing the dissection of any interface between two organisms involving numerous interactions.

Figure 1. iMAD identifies redundant relationships between Dot/Icm translocated substrates

(A) Defective intracellular growth of L. pneumophila resulting from mutations in Dot/Icm TS genes combined with depletion of host cell proteins associated with the early secretory pathway. Cultured Drosophila cells treated with dsRNA were challenged with a library of L. pneumophila transposon mutants and the relative intracellular growth under each depletion condition was determined by TraSH. A growth disadvantage is defined as a 3.7 ± 0.2-fold growth defect, equivalent to an average TraSH ratio > 1.75 ± 0.1 standard deviations from the mean, which varies depending on the dsRNA treatment. Individual genes are clustered into distinct functional groups (red brackets, I-XIV) based on common behavioral patterns across all host conditions examined. (B) Host-condition specific growth defects for L. pneumophila null mutants. Growth of wipB (lpg0642) (Group IX) (left panel) and ceg32/sidI (lpg2504) (Group X) (right panel) null mutants in cultured Drosophila cells depleted of the indicated proteins by RNAi was compared to the wild-type (WT) strain. (C) The combined deletion of bacterial genes from separate functional groups impaired intracellular replication of L. pneumophila in untreated Drosophila cells. (D) Deletion of genes belonging to the same functional group did not adversely affect bacterial growth in untreated Drosophila cells. (E) Genetic interactions define distinct relationships between different functional groups. (F) Summary of redundant relationships between individual functional groups defined by genetic interaction mapping. A solid black line indicates aggravating genetic interactions on intracellular growth of the bacterium in untreated Drosophila cells when two L. pneumophila genes, one from each of the corresponding functional groups (indicated by blue lettering), are deleted in combination. (B–E) Bacterial growth was determined by colony forming units (cfus) on solid media from lysed host cells 48 hours post infection relative to the number of cfus recovered 1 hour post infection. Data are means from at least 2 independent experiments ± standard deviation of 3 replicates. *p <0.05 relative to the wild-type (WT) strain.

Figure 2. Impaired growth of L. pneumophila mutants in natural hosts

(A) The ΔwipBΔlidA mutant shows a growth defect in A. castellanii relative to the wild-type (WT) and corresponding single deletion strains. (B) The ΔwipBΔlidA mutant is impaired in the accumulation of vacuoles containing large numbers of bacteria. A. castellanii were infected with L. pneumophila strains expressing GFP, fixed 1, 6, 8 or 10 hours post infection and the number of bacteria per phagosome was scored using fluorescence microscopy. (C) The ΔwipBΔlidA mutant is defective for recruitment of the ER-targeted fusion protein GFP-HDEL in D. discoideum. D. discoideum expressing GFP-HDEL were infected with L. pneumophila strains expressing the red fluorescent protein tdTomato for 4 hours, fixed and visualized by fluorescence microscopy (upper panel). The number of GFP-HDEL positive Legionella vacuoles was scored, counting 100 vacuoles per replicate (lower panel). (D) Defective intracellular growth of the ΔwipBΔlidA mutant in D. discoideum. (A, D) A. castellanii and D. discoideum were infected with L. pneumophila and bacterial growth was monitored as described in Fig. 1 over 3 days, equivalent to 3 consecutive rounds of replication and plotted as in Fig. 1. Data are representative of at least 2 independent experiments ± standard deviation of 3 replicates. *p <0.05 relative to the wild-type strain.

Figure 3. Hierarchial cluster analysis predicts double mutants defective in maintaining vacuole integrity

(A) Growth of the ΔwipBΔlidA mutant was reduced in bone marrow-derived murine macrophages compared to the wild-type (WT) and single deletion mutant strains. Bacterial growth was determined as described in Fig. 2. (B) ΔwipBΔlidA mutant-containing vacuoles showed enhanced recruitment of Galectin-3. Macrophages were infected with L. pneumophila for 6 hours, fixed and stained for Legionella and Galectin-3 then visualized by fluorescence microscopy (upper panel). The number of Legionella vacuoles staining positive for Galectin-3 were scored (lower panel). (C) Cells infected with the ΔwipBΔlidA mutant exhibit increased host cell death based on aberrant nuclear morphology characteristic of apoptosis. Macrophages were infected with L. pneumophila for 8 hours, fixed and stained for Legionella then treated with Hoechst stain. (D) ΔwipBΔlidA mutant bacteria exhibited aberrant morphology after challenge of macrophages. Bacteria visualized by fluorescence microscopy as in (B) showed both swelling and blebbing in vacuoles containing either single or multiple bacteria relative to the smooth rod shaped morphology of wild-type bacteria (upper panel). The number of vacuoles in which at least one bacterium exhibited aberrant morphology was scored (bottom panel). (E) The ΔwipBΔlidA mutant showed a defect in the number of vacuoles containing large numbers of bacteria in a macrophage host. Macrophages were infected with L. pneumophila for 10 hours, fixed and stained for Legionella and the number of bacteria per vacuole was scored. (A–E) Data are representative of at least 2 independent experiments ± standard deviation of 3 replicates, scoring 100 vacuoles (B, D, E) or infected cells (C) per replicate. *p <0.05 relative to the wild-type strain unless indicated otherwise.

Figure 4. Host-specific aggravating genetic interactions between mutations in genes encoding Dot/Icm translocated substrates

(A) Deletion of lidA or legA3 (Group XI) attenuated the growth defect of the ΔsdhA mutant (Group IX) in D. discoideum. (B) The ΔsdhAΔlidA mutant was more defective for growth than the ΔsdhAΔlegA3 mutant in A. castellanii. (C) A ΔmavPΔlegA3 mutant grew as well as a ΔlegA3 single mutant in bone marrow-derived murine macrophages. (D) The ΔmavPΔlegA3 mutant showed reduced growth relative to the ΔlegA3 mutant in A. castellanii. (A–D) Bacterial growth was monitored as described in Fig. 2. WT: wild-type. Data are representative of at least 2 independent experiments ± standard deviation of 3 replicates.