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.
Highlights
Coxiella burnetii is a highly infectious human pathogen responsible for a global zoonotic disease called Q fever
It has been shown that the Nine Mile strain RSA493 called clone 4 (NMII) strain is indistinguishable from the isogenic phase I strain (NMI) in tissue culture models of infection that measure the ability of C. burnetii to replicate in human cells, which include studies in primary human monocyte-derived macrophages [5,6]
We employed large-scale transposon mutagenesis to create an arrayed library of 3,237 C. burnetii transposon insertion mutants
Summary
Coxiella burnetii is a highly infectious human pathogen responsible for a global zoonotic disease called Q fever. Phase II variants of C. burnetii produce a truncated O-antigen polysaccharide and can be isolated from both infected animals and bacteria cultured ex vivo [3,4]. Most strains of C. burnetii exhibit phase variation and switch between phase I and phase II spontaneously, a phase II variant of the C. burnetii Nine Mile strain RSA493 called clone 4 (NMII) is phase locked because it has a chromosomal deletion that eliminates several genes required for the synthesis of O-antigen polysaccharide, which makes this strain incapable of causing systemic disease in guinea pig and mouse models of infection and enhances innate immune detection [3,4]. It has been shown that the NMII strain is indistinguishable from the isogenic phase I strain (NMI) in tissue culture models of infection that measure the ability of C. burnetii to replicate in human cells, which include studies in primary human monocyte-derived macrophages [5,6]. NMI and NMII encode the same array of virulence determinants that have evolved for manipulating cellular functions important for intracellular replication
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