Oral Presentation Lorne Infection and Immunity 2013

Bacterial Factors Required for the Establishment of the Coxiella burnetii Replicative Vacuole (#4)

Hayley J Newton 1 2 , Craig R Roy 1
  1. Microbial Pathogenesis, Yale University, New Haven, CT, USA
  2. Microbiology and Immunology, The Peter Doherty Institute, University of Melbourne, Parkville, Vic, Australia

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that replicates in a unique acidified vacuole derived from the host lysosomal network. In addition to a low pH, the replicative Coxiella-containing vacuole (CCV) is spacious and highly fusogenic. Coxiella encodes a Dot/Icm type IV secretion system that delivers effector proteins to the host cytosol.  The recent development of culture conditions for axenic propagation of Coxiella has enabled the application of genetic techniques to investigate the molecular strategies employed by this pathogen. A transposon insertion mutant was isolated that functionally disrupts the Dot/Icm system. Importantly, this Coxiella Dot/Icm-deficient mutant was not able to replicate intracellularly. Improved efficiency of transposon mutagenesis has allowed for the production of a library of more than 3000 Coxiella transposon mutants which have been screened for vacuole maturation and intracellular replication in HeLa cells. This screen has confirmed the requirement of the Dot/Icm apparatus with more than 50 distinct mutants, disrupting the dot/icm locus, unable to replicate intracellularly. Three mutations within the two-component response regulator predicted to control the expression of the dot/icm locus also rendered Coxiella unable to replicate intracellularly. Several other transposon mutants displayed replication defects including a mutant disrupting a conserved Dot/Icm effector. Disruption of a large, conserved hypothetical protein caused a significant defect in homotypic fusion of CCVs, with multiple CCVs present in individual infected cells. This phenotype could be complemented by introduction of the gene on a plasmid. Using a BlaM-reporter assay, the protein was shown to be a Dot/Icm effector. These data demonstrate that the intracellular replication of Coxiella requires the production of a functional Dot/Icm apparatus and that individual effectors of this system play distinct roles in the development of the replicative CCV.