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Infection and Immunity, May 2009, p. 1945-1958, Vol. 77, No. 5
0019-9567/09/$08.00+0     doi:10.1128/IAI.01365-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Resistance of Haemophilus influenzae to Reactive Nitrogen Donors and Gamma Interferon-Stimulated Macrophages Requires the Formate-Dependent Nitrite Reductase Regulator-Activated ytfE Gene

Jane C. Harrington,¹ Sandy M. S. Wong,¹ Charles V. Rosadini,¹ Oleg Garifulin,² Victor Boyartchuk,² and Brian J. Akerley^1*

Department of Molecular Genetics and Microbiology,¹ Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, Massachusetts 01655²

Received 6 November 2008/ Returned for modification 16 December 2008/ Accepted 2 March 2009

Haemophilus influenzae efficiently colonizes and persists at the human nasopharyngeal mucosa, causing disease when it spreads to other sites. Nitric oxide (NO) represents a major antimicrobial defense deployed by host cells in locations colonized by H. influenzae during pathogenesis that are likely to vary in oxygen levels. Formate-dependent nitrite reductase regulator (FNR) is an oxygen-sensitive regulator in several bacterial pathogens. We report that fnr of H. influenzae is required for anaerobic defense against exposure to NO donors and to resist NO-dependent effects of gamma interferon (IFN-)-activated murine bone marrow-derived macrophages. To understand the mechanism of resistance, we investigated the role of FNR-regulated genes in defense against NO sources. Expression analysis revealed FNR-dependent activation of nrfA, dmsA, napA, and ytfE. Nonpolar deletion mutants of nrfA and ytfE exhibited sensitivity to NO donors, and the ytfE gene was more critical for survival. Compared to the wild-type strain, the ytfE mutant exhibited decreased survival when exposed to macrophages, a defect that was more pronounced after prior stimulation of macrophages with IFN- or lipopolysaccharide. Complementation restored survival of the mutant to the level in the parental strain. Increased sensitivity of the ytfE mutant relative to that of the parent was abrogated by treatment of macrophages with a NO synthase inhibitor, implicating YtfE in resistance to a NO-dependent pathway. These results identify a requirement for FNR in positive control of ytfE and indicate a critical role for ytfE in resistance of H. influenzae to reactive nitrogen species and the antibacterial effects of macrophages.

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* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Ave., N., S6-242, Worcester, MA 01655. Phone: (508) 856-1442. Fax: (508) 856-1422. E-mail: Brian.Akerley{at}umassmed.edu

Published ahead of print on 16 March 2009.

Editor: J. B. Bliska

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Infection and Immunity, May 2009, p. 1945-1958, Vol. 77, No. 5
0019-9567/09/$08.00+0     doi:10.1128/IAI.01365-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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