Article

The Coxiella burnetii Cryptic Plasmid Is Enriched in Genes Encoding Type IV Secretion System Substrates

Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, 4301 W. Markham Street, Little Rock, AR 72205, USA.
Journal of bacteriology (Impact Factor: 2.81). 03/2011; 193(7):1493-503. DOI: 10.1128/JB.01359-10
Source: PubMed

ABSTRACT

The intracellular bacterial pathogen Coxiella burnetii directs biogenesis of a phagolysosome-like parasitophorous vacuole (PV), in which it replicates. The organism encodes a Dot/Icm
type IV secretion system (T4SS) predicted to deliver to the host cytosol effector proteins that mediate PV formation and other
cellular events. All C. burnetii isolates carry a large, autonomously replicating plasmid or have chromosomally integrated plasmid-like sequences (IPS), suggesting
that plasmid and IPS genes are critical for infection. Bioinformatic analyses revealed two candidate Dot/Icm substrates with
eukaryotic-like motifs uniquely encoded by the QpH1 plasmid from the Nine Mile reference isolate. CpeC, containing an F-box
domain, and CpeD, possessing kinesin-related and coiled-coil regions, were secreted by the closely related Legionella pneumophila Dot/Icm T4SS. An additional QpH1-specific gene, cpeE, situated in a predicted operon with cpeD, also encoded a secreted effector. Further screening revealed that three hypothetical proteins (CpeA, CpeB, and CpeF) encoded
by all C. burnetii plasmids and IPS are Dot/Icm substrates. By use of new genetic tools, secretion of plasmid effectors by C. burnetii during host cell infection was confirmed using β-lactamase and adenylate cyclase translocation assays, and a C-terminal secretion
signal was identified. When ectopically expressed in HeLa cells, plasmid effectors trafficked to different subcellular sites,
including autophagosomes (CpeB), ubiquitin-rich compartments (CpeC), and the endoplasmic reticulum (CpeD). Collectively, these
results suggest that C. burnetii plasmid-encoded T4SS substrates play important roles in subversion of host cell functions, providing a plausible explanation
for the absolute maintenance of plasmid genes by this pathogen.

    • "VCC, vacuole contenant des Coxiella; VGT, variant grande taille; VPT, variant petite taille. ces supplémentaires sont nécessaires (Broederdorf et Voth 2011). Récemment, 2 autres protéines anti-apoptotiques ont été identifiées. "

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    • "Initial studies to identify Coxiella Dot/Icm effectors used L. pneumophila as a surrogate host due to the ease with which this organism can be genetically manipulated and the functional analogy between the T4SSs (Pan et al., 2008; Voth et al., 2009; Chen et al., 2010). However, new genetic techniques have facilitated the development of methods to measure translocation by Coxiella using reporter systems such as β-lactamase and adenylate cyclase–effector fusions (Chen et al., 2010; Carey et al., 2011; Voth et al., 2011). "
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    ABSTRACT: Coxiella burnetii, the causative agent of the human disease Q fever, is a unique intracellular bacterial pathogen. Coxiella replicates to high numbers within a pathogen-derived lysosome-like vacuole, thriving within a low pH, highly proteolytic and oxidative environment. In 2009, researchers developed means to axenically culture Coxiella paving the way for the development of tools to genetically manipulate the organism. These advances have revolutionized our capacity to examine the pathogenesis of Coxiella. In recent years targeted and random mutant strains have been used to demonstrate that the Dot/Icm type IV secretion system is essential for intracellular replication of Coxiella. Current research is focused towards understanding the unique cohort of over 130 effector proteins that are translocated into the host cell. Mutagenesis screens have been employed to identify effectors that play important roles for the biogenesis of the Coxiella-containing vacuole and intracellular replication of Coxiella. A surprisingly high number of effector mutants demonstrate significant intracellular growth defects and future studies on the molecular function of these effectors will provide great insight into the pathogenesis of Coxiella. Already, this expanse of new data implicates many eukaryotic processes that are targeted by the arsenal of Coxiella effectors including autophagy, apoptosis and vesicular trafficking. This article is protected by copyright. All rights reserved.
    Preview · Article · Feb 2015 · Cellular Microbiology
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    • "Differentiated THP-1 cells were infected with C. burnetii expressing CyaA-CpeE as previously described (Voth et al., 2011). At 48 hpi, indicated cells were treated with H-89, then harvested, cAMP extracted, and samples processed using the cAMP Enzymeimmunoassay (GE Healthcare) at 72 hpi. "
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    ABSTRACT: Intracellular bacterial pathogens often subvert apoptosis signaling to regulate survival of their host cell, allowing propagation of the bacterial population. Coxiella burnetii, the intracellular agent of human Q fever, inhibits host cell apoptosis through several mechanisms, including prevention of mitochondrial cytochrome c release, triggering of an anti-apoptotic transcriptional program, and activation of pro-survival kinases. To control host cell survival, C. burnetii delivers effector proteins to the eukaryotic cytosol using a specialized Dot/Icm type IV secretion system (T4SS). Effectors are predicted to regulate activity of pro-survival host signaling proteins, such as Akt and cAMP-dependent protein kinase (PKA), to control infection. Here, we show that host PKA activity is required for C. burnetii inhibition of macrophage apoptosis. PKA is activated during infection and inhibits activity of the pro-apoptotic protein Bad via phosphorylation. Bad is also phosphorylated at an Akt-specific residue, indicating C. burnetii uses two kinases to fully inactivate Bad. Additionally, Bad and the tethering protein 14-3-3β co-localize at the C. burnetii parasitophorous vacuole (PV) membrane during infection, an event predicted to alter Bad promotion of apoptosis. Inhibiting PKA activity prevents Bad recruitment to the PV, but the protein is retained at the membrane during induction of apoptosis. Finally, PKA regulatory subunit I (RI) traffics to the PV membrane in a T4SS-dependent manner, suggesting a C. burnetii effector(s) regulates PKA-dependent activities. This study is the first to demonstrate subversion of host PKA activity by an intracellular bacterial pathogen to prevent apoptosis and survive within macrophages.
    Preview · Article · Sep 2013 · Cellular Microbiology
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