Effector Protein Translocation by the Coxiella burnetii Dot/Icm Type IV Secretion System Requires Endocytic Maturation of the Pathogen-Occupied Vacuole

Abstract

The human pathogen Coxiella burnetii encodes a type IV secretion system called Dot/Icm that is essential for intracellular replication. The Dot/Icm system delivers bacterial effector proteins into the host cytosol during infection. The effector proteins delivered by C. burnetii are predicted to have important functions during infection, but when these proteins are needed during infection has not been clearly defined. Here, we use a reporter system consisting of fusion proteins that have a β-lactamase enzyme (BlaM) fused to C. burnetii effector proteins to study protein translocation by the Dot/Icm system. Translocation of BlaM fused to the effector proteins CBU0077, CBU1823 and CBU1524 was not detected until 8-hours after infection of HeLa cells, which are permissive for C. burnetii replication. Translocation of these effector fusion proteins by the Dot/Icm system required acidification of the Coxiella-containing vacuole. Silencing of the host genes encoding the membrane transport regulators Rab5 or Rab7 interfered with effector translocation, which indicates that effectors are not translocated until bacteria traffic to a late endocytic compartment in the host cell. Similar requirements for effector translocation were discerned in bone marrow macrophages derived from C57BL/6 mice, which are primary cells that restrict the intracellular replication of C. burnetii. In addition to requiring endocytic maturation of the vacuole for Dot/Icm-mediated translocation of effectors, bacterial transcription was required for this process. Thus, translocation of effector proteins by the C. burnetii Dot/Icm system occurs after acidification of the CCV and maturation of this specialized organelle to a late endocytic compartment. This indicates that creation of the specialized vacuole in which C. burnetii replicates represents a two-stage process mediated initially by host factors that regulate endocytic maturation and then by bacterial effectors delivered into host cells after bacteria establish residency in a lysosome-derived organelle.

Right on Q: Genetics begin to Unravel Coxiella Burnetii host cell Interactions

“…The essential nature of the T4BSS for productive infection was then demonstrated by showing strains harboring Himar1 insertions in icmD or icmL or targeted deletions in dotA or dotB, had severe intracellular growth defects associated with failure to secrete effector proteins in a dot/icmdependent fashion [80,87,104]. Direct screening of potential Dot/Icm substrates in Coxiella is now feasible [60,73,87,94,107,108]. Moreover, targeted mutagenesis [73,98,107] and large-scale Tn screens [50,60] have revealed the importance of individual effectors in host cell colonization and helped resolve their function.…”

Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development

“…Upon internalization by phagocytic and nonphagocytic cells, bacteria remain confined within Coxiella-containing vacuoles (CCVs), tight-fitting compartments that mature along the endocytic pathway (1). Key to the successful colonization of host cells is the acidification of CCVs, which activates bacterial metabolism and effectors translocation by a Dot/Icm type IV secretion system (3). Bioinformatics analysis identified more than 300 Coxiella candidate effectors, a third of which have been validated for secretion (1,4).…”

Identification of Novel Coxiella burnetii Icm/Dot Effectors and Genetic Analysis of Their Involvement in Modulating a Mitogen-Activated Protein Kinase Pathway

“…burnetii utilizes an Icm/Dot type IVB secretion system, which has been shown to be required for its intracellular growth in human macrophages (5,6). The Icm/Dot secretion system was first identified in the evolutionary closely related intracellular pathogen Legionella pneumophila, the causative agent of Legionnaires' disease, where it was found to be essential for all aspects of its pathogenesis (reviewed in references 7 and 8).…”

“…This difference probably leads to their different intracellular lifestyles: C. burnetii resides within a phagosome that traffics through the endocytic pathway and matures into an acidic phagolysosome-like compartment (reviewed in references 13 and 14), whereas L. pneumophila inhibits phagosome-lysosome fusion, and its phagosome matures into an endoplasmic reticulum-like compartment (reviewed in references 7 and 8). This different intracellular habitat most likely leads to another difference in the functionality of the Icm/Dot secretion system: C. burnetii effectors were found to translocate into host cells only 8 h postinfection (after the acidification of its vacuole), in contrast to L. pneumophila effectors, which were shown to be translocated minutes postinfection (6,15).…”