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

MacDonald, Laura J.; Kurten, Richard C.; Voth, Daniel E.

doi:10.1128/iai.00101-12

Cited by 18 publications

(27 citation statements)

References 47 publications

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“…Total Bad levels remained constant throughout the infection time‐course. Akt activity is required for C. burnetii prevention of apoptosis, and is activated between 2 and 6 hpi (Voth and Heinzen, ), while PKA activation is observed from 24 to 96 hpi (Voth and Heinzen, ; MacDonald et al ., ). Densitometry analysis confirmed that the timing of increased Bad phosphorylation corresponds to kinase activation, suggesting Akt is activated first during infection and phosphorylates Bad at Ser136, followed by PKA activation and Bad Ser155 phosphorylation (Fig.…”

Section: Resultsmentioning

confidence: 97%

“…We recently demonstrated that primary human alveolar macrophages (hAMs) are a human disease‐relevant platform to investigate C. burnetii ‐regulated signalling required for infection (Graham et al ., ), and PKA is activated by virulent C. burnetii during growth in primary hAMs (MacDonald et al ., ). To determine if virulent C. burnetii modulates PKA‐dependent pro‐survival signalling, we infected primary hAMs with virulent Nine Mile I (NMI) C. burnetii and assessed the impact of PKA inhibition on apoptosis.…”

Section: Resultsmentioning

confidence: 97%

“…We recently discovered that Bad phosphorylation increases substantially during C. burnetii infection (MacDonald et al ., ), suggesting the pathogen regulates Bad activity to influence host cell survival. In the current study, we found that PKA activity is required for C. burnetii anti‐apoptotic activity, and Bad phosphorylation increases during infection at Akt‐ and PKA‐specific residues, indicating both kinases are required for prevention of apoptosis.…”

Section: Introductionmentioning

confidence: 99%

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Coxiella burnetiiexploits host cAMP-dependent protein kinase signalling to promote macrophage survival

et al. 2013

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Summary 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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Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

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Coxiella burnetiiexploits host cAMP-dependent protein kinase signalling to promote macrophage survival

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“…Previous studies demonstrated the importance of intracellular signalling cascade activation for C. burnetii PV formation and prevention of apoptosis (Voth and Heinzen, ; Hussain et al ., ; Hussain and Voth, ; MacDonald et al ., ). The organism inhibits apoptosis by activating kinases such as Akt and Erk1/2, promoting an anti‐apoptotic transcriptional programme and preventing cytochrome c release from mitochondria (Luhrmann and Roy, ; Voth et al ., ; Luhrmann et al ., ).…”

Section: Resultsmentioning

confidence: 99%

VirulentCoxiella burnetiipathotypes productively infect primary human alveolar macrophages

et al. 2013

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Summary 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. burnetii PV. Interestingly, a subset of small vacuoles harbored 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 term phosphorylation 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. Collectively, the current results demonstrate the hAM model is a human disease-relevant platform for defining novel innate immune responses to C. burnetii.

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“…To better understand the importance of autophagy in vivo, we used two models of C. burnetii-macrophage interactions. First, we used macrophage-like THP-1 cells, an established in vitro model of C. burnetii-host cell interactions (32)(33)(34). PV in THP-1 cells interacted with autophagosomes from 24 to 96 hpi, suggesting that these interactions occur throughout intracellular growth in macrophages.…”

Section: Discussionmentioning

confidence: 99%

Coxiella burnetii Type IV Secretion-Dependent Recruitment of Macrophage Autophagosomes

et al. 2014

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cCoxiella burnetii is an intracellular Gram-negative bacterium that causes human Q fever, a flu-like disease that can progress to chronic, life-threatening endocarditis. In humans, C. burnetii infects alveolar macrophages and promotes phagosomal fusion with autophagosomes and lysosomes, establishing a unique parasitophorous vacuole (PV) in which to replicate. The pathogen uses a Dot/Icm type IV secretion system (T4SS) to deliver effector proteins to the host cytoplasm, where they alter cellular processes to benefit the pathogen. The T4SS is required for PV expansion and prevention of apoptosis, but little else is known about the role of the system during intracellular growth. Recent reports suggest that C. burnetii actively recruits autophagosomes to the PV to deliver nutrients to the pathogen and provide membrane for the expanding vacuole. In this study, we examined the role of the T4SS in mediating PV interactions with autophagosomes. We found that the autophagy-related proteins LC3 and p62 localized to wild-type PV but not to T4SS mutant organism-containing phagosomes in human macrophage-like cells, primary human alveolar macrophages, and Chinese hamster ovary cells. However, while lipidated LC3 levels were elevated regardless of T4SS activity, no p62 turnover was observed during C. burnetii growth in macrophages, suggesting that the pathogen recruits preformed autophagosomes. When the T4SS was activated 24 h after infection, autophagosome recruitment ensued, indicating that autophagosome interactions are dispensable for initial PV maturation to a phagolysosome-like compartment but are involved in vacuole expansion. Together, these results demonstrate that C. burnetii actively directs PV-autophagosome interactions by using the Dot/Icm T4SS.

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

Abstract: ABSTRACTCoxiella burnetiiis 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 Show more

Cited by 18 publications

References 47 publications

Coxiella burnetiiexploits host cAMP-dependent protein kinase signalling to promote macrophage survival

Coxiella burnetiiexploits host cAMP-dependent protein kinase signalling to promote macrophage survival

VirulentCoxiella burnetiipathotypes productively infect primary human alveolar macrophages

Coxiella burnetii Type IV Secretion-Dependent Recruitment of Macrophage Autophagosomes

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