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

Newton HJ, Kohler LJ, McDonough JA, Temoche-Diaz M, Crabill E, Hartland EL, Roy CR - PLoS Pathog. (2014)

Bottom Line: These included mutants deficient in the effector proteins Cig57, CoxCC8 and Cbu1754.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America; Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia.

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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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.
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ppat-1004286-g001: 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.

Mentions: The arrayed library of C. burnetii NMII transposon mutants was analyzed using a visual assay that assessed the ability of individual mutants to form vacuoles that support intracellular replication. Specifically, HeLa 224 cells distributed in 96-well glass-bottom plates were infected with individual mutants at an MOI of approximately 500 and then the cells were incubated for 96 h. Cells were fixed and stained with antibodies specific for Coxiella (red) and LAMP1 (green), and the DNA was labeled with Hoechst dye (blue). The ability of each mutant to form a vacuole that supported intracellular replication was assessed by direct examination using fluorescence microscopy. Importantly, the parental NMII control strain and most of the C. burnetii transposon insertion mutants formed a single spacious vacuole filled with replicating bacteria (Figure 1A). Additionally, of the 459 mutants for which the transposon insertion site was determined, we found that 324 mutants (71%) did not display a discernable vacuole formation defect in this visual assay, which included several mutants having insertions in genes encoding known effectors of the Dot/Icm system (Table S5). Lastly, as described in detail below, many of the mutants that displayed vacuole formation defects had insertions in genes that would be predicted to affect intracellular replication. Thus, confidence was high that this screen would identify a unique cohort of genes important for C. burnetii replication in mammalian cells.


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

Newton HJ, Kohler LJ, McDonough JA, Temoche-Diaz M, Crabill E, Hartland EL, Roy CR - PLoS Pathog. (2014)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4117601&req=5

ppat-1004286-g001: 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.
Mentions: The arrayed library of C. burnetii NMII transposon mutants was analyzed using a visual assay that assessed the ability of individual mutants to form vacuoles that support intracellular replication. Specifically, HeLa 224 cells distributed in 96-well glass-bottom plates were infected with individual mutants at an MOI of approximately 500 and then the cells were incubated for 96 h. Cells were fixed and stained with antibodies specific for Coxiella (red) and LAMP1 (green), and the DNA was labeled with Hoechst dye (blue). The ability of each mutant to form a vacuole that supported intracellular replication was assessed by direct examination using fluorescence microscopy. Importantly, the parental NMII control strain and most of the C. burnetii transposon insertion mutants formed a single spacious vacuole filled with replicating bacteria (Figure 1A). Additionally, of the 459 mutants for which the transposon insertion site was determined, we found that 324 mutants (71%) did not display a discernable vacuole formation defect in this visual assay, which included several mutants having insertions in genes encoding known effectors of the Dot/Icm system (Table S5). Lastly, as described in detail below, many of the mutants that displayed vacuole formation defects had insertions in genes that would be predicted to affect intracellular replication. Thus, confidence was high that this screen would identify a unique cohort of genes important for C. burnetii replication in mammalian cells.

Bottom Line: These included mutants deficient in the effector proteins Cig57, CoxCC8 and Cbu1754.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, United States of America; Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia.

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.

Show MeSH
Related in: MedlinePlus