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Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing
Published on: July 6, 2016
Nitric oxide partially controls Coxiella burnetii phase II infection in mouse primary macrophages
Dario S Zamboni1, Michel Rabinovitch
1Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, UNIFESP, São Paulo, SP 04023-062 Brazil.
Abstract:
In most primary or continuous cell cultures infected with the Q-fever agent Coxiella burnetii, bacteria are typically sheltered in phagolysosome-like, large replicative vacuoles (LRVs). We recently reported that only a small proportion of mouse peritoneal macrophages (PMPhi) infected with a nonvirulent, phase II strain of C. burnetii developed LRVs and that their relative bacterial load increased only slowly. In the majority of infected PMPhi, the bacteria were confined to the small vesicles. We show here that nitric oxide (NO) induced by the bacteria partially accounts for the restricted development of LRVs in primary macrophages. Thus, (i) PMPhi and bone marrow-derived macrophages (BMMPhi) challenged with phase II C. burnetii produced significant amounts of NO; (ii) the NO synthase inhibitors aminoguanidine and N-methyl-L-arginine reduced the production of NO and increased the frequency of LRVs (although the relative bacterial loads of individual LRVs did not change, the estimated loads per well increased appreciably); (iii) gamma interferon (IFN-gamma) or the NO donor sodium nitroprusside, added to BMMPhi prior to or after infection, reduced the development and the relative bacterial loads of LRVs and lowered the yield of viable bacteria recovered from the cultures; and (iv) these effects of IFN-gamma may not be entirely dependent on the production of NO since IFN-gamma also controlled the infection in macrophages from inducible NO synthase knockout mice. It remains to be determined whether NO reduced the development of LRVs by acting directly on the bacteria; by acting on the traffic, fusion, or fission of cell vesicles; or by a combination of these mechanisms.
Insights
Nitric oxide (NO) production by Coxiella burnetii partially limits the development of large replicative vacuoles (LRVs) in macrophages. Inhibiting NO increased LRV frequency, suggesting NO
Area of Science:
- Microbiology
- Immunology
- Cell Biology
Background:
- Coxiella burnetii, the Q-fever agent, typically resides in large replicative vacuoles (LRVs) within host cells.
- Previous studies showed limited LRV development in mouse peritoneal macrophages (PMPhi) infected with a nonvirulent C. burnetii strain.
- Bacteria in most infected PMPhi were confined to smaller vesicles, with slow bacterial load increase.
Purpose of the Study:
- To investigate the role of nitric oxide (NO) in restricting LRV development in primary macrophages infected with C. burnetii.
- To elucidate the mechanisms by which NO influences C. burnetii replication within macrophages.
Main Methods:
- Infection of primary macrophages (PMPhi) and bone marrow-derived macrophages (BMMPhi) with phase II C. burnetii.
- Measurement of NO production and modulation using NO synthase inhibitors (aminoguanidine, N-methyl-L-arginine).
- Treatment with gamma interferon (IFN-gamma) or an NO donor (sodium nitroprusside).
- Analysis of LRV frequency, bacterial load, and viable bacterial yield.
- Experiments using macrophages from inducible NO synthase knockout mice.
Main Results:
- C. burnetii infection induced significant NO production in PMPhi and BMMPhi.
- NO synthase inhibition increased LRV frequency, though not individual LRV bacterial load.
- IFN-gamma and an NO donor reduced LRV development, bacterial load, and viable bacterial yield.
- IFN-gamma's anti-infective effects persisted in macrophages lacking inducible NO synthase.
Conclusions:
- Nitric oxide (NO) partially accounts for the restricted development of large replicative vacuoles (LRVs) in macrophages infected with C. burnetii.
- NO modulation impacts C. burnetii vacuole development and bacterial replication within macrophages.
- IFN-gamma controls C. burnetii infection, potentially through both NO-dependent and NO-independent pathways.
