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Virulent Coxiella burnetii pathotypes productively infect primary human alveolar macrophages.

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.?burnetii?PV. 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-? and IL-6, while virulent isolates elicited substantially reduced secretion of these cytokines. A corresponding increase in pro- and mature IL-1? occurred in hAMs infected with avirulent C.?burnetii, while little accumulation was observed following infection with virulent isolates. Finally, treatment of hAMs with IFN-? 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.

SUBMITTER: Graham JG

PROVIDER: S-EPMC3655087 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Virulent Coxiella burnetii pathotypes productively infect primary human alveolar macrophages.

Graham Joseph G JG MacDonald Laura J LJ Hussain S Kauser SK Sharma Uma M UM Kurten Richard C RC Voth Daniel E DE

Cellular microbiology 20130114 6

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 infectio ...[more]

PMID: 23279051

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Project description:Coxiella burnetii requires a type IVB secretion system (T4SS) to promote intracellular replication and virulence. We hypothesized that Coxiella employs its T4SS to secrete effectors that enable stealthy colonization of immune cells. To address this, we used RNA sequencing to compare the transcriptional response of murine bone marrow-derived macrophages (BMDM) infected with those of wild-type Coxiella and a T4SS-null mutant at 8 and 24 h postinfection. We found a T4SS-independent upregulation of proinflammatory transcripts which was consistent with a proinflammatory polarization phenotype. Despite this, infected BMDM failed to completely polarize, as evidenced by modest surface expression of CD38 and CD11c, nitrate production, and reduced proinflammatory cytokine and chemokine secretion compared to positive controls. As these BMDM permitted replication of C. burnetii, we employed them to identify T4SS effectors that are essential in the specific cellular context of a primary macrophage. We found five Himar1 transposon mutants in T4SS effectors that had a replication defect in BMDM but not J774A.1 cells. The mutants were also attenuated in a SCID mouse model of infection. Among these candidate virulence factors, we found that CBU1639 contributed to the inhibition of macrophage proinflammatory responses to Coxiella infection. These data demonstrate that while T4SS is dispensable for the stealthy invasion of primary macrophages, Coxiella has evolved multiple T4SS effectors that specifically target macrophage function to proliferate within that specific cellular context. IMPORTANCE Coxiella burnetii, the causative agent of Q fever, preferentially infects macrophages of the respiratory tract when causing human disease. This work describes how primary macrophages respond to C. burnetii at the earliest stages of infection, before bacterial replication. We found that while infected macrophages increase expression of proinflammatory genes after bacterial entry, they fail to activate the accompanying antibacterial functions that might ultimately control the infection. This disconnect between initial response and downstream function was not mediated by the bacterium's type IVB secretion system, suggesting that Coxiella has other virulence factors that dampen host responses early in the infection process. Nevertheless, we were able to identify several type IVB secreted effectors that were specifically required for survival in macrophages and mice. This work is the first to identify type IVB secretion effectors that are specifically required for infection and replication within primary macrophages.

Project description:Coxiella burnetii is an intracellular bacterial pathogen that causes human Q fever, an acute flu-like illness that can progress to chronic endocarditis and liver and bone infections. Humans are typically infected by aerosol-mediated transmission, and C. burnetii initially targets alveolar macrophages wherein the pathogen replicates in a phagolysosome-like niche known as the parasitophorous vacuole (PV). C. burnetii manipulates host cAMP-dependent protein kinase (PKA) signaling to promote PV formation, cell survival, and bacterial replication. In this study, we identified the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) as a PKA substrate that is increasingly phosphorylated at S157 and S239 during C. burnetii infection. Avirulent and virulent C. burnetii triggered increased levels of phosphorylated VASP in macrophage-like THP-1 cells and primary human alveolar macrophages, and this event required the Cα subunit of PKA. VASP phosphorylation also required bacterial protein synthesis and secretion of effector proteins via a type IV secretion system, indicating the pathogen actively triggers prolonged VASP phosphorylation. Optimal PV formation and intracellular bacterial replication required VASP activity, as siRNA-mediated depletion of VASP reduced PV size and bacterial growth. Interestingly, ectopic expression of a phospho-mimetic VASP (S239E) mutant protein prevented optimal PV formation, whereas VASP (S157E) mutant expression had no effect. VASP (S239E) expression also prevented trafficking of bead-containing phagosomes to the PV, indicating proper VASP activity is critical for heterotypic fusion events that control PV expansion in macrophages. Finally, expression of dominant negative VASP (S157A) in C. burnetii-infected cells impaired PV formation, confirming importance of the protein for proper infection. This study provides the first evidence of VASP manipulation by an intravacuolar bacterial pathogen via activation of PKA in human macrophages.

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Virulent Coxiella burnetii pathotypes productively infect primary human alveolar macrophages.

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Virulent Coxiella burnetii pathotypes productively infect primary human alveolar macrophages.

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