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Coxiella burnetii Blocks Intracellular Interleukin-17 Signaling in Macrophages.

ABSTRACT: Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever. Successful host cell infection requires the Coxiella type IVB secretion system (T4BSS), which translocates bacterial effector proteins across the vacuole membrane into the host cytoplasm, where they manipulate a variety of cell processes. To identify host cell targets of Coxiella T4BSS effector proteins, we determined the transcriptome of murine alveolar macrophages infected with a Coxiella T4BSS effector mutant. We identified a set of inflammatory genes that are significantly upregulated in T4BSS mutant-infected cells compared to mock-infected cells or cells infected with wild-type (WT) bacteria, suggesting that Coxiella T4BSS effector proteins downregulate the expression of these genes. In addition, the interleukin-17 (IL-17) signaling pathway was identified as one of the top pathways affected by the bacteria. While previous studies demonstrated that IL-17 plays a protective role against several pathogens, the role of IL-17 during Coxiella infection is unknown. We found that IL-17 kills intracellular Coxiella in a dose-dependent manner, with the T4BSS mutant exhibiting significantly more sensitivity to IL-17 than WT bacteria. In addition, quantitative PCR confirmed the increased expression of IL-17 downstream signaling genes in T4BSS mutant-infected cells compared to WT- or mock-infected cells, including the proinflammatory cytokine genes Il1a, Il1b, and Tnfa, the chemokine genes Cxcl2 and Ccl5, and the antimicrobial protein gene Lcn2 We further confirmed that the Coxiella T4BSS downregulates macrophage CXCL2/macrophage inflammatory protein 2 and CCL5/RANTES protein levels following IL-17 stimulation. Together, these data suggest that Coxiella downregulates IL-17 signaling in a T4BSS-dependent manner in order to escape the macrophage immune response.

SUBMITTER: Clemente TM

PROVIDER: S-EPMC6204741 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Coxiella burnetii Blocks Intracellular Interleukin-17 Signaling in Macrophages.

Clemente Tatiana M TM Mulye Minal M Justis Anna V AV Nallandhighal Srinivas S Tran Tuan M TM Gilk Stacey D SD

Infection and immunity 20180921 10

Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever. Successful host cell infection requires the Coxiella type IVB secretion system (T4BSS), which translocates bacterial effector proteins across the vacuole membrane into the host cytoplasm, where they manipulate a variety of cell processes. To identify host cell targets of Coxiella T4BSS effector proteins, we determined the transcriptome of murine alveolar macrophages infected with a ...[more]

PMID: 30061378

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Project description:Coxiella burnetii is a highly infectious pathogen that causes Q fever, a leading cause of culture-negative endocarditis. Coxiella first targets alveolar macrophages and forms a phagolysosome-like compartment called the C oxiella - C ontaining V acuole (CCV). Successful host cell infection requires the Type 4B Secretion System (T4BSS), which translocates bacterial effector proteins across the CCV membrane into the host cytoplasm, where they manipulate numerous cell processes. Our prior transcriptional studies revealed that Coxiella T4BSS blocks IL-17 signaling in macrophages. Given that IL-17 is known to protect against pulmonary pathogens, we hypothesize that C. burnetii T4BSS downregulates intracellular IL-17 signaling to evade the host immune response and promote bacterial pathogenesis. Using a stable IL-17 promoter reporter cell line, we confirmed that Coxiella T4BSS blocks IL-17 transcription activation. Assessment of the phosphorylation state of NF-κB, MAPK, and JNK revealed that Coxiella downregulates IL-17 activation of these proteins. Using ACT1 knockdown and IL-17RA or TRAF6 knockout cells, we next determined that IL17RA-ACT1-TRAF6 pathway is essential for the IL-17 bactericidal effect in macrophages. In addition, macrophages stimulated with IL-17 generate higher levels of reactive oxygen species, which is likely connected to the bactericidal effect of IL-17. However, C. burnetii T4SS effector proteins block the IL-17-mediated oxidative stress, suggesting that Coxiella blocks IL-17 signaling to avoid direct killing by the macrophages.ImportanceBacterial pathogens are constantly evolving mechanisms to modulate the hostile host environment encountered during infection. Coxiella burnetii , the causative agent of Q fever, is a fascinating example of intracellular parasitism. Coxiella survives in a phagolysosome-like vacuole and uses the Dot/Icm type IVB secretion system (T4BSS) to deliver bacterial effector proteins into the host cell cytoplasm to manipulate several host cell functions. We recently demonstrated that Coxiella T4BSS blocks the IL-17 signaling in macrophages. Here, we found that Coxiella T4BSS inhibits IL-17 activation of the NF-κB and MAPK pathways and blocks IL-17-mediated oxidative stress. These findings reveal a novel strategy employed by intracellular bacteria to escape the immune response during initial stages of infection. Further identification of virulence factors involved in this mechanism will bring to light new therapeutic targets to prevent Q fever development into a chronic life-threatening endocarditis.

Project description: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.

Project description:Coxiella burnetii, the etiological agent of Q fever, undergoes a unique biphasic developmental cycle where bacteria transition from a replicating (exponential-phase) large cell variant (LCV) form to a nonreplicating (stationary-phase) small cell variant (SCV) form. The alternative sigma factor RpoS is an essential regulator of stress responses and stationary-phase physiology in several bacterial species, including Legionella pneumophila, which has a developmental cycle superficially similar to that of C. burnetii Here, we used a C. burnetii ?rpoS mutant to define the role of RpoS in intracellular growth and SCV development. Growth yields following infection of Vero epithelial cells or THP-1 macrophage-like cells with the rpoS mutant in the SCV form, but not the LCV form, were significantly lower than that of wild-type bacteria. RNA sequencing and whole-cell mass spectrometry of the C. burnetii ?rpoS mutant revealed that a substantial portion of the C. burnetii genome is regulated by RpoS during SCV development. Regulated genes include those involved in stress responses, arginine transport, peptidoglycan remodeling, and synthesis of the SCV-specific protein ScvA. Genes comprising the dot/icm locus, responsible for production of the Dot/Icm type 4B secretion system, were also dysregulated in the rpoS mutant. These data were corroborated with independent assays demonstrating that the C. burnetii ?rpoS strain has increased sensitivity to hydrogen peroxide and carbenicillin and a thinner cell wall/outer membrane complex. Collectively, these results demonstrate that RpoS is an important regulator of genes involved in C. burnetii SCV development and intracellular growth.IMPORTANCE The Q fever bacterium Coxiella burnetii has spore-like environmental stability, a characteristic that contributes to its designation as a potential bioweapon. Stability is likely conferred by a highly resistant, small cell variant (SCV) stationary-phase form that arises during a biphasic developmental cycle. Here, we define the role of the alternative sigma factor RpoS in regulating genes associated with SCV development. Genes involved in stress responses, amino acid transport, cell wall remodeling, and type 4B effector secretion were dysregulated in the rpoS mutant. Cellular impairments included defects in intracellular growth, cell wall structure, and resistance to oxidants. These results support RpoS as a central regulator of the Coxiella developmental cycle and identify developmentally regulated genes involved in morphological differentiation.

Dataset Information

Coxiella burnetii Blocks Intracellular Interleukin-17 Signaling in Macrophages.

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Coxiella burnetii Blocks Intracellular Interleukin-17 Signaling in Macrophages.

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