Haemophilus influenzae efficiently and chronically colonizes the human nasopharyngeal mucosa, and is capable of causing invasive disease including otitis media, pneumonia, and, more rarely, meningitis. A number of factors involved in H. influenzae virulence have been identified in the pre-genomic era. Taking advantage of the genome sequence and the advent of new technologies, such as global expression profiling, we intend to advance understanding of critical virulence characteristics of this organism. Lipopolysaccharide (LPS) structural modifications are essential virulence determinants for H. influenzae. Using expression profiling with DNA microarrays, complemented by classical approaches, we have recently uncovered a previously unappreciated link between redox regulation and LPS modifications in H. influenzae. In addition, we have isolated a mariner transposon insertion mutation in H. influenzae that disrupts redox control over one such modification (addition of a phosphorylcholine epitope, termed ChoP, to the LPS) and also results in a pronounced colonization defect in an animal model of H. influenzae infection. These observations are of potential significance for in vivo modulation of the LPS structure by environmental signals. We propose to use such signaling and regulatory mutants generated in our laboratory to examine the role of redox signaling in controlling virulence genes in H. influenzae. Global genomic approaches we have developed for studies of H. influenzae will facilitate our analysis of how LPS modifications are modulated in response to environmental conditions. We will also determine whether other genes that play a role in pathogenesis are coregulated, inversely regulated, or constitutively transcribed under the varied redox conditions that affect LPS modification. We believe that these studies will provide important insights into the relationship between physiological adaptations to the host environment and the coordinated production of bacterial cell-surface structures critical for interactions with host cells or for evading the immune response. Specifically, we will: 1. Characterize the redox control mechanisms involved in the regulation of the ChoP cell surface LPS modification. 2. Investigate the role of signaling pathways in H. influenzae in the context of epithelial cell interactions and in a model of respiratory tract infection. 3. Examine coordinate regulation of virulence factors by redox signaling systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI049437-04
Application #
6743175
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Klein, David L
Project Start
2002-06-01
Project End
2007-05-31
Budget Start
2004-06-01
Budget End
2005-05-31
Support Year
4
Fiscal Year
2004
Total Cost
$387,592
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Genetics
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
Rosadini, Charles V; Ram, Sanjay; Akerley, Brian J (2014) Outer membrane protein P5 is required for resistance of nontypeable Haemophilus influenzae to both the classical and alternative complement pathways. Infect Immun 82:640-9
Wong, Sandy M; Bernui, Mariana; Shen, Hao et al. (2013) Genome-wide fitness profiling reveals adaptations required by Haemophilus in coinfection with influenza A virus in the murine lung. Proc Natl Acad Sci U S A 110:15413-8
Wong, Sandy M S; St Michael, Frank; Cox, Andrew et al. (2011) ArcA-regulated glycosyltransferase lic2B promotes complement evasion and pathogenesis of nontypeable Haemophilus influenzae. Infect Immun 79:1971-83
Wong, Sandy M S; Gawronski, Jeffrey D; Lapointe, David et al. (2011) High-throughput insertion tracking by deep sequencing for the analysis of bacterial pathogens. Methods Mol Biol 733:209-22
Harrington, Jane C; Wong, Sandy M S; Rosadini, Charles V et al. (2009) Resistance of Haemophilus influenzae to reactive nitrogen donors and gamma interferon-stimulated macrophages requires the formate-dependent nitrite reductase regulator-activated ytfE gene. Infect Immun 77:1945-58
Gawronski, Jeffrey D; Wong, Sandy M S; Giannoukos, Georgia et al. (2009) Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung. Proc Natl Acad Sci U S A 106:16422-7
Vijayalakshmi, J; Akerley, Brian J; Saper, Mark A (2008) Structure of YraM, a protein essential for growth of Haemophilus influenzae. Proteins 73:204-17
Rosadini, Charles V; Wong, Sandy M S; Akerley, Brian J (2008) The periplasmic disulfide oxidoreductase DsbA contributes to Haemophilus influenzae pathogenesis. Infect Immun 76:1498-508
Wong, Sandy M S; Akerley, Brian J (2008) Identification and analysis of essential genes in Haemophilus influenzae. Methods Mol Biol 416:27-44
Wong, Sandy M S; Alugupalli, Kishore R; Ram, Sanjay et al. (2007) The ArcA regulon and oxidative stress resistance in Haemophilus influenzae. Mol Microbiol 64:1375-90

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