D-083. FNR, an Anaerobically Active Regulator, Confers Nitric Oxide Resistance in Haemophilus Influenzae

J. C. Harrington, B. J. Akerley;
Univ. of Massachusetts Med. Sch., Worcester, MA.

Haemophilus influenzae encounters niches within the human host that are predicted to differ in oxygen availability. Previous data from our lab have indicated that an altered redox condition could serve as a signal recognized by H. influenzae to optimize its survival within host microenvironments. We examined the H. influenzae FNR homologue, whose counterpart in E. coli is considered to be a direct sensor of oxygen. To study FNR, we targeted a suspected FNR-regulated gene, nrfA, which encodes nitrite reductase, a periplasmic cytochrome-c involved in anaerobic respiration, as a read-out for FNR activity. Using Western blot detection of an epitope-tagged reporter protein fused to the endogenous nrfA promoter (Pnrf-hel), we demonstrate that FNR is required for activation of nrfA promoter. We further show that Pnrf-hel expression increases as oxygen becomes depleted. The nrfA of E. coli has been implicated in resistance to the reactive nitrogen species, nitric oxide (NO), which is produced by innate immune cells during infection. We show that a mutant lacking FNR is more sensitive to NO exposure than wild type H. influenzae after anaerobic pre-growth. A mutant lacking nrfA has additionally been tested and initial experiments have shown the nrfA mutant has a lesser NO sensitivity phenotype as compared to the fnr mutant, suggesting that other factors could be involved in FNR-mediated NO resistance in H. influenzae. Upon examination of potential factors, we discovered another regulator that contributes to defense against NO challenge.