Host pathways important for Coxiella burnetii infection revealed by genome-wide RNA interference screening

Abstract

Coxiella burnetii is an intracellular pathogen that replicates within a lysosome-like vacuole. A Dot/Icm type IVB secretion system is used by C. burnetii to translocate effector proteins into the host cytosol that likely modulate host factor function. To identify host determinants required for C. burnetii intracellular growth, a genome-wide screen was performed using gene silencing by small interfering RNA (siRNA). Replication of C. burnetii was measured by immunofluorescence microscopy in siRNA-transfected HeLa cells. Newly identified host factors included components of the retromer complex, which mediates cargo cycling between the endocytic pathway and the Golgi apparatus. Reducing the levels of the retromer cargo-adapter VPS26-VPS29-VPS35 complex or retromer-associated sorting nexins abrogated C. burnetii replication. Several genes, when silenced, resulted in enlarged vacuoles or an increased number of vacuoles within C. burnetii-infected cells. Silencing of the STX17 gene encoding syntaxin-17 resulted in a striking defect in homotypic fusion of vacuoles containing C. burnetii, suggesting a role for syntaxin-17 in regulating this process. Lastly, silencing host genes needed for C. burnetii replication correlated with defects in the translocation of Dot/Icm effectors, whereas, silencing of genes that affected vacuole morphology, but did not impact replication, did not affect Dot/Icm translocation. These data demonstrate that C. burnetii vacuole maturation is important for creating a niche that permits Dot/Icm function. Thus, genome-wide screening has revealed host determinants involved in sequential events that occur during C. burnetii infection as defined by bacterial uptake, vacuole transport and acidification, activation of the Dot/Icm system, homotypic fusion of vacuoles, and intracellular replication.

Importance: Q fever in humans is caused by the bacterium Coxiella burnetii. Infection with C. burnetii is marked by its unique ability to replicate within a large vacuolar compartment inside cells that resembles the harsh, acidic environment of a lysosome. Central to its pathogenesis is the delivery of bacterial effector proteins into the host cell cytosol by a Dot/Icm type IVB secretion system. These proteins can interact with and manipulate host factors, thereby leading to creation and maintenance of the vacuole that the bacteria grow within. Using high-throughput genome-wide screening in human cells, we identified host factors important for several facets of C. burnetii infection, including vacuole transport and membrane fusion events that promote vacuole expansion. In addition, we show that maturation of the C. burnetii vacuole is necessary for creating an environment permissive for the Dot/Icm delivery of bacterial effector proteins into the host cytosol.

FIG 1

Genome-wide siRNA screen to identify host factors involved in C. burnetii infection. (A) Mock-transfected or Rab7A siRNA-treated HeLa cells were infected with C. burnetii and imaged at 4 days postinfection (p.i.). Shown is a 10× objective field of cells indicating immunolabeling of C. burnetii (green) and host nuclei with Hoechst (blue). The insets show closeups of a portion of the field. (B) Schematic representation of the screening procedure. HeLa cells were transfected with siRNA SMARTPools for 2 days and incubated with C. burnetii (Nine Mile strain phase II) for 4 days. Samples were fixed, immunostained, and imaged by automated microscopy as described here (see Materials and Methods). (C) Quantitative imaging of positive (mock infection)- and negative (mock infection plus chloramphenicol [chlor])-control phenotypes for C. burnetii replication. Samples were fixed and stained with Hoechst and a FITC-conjugated polyclonal antibody directed against C. burnetii. Segmented fluorescent images were analyzed using Metamorph software by quantifying the total numbers and sizes of the C. burnetii vacuoles in each image. (D) Pie chart based on GeneOntology (GO) index showing the biological process categories of host factors that were overrepresented within the population of siRNA screen hits that showed a defect in C. burnetii replication. Categories names are shown with the number of host factors identified within the category and the P value calculated using the binominal statistical test within the Panther classification system.

FIG 2

Representative C. burnetii fluorescence phenotypes identified in the siRNA screen. (A to D) Representative fluorescent images (10×) of siRNA-treated cells showing (A) fewer and smaller or (C) more and/or larger C. burnetii vacuoles at 4 days p.i. compared to mock treatment and (B and D) quantification of the fluorescent image phenotypes. Shown are C. burnetii-stained vacuoles and Hoechst-stained cell nuclei. (A) Rab5A, Rab7A, VPS16, VPS35, and ATP6V1D SMARTPool-treated cells, and Z-values corresponding to a normalized vacuole count compared to mock treatment. chlor, chloramphenicol. (B) Quantification is represented as the normalized C. burnetii vacuole count (total vacuoles/total nuclei per image) graphed as a ratio of the siRNA-treated value (Treated) versus the average value from mock-transfected wells (Untreated). (C) RAB40C (larger vacuoles), USP10 (more and larger vacuoles), and STX17 (more vacuoles) SMARTPool-treated cells, and Z-values corresponding to normalized vacuole count/percentage of large C. burnetii vacuoles. (D) Quantification is represented as both the normalized C. burnetii vacuole count (light gray) and the percentage of large C. burnetii vacuoles (dark gray). Shown are representative graphs with results from a single experiment presented as means + standard deviations. (E) Inside-out staining of C. burnetii-infected VPS35, Rab7A, and STX17 siRNA knockdowns compared to mock-transfected cells. After siRNA transfection and incubation for 3 days, cells were infected at an approximate multiplicity of infection (MOI) of 100 or 1,000 with C. burnetii for 4 h and processed for inside-out staining as described here (see Materials and Methods). Fluorescent microscopy was used to determine the percentages of cells infected.

FIG 3

siRNA targeting host VPS35 decreases C. burnetii replication in HeLa cells. (A) Fluorescent C. burnetii-labeled (green) and Hoechst-labeled (blue) images for the 4 individual siRNA targeting VPS35 and the parent SMARTPool compared to mock-transfected cells, and corresponding Z-values for normalized vacuole count. (B) Quantification of the fluorescent image phenotypes for the VPS35 siRNA-treated cells (total vacuole count normalized to counts of nuclei). The data are graphed as a ratio of the siRNA-treated value (Treated) versus the average value from mock-transfected wells (Untreated). Shown is a representative graph with results from a single experiment presented as means + standard deviations. (C) qRT-PCR measuring VPS35 mRNA levels in HeLa cells 3 days posttransfection for VPS35 siRNA-treated cells. Data are normalized to GAPDH and shown as fold expression level compared to mock-transfected cells. Results of an immunoblot analysis of siRNA-transfected HeLa cell lysates 3 days posttransfection measuring VPS35 and calnexin total protein levels using specific antibodies are shown. VPS35 protein expression levels were quantified by optical densitometry from immunoblots and shown below the immunoblot as fold change compared to mock-transfected cells. (D) Total fluorescence measurement of live HeLa cells infected with C. burnetii expressing mCherry. Cells subjected to mock transfection or transfected with VPS35 siRNA were incubated for 3 days before infection. The same wells were measured for total fluorescence emission at 610 nm once a day for 8 days postinfection.

FIG 4

siRNA targeting the host retromer complex decreases C. burnetii replication in HeLa cells. (A) Quantification of C. burnetii::Tn mCherry fluorescent image phenotypes for cells treated with each of 3 different siRNA against individual members of the host retromer complex (VPS26A, VPS26B, VPS35, VPS29, SNX1, SNX2, SNX3, SNX5, and SNX6) compared to mock-transfected cells. The data are graphed as a ratio of the siRNA-treated value versus the average value from mock-transfected wells at 4 days p.i. for the total vacuole count normalized to counts of nuclei. (B) qRT-PCR measuring individual retromer component mRNA levels in HeLa cells 3 days posttransfection for each of 3 different siRNA targeting the corresponding retromer complex member. Data are normalized to GAPDH and shown as fold expression level compared to the mock-transfected cells.

FIG 5

Depletion of syntaxin-17 increases the number of C. burnetii-containing vacuoles per cell. (A) Quantification of the fluorescent image phenotypes for the STX17 siRNA-treated cells. The data are graphed as a ratio of the siRNA-treated value versus the average value from mock-transfected wells (untreated). Shown are two phenotypic categories: total vacuole counts normalized to counts of nuclei (left) and the percentages of cells with large vacuoles (>50-pixel-sized objects) (right). Shown is a representative graph with results from a single experiment presented as means + standard deviations. (B) Left: results of an immunoblot analysis of siRNA-transfected HeLa cell lysates 3 days posttransfection measuring STX17 total protein levels using a STX17-specific antibody. The asterisk (*) represents a nonspecific band. STX17 protein expression levels were quantified by optical densitometry from immunoblots and shown below the immunoblot as fold change compared to mock-transfected cells. Right: qRT-PCR measuring STX17 mRNA levels in HeLa cells 6 days posttransfection for the 4 individual siRNA targeting STX17 and the parent SMARTPool. Data are normalized to GAPDH and shown as fold expression level compared to mock-transfected cells. (C) Fluorescent C. burnetii-stained images (10×) for STX17 SMARTPool-transfected HeLa cells compared to mock-transfected cells at 4 days p.i. (top images) and 40× phase-contrast images showing spacious C. burnetii vacuoles (arrowheads) for STX17 SMARTPool-transfected HeLa cells compared to mock-transfected cells at 2 days p.i. (bottom images). (D and E) Single-cell quantification of C. burnetii vacuole counts in STX17-transfected HeLa cells compared to STX18-transfected cells and mock-transfected cells at 2 days p.i. (D) The percentages of cells with one or more C. burnetii vacuoles were counted. (E) Subpopulations of infected cells from panel D that had two or more C. burnetii vacuoles per cell were counted.

FIG 6

Depletion of syntaxin-17 results in multiple C. burnetii vacuoles per cell. (A) Immunolabeling of STX17 SMARTPool-transfected HeLa cells compared to mock-transfected cells at 2 days p.i. with C. burnetii. Cells were immunostained for LAMP-1 (red) and C. burnetii (green). Cell nuclei and C. burnetii were Hoechst stained. (B) Ectopic expression of 3× Flag-STX17 in uninfected HeLa cells or HeLa cells persistently infected with C. burnetii. At 24 h posttransfection, cells were immunostained for calnexin (red) and 3× Flag (green). Cell nuclei and C. burnetii were Hoechst stained. Arrow = C. burnetii-containing vacuole.

FIG 7

The effect of siRNA depletion of host genes on translocation of a C. burnetii Dot/Icm effector protein. HeLa cells (Rab7A, VPS35, and STX17) treated with siRNA for 3 days were infected with C. burnetii expressing a plasmid carrying beta-lactamase (BlaM) alone or BlaM fused to the amino terminus of the C. burnetii Dot/Icm effector CBU0077 (20). At 24 h p.i., cells were incubated with the CCF4-AM substrate and assayed for translocation by (A) image analysis and (B) total fluorescence measurement. (A) The mean percentages of translocation-positive cells (lower right corner of images) were determined by visual observation of blue fluorescence emission at 460 nm indicative of CCF4-AM cleavage by translocated BlaM. (B) Translocation of BlaM and BlaM-CBU0077 was determined by measuring the change in the 460-nm/535-nm fluorescence emission ratio resulting from CCF4-AM cleavage. (C) BlaM-CBU0077 translocation from C. burnetii was measured in cells treated with each of 3 different siRNA targeting separate components of the retromer complex. (D) Left: translocation assay using 3 C. burnetii effectors (CBU0077, CBU1823, CBU1524) fused to BlaM. BlaM-effector translocation from C. burnetii was measured in cells treated with SMARTPool siRNA targeting Rab7A and VPS16. Right: qRT-PCR measuring Rab7A and VPS16 mRNA levels in HeLa cells 3 days posttransfection for SMARTPool siRNA targeting Rab7A and VPS16. Data are normalized to GAPDH and shown as fold expression levels compared to mock-transfected cells.