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

Hayley J Newton,Craig R Roy,Justin A Mcdonough,Yousef Abu Kwaik

doi:10.1371/journal.pone.0054566

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.

Highlights

Intracellular bacterial pathogens direct distinct and unique interactions with the eukaryotic host to establish a replicative niche and avoid being killed
Immunoblot analysis confirmed that the b-lactamase enzyme (BlaM) and the BlaM-effector fusion proteins were produced by C. burnetii grown in ACCM-2, which demonstrates that these proteins are available for translocation before bacteria make contact with the host cell (Figure S1)
This question was addressed using macrophages derived from C57BL/ 6 mice (BMMs), which restrict the intracellular replication of C. burnetii Nine Mile phase II bacteria [31]

Summary

Introduction

Intracellular bacterial pathogens direct distinct and unique interactions with the eukaryotic host to establish a replicative niche and avoid being killed. Specialized secretion systems that translocate a cohort of virulence proteins called effectors into the host cytosol are often essential for establishing a replicative compartment [1]. These effectors can modulate a wide range of eukaryotic processes, including vesicular trafficking, cytoskeletal dynamics and innate signaling pathways [2]. A virulence-associated T4BSS system called Dot/Icm was first described in the intracellular pathogen Legionella pneumophila and was shown to be essential for replication of L. pneumophila inside eukaryotic host cells [5,6]. The Legionella Dot/Icm system is assembled before bacteria engage host cells and initiates effector translocation once cell contact is sensed, which is central to the ability of Legionella to rapidly avoid endocytic maturation processes that occur within minutes of bacterial uptake [8]

Methods

Results

Conclusion