A screen of Coxiella burnetii mutants reveals important roles for Dot/Icm effectors and host autophagy in vacuole biogenesis - PubMed (original) (raw)

A screen of Coxiella burnetii mutants reveals important roles for Dot/Icm effectors and host autophagy in vacuole biogenesis

Hayley J Newton et al. PLoS Pathog. 2014.

Abstract

Coxiella burnetii is an intracellular pathogen that replicates in a lysosome-derived vacuole. The molecular mechanisms used by this bacterium to create a pathogen-occupied vacuole remain largely unknown. Here, we conducted a visual screen on an arrayed library of C. burnetii NMII transposon insertion mutants to identify genes required for biogenesis of a mature Coxiella-containing vacuole (CCV). Mutants defective in Dot/Icm secretion system function or the PmrAB regulatory system were incapable of intracellular replication. Several mutants with intracellular growth defects were found to have insertions in genes encoding effector proteins translocated into host cells by the Dot/Icm system. These included mutants deficient in the effector proteins Cig57, CoxCC8 and Cbu1754. Mutants that had transposon insertions in genes important in central metabolism or encoding tRNA modification enzymes were identified based on the appearance filamentous bacteria intracellularly. Lastly, mutants that displayed a multi-vacuolar phenotype were identified. All of these mutants had a transposon insertion in the gene encoding the effector protein Cig2. Whereas vacuoles containing wild type C. burnetii displayed robust accumulation of the autophagosome protein LC3, the vacuoles formed by the cig2 mutant did not contain detectible amounts of LC3. Furthermore, interfering with host autophagy during infection by wild type C. burnetii resulted in a multi-vacuolar phenotype similar to that displayed by the cig2 mutant. Thus, a functional Cig2 protein is important for interactions between the CCV and host autophagosomes and this drives a process that enhances the fusogenic properties of this pathogen-occupied organelle.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1. Transposon mutants of C. burnetii display different intracellular phenotypes.

Transposon mutants were subject to a vacuole formation assay in which 96-well plates of HeLa 229 cells were infected, at an MOI of approximately 500, with individual transposon mutants. Following a 96 h infection period, the infection was fixed and stained with anti-Coxiella (red), anti-LAMP1 (green) and Hoechst dye (blue) and observed with low magnification fluorescence microscopy. (A) The parental strain C. burnetii NMII displayed a large CCV in the majority of HeLa cells. (B) A cohort of mutants showed no intracellular replication as demonstrated by dotA::Tn, (C) another category produced smaller replicative vacuoles such as that seen with cig57::Tn, (D) transposon insertions that disrupted cig2 resulted in the appearance of multiple vacuoles in a single cell, and (E) a small proportion of mutants, such as gidA::Tn, displayed CCVs with an abnormal shape due to filamentous replication of the C. burnetii.

Figure 2. The Dot/Icm system and the PmrAB system are essential for intracellular replication and delivery of C. burnetii effector proteins.

(A) Indicated are the locations of transposon insertions in the chromosomal regions encoding the Dot/Icm system from 1559100 bp to 1591800 bp, and (B) the PmrAB two-component regulatory system from 1176500 to 1179500 bp. The site of each transposon insertion is represented by an arrow. The mutants that could not replicate intracellularly were assigned red arrows, mutants that produced normal CCVs were assigned black arrows, and mutants that formed small vacuoles were assigned grey arrows. (C) The plasmids pBlaM and pBlaM-77 were introduced into C burnetii NMII (black bars) and pmrA::Tn (grey bars) to determine whether the PmrAB system was essential for effector translocation. Cleavage of the fluorescent β-lactamase substrate CC4F-AM was determined by calculating the ratio of fluorescence at 460 nm to 535 nm relative to uninfected cells. The graph shows the mean ± SD calculated for three independent samples.

Figure 3. The Dot/Icm effector Cig57 is required for efficient intracellular replication of C. burnetii.

(A) Intracellular replication of C. burnetii NMII (black squares), cig57::Tn (black triangles) and cig57::Tn pFLAG-Cig57 (open circles). The fold-increase in GEs relative to the inoculum was determined by _dotA_-specific qPCR and is represented here as the mean ± SD of 3 independent infections at days 1, 3, 5 and 7 post-infection. (B,C) Representative micrographs of HeLa cells infected for either 3 days (B) or 5 days (C) with C. burnetii NMII, cig57::Tn or cig57::Tn pFLAG-Cig57. Cell with stained with anti-Coxiella antibody (red), anti-LAMP1 antibody (green) and Hoechst dye (blue). Note that the vacuoles containing cig57::Tn were smaller and contained few bacteria at each time point compared to the control NMII strain or the complemented cig57::Tn pFLAG-Cig57 strain. Scale bars represent 10 µm.

Figure 4. The Dot/Icm effector Cig2 is necessary for homotypic fusion of CCVs.

(A) HeLa cells were infected with C. burnetii NMII strains at an MOI of 500. Five days post-infection the infections were fixed and stained with anti-Coxiella (red), anti-LAMP1 (green) and Hoechst dye (blue). Representative micrographs demonstrate the multiple vacuole phenotype observed for the cig2::Tn mutant strain. * indicates the location of individual CCVs within a chosen cell. These CCVs were identified both by LAMP1 staining and phase contrast microscopy. Scale bars represent 10 µm. (B) The average number of vacuoles containing C. burnetii NMII (black bars), cig2::Tn (white bars) and cig2::Tn pFLAG:Cig2 (grey bars) was determined at day 3 post-infection for at least 100 infected cells on duplicate coverslips. Data are the mean ± SD from 3 independent experiments. (C) Replication of cig2::Tn (open triangles) and the parental C. burnetii NMII strain (black squares) was determined by measuring genome equivalents over a 7 day infection period. (D) Micrographs from translocation assays using C. burnetii NMII and the icmL::Tn strains producing either BlaM alone (pJB-Cm:BlaM) or BlaM-Cig2 (pJB-Cm:BlaM-Cig2). Fluorescence intensity at 535 nm of uncleaved CCF4-AM is shown in green. Fluorescence intensity of cleaved CCF4-AM at 460 nm generated by the enzymatic activity of BlaM fusion proteins delivered into the host cell cytosol is shown in blue. These images are representative of 3 independent experiments.

Figure 5. Autophagy is required for homotypic fusion of CCVs.

(A) HeLa cells in which the indicated genes were silenced using siRNA were infected with C. burnetii and fixed 3 days post-infection before immunostaining with anti-C. burnetii (red) and anti-LAMP1 (green) antibodies. DNA was stained with Hoechst (blue). Shown are representative micrographs for mock-transfected cells and cells transfected with siRNA specific to human ATG5, ATG12, STX17, and STX18. (B) Single-cell quantification of C. burnetii vacuole counts in ATG5, ATG12, and STX17 siRNA-transfected HeLa cells compared to STX18-transfected cells and mock-transfected cells at 3 days post-infection. The percentages of infected cells with two or more C. burnetii vacuoles were counted. Shown is the mean ± SD from 3 independent experiments. (C) HeLa cells infected with C. burnetii expressing the L. pneumophila effector RavZ from a plasmid (3×FL-RavZ) were assayed 3 days after infection and scored for the multi-vacuolar phenotype. As controls, C. burnetii expressing empty vector (3×FL) or a point mutant of RavZ defective in autophagy inhibition (3×FL-RavZC258A) were used. Shown are representative fluorescent images for each C. burnetii infection. (D) Immunoblots from uninfected or _C. burnetii_-infected HeLa cell lysates showing bands for non-lipidated (LC3-I) and lipidated (LC3-II) forms of LC3 and actin. Cells were untreated (top images) or treated (bottom images) with bafilomycin A1 and rapamycin for 2 h prior to lysis. Cells were infected with a Dot/Icm-deficient strain of C. burnetii (icmL::Tn), infected with parental C. burnetii NMII (no vector), or C. burnetii NMII with vector alone or vectors producing the indicated RavZ proteins. (E) Single-cell quantification of C. burnetii vacuole counts in cells at 3 days post-infection. The percentages of infected cells with two or more C. burnetii vacuoles were counted. Shown is the mean ± SD from three independent experiments.

Figure 6. The effector Cig2 is important for CCV fusion with autophagosomes.

(A) HeLa cells were infected with C. burnetii NMII, cig2::Tn or cig2::Tn pFLAG:Cig2 for 5 days and stained with anti-LC3 (green) and anti-Coxiella (red) antibodies. (B) The association of LC3 with the CCV was quantified by observing 100 infected cells per sample in 3 independent experiments. (C) LC3 and actin immunoblots from HeLa cells infected with parental C. burnetii NMII, the cig2::Tn mutant, the parental C. burnetii NMII producing either RavZ or RavZC258A, or uninfected cells. Cells were untreated (top image), or treated (bottom image) with bafilomycin A1 and rapamycin for 2 h prior to lysis. (D) J774A.1 cells were infected with C. burnetii NMII, cig2::Tn, or cig2::Tn pFLAG:Cig2 for 36 h before loading with 50 µg/ml DQ Green BSA for 16 h. Micrographs show phase contrast images to reveal vacuoles and the green fluorescence that results from DQ Green BSA degradation by proteases in lysosomal compartments.

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A screen of Coxiella burnetii mutants reveals important roles for Dot/Icm effectors and host autophagy in vacuole biogenesis - PubMed (original) (raw)