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The Coxiella burnetii Dot/Icm system delivers a unique repertoire of type IV effectors into host cells and is required for intracellular replication.

Carey KL, Newton HJ, Lührmann A, Roy CR - PLoS Pathog. (2011)

Bottom Line: The identification of a transposon insertion mutation that disrupts the dot/icm locus was used to validate that this apparatus was essential for translocation of effectors.Importantly, this C. burnetii Dot/Icm-deficient mutant was found to be defective for intracellular replication.Thus, these data indicate that C. burnetii encodes a unique subset of bacterial effector proteins translocated into host cells by the Dot/Icm apparatus, and that the cumulative activities exerted by these effectors enables C. burnetii to successfully establish a niche inside mammalian cells that supports intracellular replication.

View Article: PubMed Central - PubMed

Affiliation: Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT
Coxiella burnetii, the causative agent of human Q fever, is an intracellular pathogen that replicates in an acidified vacuole derived from the host lysosomal network. This pathogen encodes a Dot/Icm type IV secretion system that delivers bacterial proteins called effectors to the host cytosol. To identify new effector proteins, the functionally analogous Legionella pneumophila Dot/Icm system was used in a genetic screen to identify fragments of C. burnetii genomic DNA that when fused to an adenylate cyclase reporter were capable of directing Dot/Icm-dependent translocation of the fusion protein into mammalian host cells. This screen identified Dot/Icm effectors that were proteins unique to C. burnetii, having no overall sequence homology with L. pneumophila Dot/Icm effectors. A comparison of C. burnetii genome sequences from different isolates revealed diversity in the size and distribution of the genes encoding many of these effectors. Studies examining the localization and function of effectors in eukaryotic cells provided evidence that several of these proteins have an affinity for specific host organelles and can disrupt cellular functions. The identification of a transposon insertion mutation that disrupts the dot/icm locus was used to validate that this apparatus was essential for translocation of effectors. Importantly, this C. burnetii Dot/Icm-deficient mutant was found to be defective for intracellular replication. Thus, these data indicate that C. burnetii encodes a unique subset of bacterial effector proteins translocated into host cells by the Dot/Icm apparatus, and that the cumulative activities exerted by these effectors enables C. burnetii to successfully establish a niche inside mammalian cells that supports intracellular replication.

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CBU0635 interferes with host protein secretion.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 ARF1T31N 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 ARF1T31N (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 ARF1T31N (grey circles). A similar defect in SEAP secretion was observed in cells producing ARF1T31N as in cells producing CBU0635.
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ppat-1002056-g004: CBU0635 interferes with host protein secretion.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 ARF1T31N 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 ARF1T31N (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 ARF1T31N (grey circles). A similar defect in SEAP secretion was observed in cells producing ARF1T31N as in cells producing CBU0635.

Mentions: External/Internal SEAP activity (Figure 4).


The Coxiella burnetii Dot/Icm system delivers a unique repertoire of type IV effectors into host cells and is required for intracellular replication.

Carey KL, Newton HJ, Lührmann A, Roy CR - PLoS Pathog. (2011)

CBU0635 interferes with host protein secretion.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 ARF1T31N 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 ARF1T31N (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 ARF1T31N (grey circles). A similar defect in SEAP secretion was observed in cells producing ARF1T31N as in cells producing CBU0635.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102713&req=5

ppat-1002056-g004: CBU0635 interferes with host protein secretion.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 ARF1T31N 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 ARF1T31N (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 ARF1T31N (grey circles). A similar defect in SEAP secretion was observed in cells producing ARF1T31N as in cells producing CBU0635.
Mentions: External/Internal SEAP activity (Figure 4).

Bottom Line: The identification of a transposon insertion mutation that disrupts the dot/icm locus was used to validate that this apparatus was essential for translocation of effectors.Importantly, this C. burnetii Dot/Icm-deficient mutant was found to be defective for intracellular replication.Thus, these data indicate that C. burnetii encodes a unique subset of bacterial effector proteins translocated into host cells by the Dot/Icm apparatus, and that the cumulative activities exerted by these effectors enables C. burnetii to successfully establish a niche inside mammalian cells that supports intracellular replication.

View Article: PubMed Central - PubMed

Affiliation: Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America.

ABSTRACT
Coxiella burnetii, the causative agent of human Q fever, is an intracellular pathogen that replicates in an acidified vacuole derived from the host lysosomal network. This pathogen encodes a Dot/Icm type IV secretion system that delivers bacterial proteins called effectors to the host cytosol. To identify new effector proteins, the functionally analogous Legionella pneumophila Dot/Icm system was used in a genetic screen to identify fragments of C. burnetii genomic DNA that when fused to an adenylate cyclase reporter were capable of directing Dot/Icm-dependent translocation of the fusion protein into mammalian host cells. This screen identified Dot/Icm effectors that were proteins unique to C. burnetii, having no overall sequence homology with L. pneumophila Dot/Icm effectors. A comparison of C. burnetii genome sequences from different isolates revealed diversity in the size and distribution of the genes encoding many of these effectors. Studies examining the localization and function of effectors in eukaryotic cells provided evidence that several of these proteins have an affinity for specific host organelles and can disrupt cellular functions. The identification of a transposon insertion mutation that disrupts the dot/icm locus was used to validate that this apparatus was essential for translocation of effectors. Importantly, this C. burnetii Dot/Icm-deficient mutant was found to be defective for intracellular replication. Thus, these data indicate that C. burnetii encodes a unique subset of bacterial effector proteins translocated into host cells by the Dot/Icm apparatus, and that the cumulative activities exerted by these effectors enables C. burnetii to successfully establish a niche inside mammalian cells that supports intracellular replication.

Show MeSH
Related in: MedlinePlus