CHO-FcγRII cells were infected with Dot/Icm-sufficient LP01 strain of L. pneumophila (black) or the isogenic ΔdotA mutant (grey) expressing Cya fusions to the indicated C. burnetii proteins. (A) Fusions to Cya of the indicated full-length derivatives of C. burnetii NM proteins identified in the genetic screen were tested for translocation (B). Fusions to Cya of the indicated full-length derivatives of C. burnetii NM proteins identified based on homology or proximity to proteins identified in the screen were tested for translocation. Cya indicates empty vector control. Cya-RalF was used as a positive control. After 1 h, host cells were lysed and cAMP was extracted. Total cAMP levels resulting from translocation of protein fusions were quantified using an enzyme-immunoassay system, and are shown in fmol. Results represent average values +/− SD of experiments performed in triplicate.

The schematic shows the eighteen NM effectors identified in this study represented in blue. Below each NM effector is a representation of the size of the homologous reading frame encoded by the genes in the other sequenced strains of C. burnetii. The size of the predicted proteins is represented in kDa in brackets following the gene designation. Black lines represent the presence of homologous DNA that does not encode an open reading frame due to small deletions (vertical black line) and stop codons (red cross). In cases where multiple deletions occur the first deletion, representing the site of the frameshift, is displayed. The locations of conserved domains in each protein identified in a SMART database search are indicated according to the key provided.

Immunofluorescence micrographs show the representative localization profiles of the indicated FLAG-tagged C. burnetii effectors (Green) and the indicated host organelles (Red) after ectopic production of the proteins in HeLa 229 cells. (A) CbuK1976 and CBU1524 show localization to the nucleus, but the NM protein CBU0080 is distributed throughout the cytoplasm. The schematic shows that CBU0080 does not contain the helix-loop-helix motif (octagon) found in the homolog CbuK1976, which could explain the differential localization phenotypes observed (B) Cells stained for FLAG-tagged CBU0077 and LAMP1 show colocalization of the effector on lysosome-derived organelles, and cells stained for CBU0635 and GM130 show distribution of the protein to a perinuclear region containing the Golgi apparatus. (C). Cells stained for FLAG-tagged effectors and the TOM22 protein show colocalization of the effectors on mitochondria. The NM protein CBU1825 and the homologous Dugway protein CBUD0054 showed similar localization patterns. The NM protein CBU1532 and the Dugway homolog CBUD0454 showed slightly different mitochondrial straining patterns, with CBU1532 production leading to cell rounding and mitochondria aggregation. Scale bars represent 10 µm.

The impact of C. burnetii Dot/Icm effectors on the host cell secretory pathway was examined by monitoring secretion of the SEAP protein into the culture supernatant. (A) HEK 293 cells were co-transfected with pSEAP and the plasmid encoding the indicated effector protein or the GDP-locked ARF1_T31N protein (pArf1-T31N) or empty vector (pFLAG). External and internal SEAP activity was measured. The grey bars show that there was a significant decrease in the external/internal ratio of SEAP activity observed upon ectopic expression of ARF1_T31N (P = 0.0012) or CBU0635 (P = 0.0057) in comparison to HEK 293 cells transfected with pFLAG alone (black bar). Expression of all other C. burnetii effectors did not significantly the ratio of SEAP activity (white bars). (B). SEAP activity (y-axis) was measured at the indicated times after cells were washed and new culture medium was added (x-axis) Data show SEAP ratios for cells with vector alone (pFLAG; black triangles), cells producing CBU0635 (pCBU0635; open squares) and cells producing ARF1_T31N (grey circles). A similar defect in SEAP secretion was observed in cells producing ARF1_T31N as in cells producing CBU0635.

(A) Yeast strains were grown in YNB supplemented with 2% galactose to induce the expression of the indicated effector proteins listed in the legend on the right. The plots show yeast replication as determined by measuring the optical density of the culture at 600 nm (OD 600 nm, y-axis) every 30 min (x-axis) for a period of 36 h. Growth of S. cerevisiae expressing C. burnetii effectors were compared to controls that included S. cerevisiae containing the vector control (pYES2; open squares) and S. cerevisiae producing the L. pneumophila effector YlfA (pJG4-5:YlfA; red triangles). Effectors that resulted in a delay in the doubling rate of S. cerevisiae are highlighted with red symbols. (B) The doubling time of S. cerevisiae producing the indicated effector proteins was calculated during the exponential phase of growth from the growth curves shown in panel A. Effectors that resulted in a significant increase in doubling time compared to S. cerevisiae pYES2 are highlighted in open boxes (P<0.01). Data represent the mean doubling time ± SD determined from at least 3 independent growth curves.

(A) A clone of C. burnetii with a 3.69 kb transposon inserted in the icmL gene (dotI/icmL.1) was isolated after enrichment on medium containing kanamycin. The schematic shows the predicted location and orientation of the transposon in icmL as determined by sequence analysis. The location of probes and restriction sites used to validate the site of transposon insertion by PCR and Southern hybridization are indicated. (B) As indicated, genomic DNA digested with HindIII isolated from the C. burnetii NM strain and the isogenic icmL::Tn mutant was analyzed by Southern hybridization using a probe derived from the icmL gene and a probe derived from the transposon. As predicted from the schematic in panel A, the icmL probe identified a single band of 4.2 kb in the NM strain, and two bands of 3.3 kb and 2.5 kb in the icmL::Tn mutant because of a new HindIII site introduced into the icmL locus by the transposon. When a probe homologous to the transposon was used (Tn probe), a single band was identified in the icmL::Tn mutant, indicating that this strain has a single insertion of the transposon in the chromosome. (C) PCR amplification of genomic DNA from NM and the icmL::Tn mutant using primer sets shown in panel A confirm the predicted location and orientation of the transposon insertion in icmL, and that the transposon is retained by the icmL::Tn strain after introduction of a plasmid encoding icmL (pIcmL) or a plasmid encoding the entire operon harboring icmL (pQC). (D,E) The ability of the icmL::Tn mutant to replicate in HeLa (D) and Vero (E) cells was determined by measuring genomic equivalents (y-axis) at the indicated times after infection (x-axis). There was a 50 to 100-fold increase over 7 days in C. burnetii NM genomic units (black squares). No significant increase in genomic units was detected for the icmL::Tn mutant (white squares) or C. burnetii NM treated with 10 µg/ml chloramphenicol (gray squares). (F,G) The ability of the icmL::Tn mutant to replicate in HeLa (F) and Vero (G) cells was determined by measuring genomic equivalents (y-axis) at the indicated times after infection (x-axis). Replication was observed for C. burnetii NM containing pBlaM (black squares) and the icmL::Tn mutant containing the plasmid pQC (black triangles). The icmL::Tn mutant containing the vector pBlaM (white squares) or for the icmL::Tn mutant containing pIcmL (white triangles). Graphs represent the mean ± SD of at least three independent experiments. (H) Fluorescence micrographs were acquired after infection of HeLa cells for 5 days by the strains of C. burnetii indicated. An anti-Coxiella antibody (green) was used to visualize intracellular bacteria and the nucleus of the host cell was visualized by DAPI staining (blue). Replicating bacteria in large vacuoles were observed for cells infected with C. burnetii NM, C. burnetii NM containing pBlaM and the icmL::Tn mutant containing pQC. Only individual bacteria were observed inside the host cells infected with the icmL::Tn mutant, C. burnetii NM in medium with chloramphenicol, icmL::Tn containing pBlaM and icmL::Tn containing pIcmL (indicated with arrows). These are representative images from at least three independent experiments.

(A) HeLa cells either infected with C. burnetii NM or uninfected were incubated with or without chloramphenicol (CM) and cellular lysates were separated into supernatant and pellet fractions. Immunoblot analysis was used to detect CBU0077 (top panel; α-77) and EF-Ts (bottom panel; α-EF-Ts). The CBU0077 protein was detected in both the supernatant fraction containing proteins secreted during infection and in the pellet fraction containing intact bacteria. The cell associated EF-Ts protein was detected only in the pellet fraction containing intact bacterial cells. The addition of chloramphenicol resulted in a slight drop in levels of CBU0077 detected in the supernatant, suggesting the secreted protein has a relatively long half-life. Similar results were obtained from three independent experiments. (B) C. burnetii NM (black bars) and the icmL::Tn mutant (white bars) containing either pBlaM or pBlaM-77 were used to infect HeLa cells for the times indicated (x-axis). Translocation of the BlaM and BlaM-CBU0077 fusion protein by the respective strains was determined by measuring the change in the 460 nm/535 nm fluorescence emission ratio resulting from cleavage of the CCF4-AM substrate (y-axis). Results represent the mean ± SD obtained from triplicate samples. (C, D) HeLa cells were infected in parallel with C. burnetii NM producing BlaM-CBU0077 or the icmL::Tn strain producing BlaM-CBU0077 at the multiplicities of infection (MOI) indicated on the x-axis. Fluorescence microscopy was used to determine the percent of cells infected by the two strains (C) and the number of cells that stained positive for BlaM translocation (D) Results represent the mean ± SD obtained from triplicate samples. (E) Fluorescence micrographs of HeLa cells that were infected for 24 h with either C. burnetii NM or the icmL::Tn strains producing either BlaM alone or BlaM fusions to CBU0077 (pBlaM-77), CBU0635 (pBlaM-0635) or CBU1524 (pBlaM-1524). Emission at 535 nm (green) identifies the CCF4-AM-loaded cells and emission at 460 nm (blue) indicates cleavage of CCF4-AM in the cytosol of the infected cell resulting from translocation of the BlaM fusion protein. The percent of cells that were BlaM positive (blue) was determined for each infection condition and the mean ± SD from three independent experiments is displayed in the bottom right corner of each representative micrograph.