Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella-containing vacuole

Abstract

Legionella pneumophila requires the Dot/Icm translocation system to replicate in a vacuolar compartment within host cells. Strains lacking the translocated substrate SdhA form a permeable vacuole during residence in the host cell, exposing bacteria to the host cytoplasm. In primary macrophages, mutants are defective for intracellular growth, with a pyroptotic cell death response mounted due to bacterial exposure to the cytosol. To understand how SdhA maintains vacuole integrity during intracellular growth, we performed high-throughput RNAi screens against host membrane trafficking genes to identify factors that antagonise vacuole integrity in the absence of SdhA. Depletion of host proteins involved in endocytic uptake and recycling resulted in enhanced intracellular growth and lower levels of permeable vacuoles surrounding the ΔsdhA mutant. Of interest were three different Rab GTPases involved in these processes: Rab11b, Rab8b and Rab5 isoforms, that when depleted resulted in enhanced vacuole integrity surrounding the sdhA mutant. Proteins regulated by these Rabs are responsible for interfering with proper vacuole membrane maintenance, as depletion of the downstream effectors EEA1, Rab11FIP1, or VAMP3 rescued vacuole integrity and intracellular growth of the sdhA mutant. To test the model that specific vesicular components associated with these effectors could act to destabilise the replication vacuole, EEA1 and Rab11FIP1 showed increased density about the sdhA mutant vacuole compared with the wild type (WT) vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant redistribution. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilise vacuole integrity during infection.

Keywords: Legionella; RNAi; Rab proteins; intracellular growth; vesicle trafficking.

Figure 1

RNAi screens identify membrane trafficking proteins that antagonise L. pneumophila ΔsdhA intracellular growth and vacuole integrity. (a) Identification of siRNA that enhance intracellular growth. RAW 264.7 macrophages were seeded in 96‐well plates and transfected the following day with a siRNA library directed against transcripts encoding membrane trafficking proteins. Transfected cells were challenged with L. pneumophila ΔsdhA Lux⁺ and luminescence was measured at 12 and 24 hr post infection (hpi). (b) Identification of shRNAs that decrease vacuole permeability in primary macrophages. A/J bone marrow‐derived progenitors were seeded in 96‐well plates. Cells were transduced with shRNAs targeting genes identified in the siRNA screen (a). Terminally differentiated BMDM transductants were challenged with ΔsdhA for 6 hr. Plates were fixed and stained for cytosol‐detected L.p. and total L.p.(c) Results of high‐throughput siRNA screen for enhanced intracellular growth of L. pneumophila ΔsdhA Lux⁺. Experimental design, Figure 1a, performing luminescence readings at 12 or 24 hpi, with least three replicate measurements for each gene target being performed Luminescence from each experimental well was normalised to the average luminescence of the non‐targeting siRNA‐treated control wells of the same plate. Normalised ZMAD values are shown colour‐coded for each siRNA of the library. A cut‐off of ZMAD ≥1.5 was used to select potential candidates. (d) Candidates identified in Figure 2a and additional targets not covered in the original screen were tested in a high‐throughput shRNA screen, performing assay detailed in Figure 1b, detecting cytosolic exposure of bacteria. Three replicates for each knockdown condition were performed, capturing 16 images/well with automated microscopy. Image capture, analysis and normalisation of data were performed as described (Experimental Procedures). (e) Depletion of transcripts for a subset of Rab11 effectors results in increased vacuole integrity during L. pneumophila ΔsdhA intracellular growth. Assay conditions as in Figure 2b. Statistical analyses were performed on normalised data by unpaired t test (*p < .05) (Experimental Procedures)

Figure 2

Depletion of proteins that control early endosome dynamics results in increased vacuole integrity after BMDM challenge with L. pneumophila ΔsdhA. (a) Depletion of Rab5 isoforms partially rescues ΔsdhA vacuole integrity. A/J bone marrow‐derived macrophages were nucleofected with noted siRNAs. Knockdown efficiency was assessed by immunoblots with noted antibodies (left panels). Nucleofected macrophages were challenged with either WT or ΔsdhA Legionella, fixed at 6 hpi, and immunostained to determine cytosol exposure. Percent of cytosol‐detected bacteria was quantified, as described (Experimental Procedures). (b) Growth of noted L pneumophila strains in nucleofected BMDM. After BMDMs were challenged for 14 hr, the cells were fixed and probed to determine yield of bacteria in each macrophage. Number of bacteria per vacuole were quantified microscopically, and binned into four groups, displaying the binned vacuole size based on the graph legend (Experimental Procedures; [Luo & Isberg, 2004]). (c) Depletion of downstream Rab5 effector EEA1 rescues ΔsdhA vacuole integrity and growth defects. Nucleofection, vacuole integrity and bacterial yields determined as in panels (a and b). Statistical analyses were performed on normalised data by unpaired t test (*<.05; **<.01; ***<.001; Experimental Procedures)

Figure 3

The SdhA protein interferes with contact of early endosomal compartments with the LCV. (a) Representative immunofluorescence microscopy images of the Legionella‐containing vacuole in infected BMDMs at 4hpi, challenged with noted bacterial strains. Cells were probed with anti‐L. pneumophila before and after permeabilisation, to determine vacuole integrity (Experimental Procedures) as well as anti‐SidC and anti‐EEA1 after permeabilisation. EEA1 and SidC are pseudo‐coloured in green and red, respectively. Insets are magnified 3.25‐fold from the original image by changing resolution. Other panels are identical resolution to original grabbed images. (b) Images from (a) were analysed to quantify EEA1 events (Experimental Procedures). Total of 146 vacuoles from two experiments were analysed. (c) Frequency distribution of events from (b). Interquartile range was calculated from integer values. (d) Lack of correlation between intact or cytosol‐detected (permeable) ΔsdhA vacuoles and distribution of EEA1‐containing compartments at the vacuole. (e) BMDMs nucleofected with siRNA were challenged with noted L. pneumophila strains and stained as in (a). Images were captured and analysed after capture to determine localisation of EEA1 near the vacuole) (Experimental Procedures). Total of 182 vacuoles from two experiments were analysed. (f) Frequency distribution of localisation events from (e). Interquartile range was calculated from integer values. Statistical tests were as in Figure 2

Figure 4

Depletion of proteins associated with recycling dynamics results in increased vacuole integrity after BMDM challenge with L. pneumophila ΔsdhA. (a) Specific depletion of Rab11b rescues ΔsdhA vacuole integrity. A/J bone marrow‐derived macrophages were siRNAs treated as in Figure 2, and knockdown efficiency was assessed (left panels). Nucleofected macrophages were challenged with either WT or ΔsdhA Legionella and percent of cytosol‐detected bacteria was quantified (Experimental Procedures). (b) Growth of noted L pneumophila strains in nucleofected BMDM for 14 hr. and quantified as described (Figure 2; Experimental Procedures; [Luo & Isberg, 2004]). (c) Depletion of Rab11 effector Fab11FIP1 rescues ΔsdhA vacuole integrity and growth defects. Nucleofection, vacuole integrity and bacterial yields determined as in panels (A and B). Statistical analyses were performed on normalised data by unpaired t test (*<.05; **<.01; ***<.001; Experimental Procedures)

Figure 5

Depletion of anterograde transport Rab8b and a downstream effector reduces bacterial cytosolic exposure. (a) Depletion of Rab8b rescues ΔsdhA vacuole integrity. A/J bone marrow‐derived macrophages were siRNAs treated and knockdown efficiency was assessed (left panels). Nucleofected macrophages were challenged with either WT or ΔsdhA Legionella and percent of cytosol‐detected bacteria was quantified (Experimental Procedures). (b) Growth of noted L pneumophila strains in nucleofected BMDM for 14 hr and quantified as described (Figure 2; Experimental Procedures; [Luo & Isberg, 2004]). (c) Depletion of Rab8 and Rab11 effector VAMP3 rescues ΔsdhA vacuole integrity and growth defects. Nucleofection, vacuole integrity and bacterial yields determined as in panels (A and B). Statistical analyses were performed on normalised data by unpaired t test (*<.05; **<.01; ***<.001; Experimental Procedures)

Figure 6

SdhA functions to interfere with Rab11FIP1‐LCV interactions. (a) Representative immunofluorescence microscopy images of the Legionella‐containing vacuole in infected BMDMs at 4hpi, challenged with noted bacterial strains. Cells were probed with anti‐L. pneumophila before and after permeabilisation, to determine vacuole integrity (Experimental Procedures) as well as anti‐Rab11FIP1 and SidC after permeabilisation. Rab11FIP1 and SidC are pseudo‐coloured in green and red, respectively. Insets are magnified 3.25‐fold from the original image by changing resolution. Other panels are identical resolution to original grabbed images. (b) Images from (a) were analysed in Volocity to quantify localisation events of Rab11FIP1. Total of 181 vacuoles from two experiments were analysed. (c) Frequency distribution of localisation events from (b). Interquartile range was calculated from integer values. (d) Lack of correlation between intact or cytosol‐detected (permeable) ΔsdhA vacuoles and density of Rab11FIP1 near the vacuole. (e) BMDMs were nucleofected with siRNA, were challenged with noted L. pneumophila strains and stained as in (a). Images were captured and analysed after capture to quantify density of Rab11FIP‐associated compartments at the vacuole (Experimental Procedures). Total of 158 vacuoles from two experiments were analysed. (f) Frequency distribution of events from (e). Interquartile range was calculated from integer values