Coxiella burnetii Alters Cyclic AMP-Dependent Protein Kinase Signaling during Growth in Macrophages

Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA.
Infection and immunity (Impact Factor: 3.73). 04/2012; 80(6):1980-6. DOI: 10.1128/IAI.00101-12
Source: PubMed


Coxiella burnetii is the bacterial agent of human Q fever, an acute, flu-like illness that can present as chronic endocarditis in immunocompromised
individuals. Following aerosol-mediated transmission, C. burnetii replicates in alveolar macrophages in a unique phagolysosome-like parasitophorous vacuole (PV) required for survival. The
mechanisms of C. burnetii intracellular survival are poorly defined and a recent Q fever outbreak in the Netherlands emphasizes the need for better
understanding this unique host-pathogen interaction. We recently demonstrated that inhibition of host cyclic AMP-dependent
protein kinase (PKA) activity negatively impacts PV formation. In the current study, we confirmed PKA involvement in PV biogenesis
and probed the role of PKA signaling during C. burnetii infection of macrophages. Using PKA-specific inhibitors, we found the kinase was needed for biogenesis of prototypical PV
and C. burnetii replication. PKA and downstream targets were differentially phosphorylated throughout infection, suggesting prolonged regulation
of the pathway. Importantly, the pathogen actively triggered PKA activation, which was also required for PV formation by virulent
C. burnetii isolates during infection of primary human alveolar macrophages. A subset of PKA-specific substrates were differentially
phosphorylated during C. burnetii infection, suggesting the pathogen uses PKA signaling to control distinct host cell responses. Collectively, the current
results suggest a versatile role for PKA in C. burnetii infection and indicate virulent organisms usurp host kinase cascades for efficient intracellular growth.

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    • "Coxiella burnetii phase II (RSA439) organisms (NMII) were cultured in Vero cells [American Type Culture Collection (ATCC)], and purified as previously described (Coleman et al., 2004). C. burnetii Nine Mile phase I (RSA493) organisms (NMI) were cultured in acidified citrate cysteine medium (ACCM) and purified as previously described (Omsland et al., 2009; MacDonald et al., 2012). For R1 localization assays, NMII wild-type and icmD::Tn mutant organisms were cultured in ACCM medium as described previously (Omsland et al., 2009), with mutant cultures containing kanamycin (350 μg ml "
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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.
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    ABSTRACT: The intracellular bacterial pathogen Coxiella burnetii is a category B select agent that causes human Q fever. In vivo, C.burnetii targets alveolar macrophages wherein the pathogen replicates in a lysosome-like parasitophorous vacuole (PV). In vitro, C.burnetii infects a variety of cultured cell lines that have collectively been used to model the pathogen's infectious cycle. However, differences in the cellular response to infection have been observed, and virulent C.burnetii isolate infection of host cells has not been well defined. Because alveolar macrophages are routinely implicated in disease, we established primary human alveolar macrophages (hAMs) as an in vitro model of C.burnetii-host cell interactions. C.burnetii pathotypes, including acute disease and endocarditis isolates, replicated in hAMs, albeit with unique PV properties. Each isolate replicated in large, typical PV and small, non-fused vacuoles, and lipid droplets were present in avirulent C.burnetiiPV. Interestingly, a subset of small vacuoles harboured single organisms undergoing degradation. Prototypical PV formation and bacterial growth in hAMs required a functional type IV secretion system, indicating C.burnetii secretes effector proteins that control macrophage functions. Avirulent C.burnetii promoted sustained activation of Akt and Erk1/2 pro-survival kinases and short-termphosphorylation of stress-related p38. Avirulent organisms also triggered a robust, early pro-inflammatory response characterized by increased secretion of TNF-α and IL-6, while virulent isolates elicited substantially reduced secretion of these cytokines. A corresponding increase in pro- and mature IL-1β occurred in hAMs infected with avirulent C.burnetii, while little accumulation was observed following infection with virulent isolates. Finally, treatment of hAMs with IFN-γ controlled intracellular replication, supporting a role for this antibacterial insult in the host response to C.burnetii.
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