Fig 5.

Individual C. burnetii effector proteins are essential for intracellular replication and CCV formation. NMII C. burnetii was transformed with the Himar1 transposon, and the resulting clonal substrate mutants were used to infect J774A.1 cells. (A) Tn insertion into Cbu0041 (cirA), Cbu0388, Cbu0425 (cirB), Cbu0937 (cirC), Cbu2052 (cirD), and Cbu2059 (cirE) resulted in an inability to replicate intracellularly, however. (B) Tn insertion into Cbu0012, Cbu1198, Cbu1457, and Cbu2013 resulted in reduced intracellular replication compared to RSA439 levels. (C) To determine if coinfection with parental NMII strain would rescue the growth defect of the Tn mutants, HeLa cells were infected with an MOI of 100 of each strain and incubated for 7 days. Images were captured using a Nikon A1 confocal microscope, and at least 100 infected cells were imaged per experiment. Coinfection with the wild type resulted in significant replication for both the wild-type and Tn substrate mutant strains, whereas cells infected only with the substrate mutant did not exhibit significant replication. Scale bars represent 6 μM.