Nitric Oxide InhibitsCoxiella burnetiiReplication and Parasitophorous Vacuole Maturation

Publisher Website
Google Scholar
Abstract
Nitric oxide is a recognized cytotoxic effector against facultative and obligate intracellular bacteria. This study examined the effect of nitric oxide produced by inducible nitric oxide synthase (iNOS) up-regulated in response to cytokine stimulation, or by a synthetic nitric oxide donor, on replication of obligately intracellularCoxiella burnetiiin murine L-929 cells. Immunoblotting and nitrite assays revealed thatC. burnetiiinfection of L-929 cells augments expression of iNOS up-regulated in response to gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Infection in the absence of cytokine stimulation did not result in demonstrable up-regulation of iNOS expression or in increased nitrite production. Nitrite production by cytokine-treated cells was significantly inhibited by the iNOS inhibitorS-methylisothiourea (SMT). Treatment of infected cells with IFN-γ and TNF-α or the synthetic nitric oxide donor 2,2′-(hydroxynitrosohydrazino)bis-ethanamine (DETA/NONOate) had a bacteriostatic effect onC. burnetiireplication. Inhibition of replication was reversed upon addition of SMT to the culture medium of cytokine-treated cells. Microscopic analysis of infected cells revealed that nitric oxide (either cytokine induced or donor derived) inhibited formation of the mature (large) parasitophorous vacuole that is characteristic ofC. burnetiiinfection of host cells. Instead, exposure of infected cells to nitric oxide resulted in the formation of multiple small, acidic vacuoles usually containing oneC. burnetiicell. Removal of nitrosative stress resulted in the coalescence of small vacuoles to form a large vacuole harboring multipleC. burnetiicells. These experiments demonstrate that nitric oxide reversibly inhibits replication ofC. burnetiiand formation of the parasitophorous vacuole.