Hybrid Cluster Proteins and Flavodiiron Proteins Afford Protection to Desulfovibrio vulgaris upon Macrophage Infection

Abstract

bDesulfovibrio species are Gram-negative anaerobic sulfate-reducing bacteria that colonize the human gut. Recently, Desulfovibrio spp. have been implicated in gastrointestinal diseases and shown to stimulate the epithelial immune response, leading to increased production of inflammatory cytokines by macrophages. Activated macrophages are key cells of the immune system that impose nitrosative stress during phagocytosis. Hence, we have analyzed the in vitro and in vivo responses of Desulfovibrio vulgaris Hildenborough to nitric oxide (NO) and the role of the hybrid cluster proteins (HCP1 and HCP2) and rubredoxin oxygen oxidoreductases (ROO1 and ROO2) in NO protection. Among the four genes, hcp2 was the gene most highly induced by NO, and the hcp2 transposon mutant exhibited the lowest viability under conditions of NO stress. Studies in murine macrophages revealed that D. vulgaris survives incubation with these phagocytes and triggers NO production at levels similar to those stimulated by the cytokine gamma interferon (IFN-␥). Furthermore, D. vulgaris hcp and roo mutants exhibited reduced viability when incubated with macrophages, revealing that these gene products contribute to the survival of D. vulgaris during macrophage infection. Desulfovibrio spp. are anaerobic sulfate-reducing bacteria (SRB) that occur in several environmental niches, such as marine and freshwater sediments, as well as in humans as part of the normal oral cavity and gut flora. In particular, four Desulfovibrio spp., namely, D. fairfieldensis, D. desulfuricans, D. piger, and D. vulgaris, were detected in healthy humans (1-3). Furthermore, Desulfovibrio spp. have also been implicated in gastrointestinal diseases, such as inflammatory bowel diseases and periondontitis, since Desulfovibrio strains were isolated from biopsy specimens of patients with ulcerative colitis, brain, abdominal wall, and liver abscesses, and appendicitis (4-6).Recently, D. desulfuricans and D. fairfieldensis were shown to be able to invade nonprofessional phagocytic cells such as the oral epithelial cells and to stimulate the epithelial immune response by increasing the production of inflammatory interleukins (7). Nonetheless, the ability of Desulfovibrio spp. to survive professional phagocytes, such as macrophages, remains to be evaluated.Two of the main weapons of the innate immune system to eradicate pathogens are the generation of reactive oxygen species (ROS) and the generation of reactive nitrogen species (RNS), which are derived from the superoxide and nitric oxide produced by the NADPH oxidase and the mammalian inducible nitric oxide synthase (iNOS), respectively (8, 9). These chemicals can inflict serious damage in bacteria, which employ the expression of several detoxification systems to avoid such damage (9). The flavodiiron proteins (FDP) constitute a large family of enzymes widespread among archaea and bacteria, including in Desulfovibrio spp. They are believed to contribute to bacterial survival under oxidative and nitrosative stress conditions (10). FDPs a...

Regulation of Nitrite Stress Response in Desulfovibrio vulgaris Hildenborough, a Model Sulfate-Reducing Bacterium

“…4B). To confirm that the cells were experiencing NO stress, we examined expression of one of the hybrid cluster protein-encoding genes, hcp2, that has been shown previously to be induced by NO stress (41). We observed that the hcp2 gene was strongly induced in all three strains in the presence of GSNO (Fig.…”

“…The NO donor S-nitrosoglutathione (GSNO) was added at 10 M to mid-log-phase cells. This concentration was previously shown to cause moderate inhibition of growth in D. vulgaris (41). Addition of GSNO did not affect nrfH transcription in the WT, the nrfR mutant, or the JW9382 strain (Fig.…”

“…A description of the library is in preparation. The library has been used in other studies (39)(40)(41).…”

13. Reactivity of iron-sulfur clusters with nitric oxide

“…The so called hybrid cluster protein (Hcp) is found in many anaerobic or facultatively anaerobic bacteria, where it functions in response to nitrosative stress [217][218][219][220]. Hcp shares structural homology with anaerobic carbon monoxide dehydrogenases and contain two types of iron sulfur clusters; the first occurs as either a [2Fe-2S] or [4Fe-4S] cluster, while the second, socalled "hybrid cluster", is a unique cluster type, the precise form of which is dependent on the oxidation state.…”

Bacteriostatic and bactericidal effects of free nitrous acid on model microbes in wastewater treatment

“…In other studies, HCP and rebredoxin oxygen oxidoreductases (ROO1 and ROO2) in D. vulgaris were demonstrated to play a role in NO protection and contributed to the survival of D. vulgaris during macrophage infection (Figueiredo et al 2013). …”