H. influenzae is the most common cause of pyogenic meningitis in the United States. The peak incidence of H. influenzae meningitis is 6-9 months of age, and neurological sequelae are detected in approximately 50% of survivors. To expand our understanding of the molecular genetics of H. influenzae, we have developed a novel method for insertiona mutagenesis of the chromosome of H. influenzae. A mutagenizing elements, """"""""TSTE"""""""", has been constructed by ligation of the portion of the transposon Tn5 encoding kanamycin resistance with a synthetic oligonucleotide which includes the uptake sequence which enhances natural transformation of H. influenzae by several orders of magnitude. Chromosomal DNA fragment will be circularized by self-ligation. After digestion with a restriction enzyme which produces compatible sticky ends, the """"""""TSTE"""""""" element will be ligated, and the interrupted DNA fragments will be transformed back into the original strain. Kanamycin resistance transformants will have insertional mutations in the chromosome. The """"""""TSTE"""""""" insertional mutagenesis will be applied to genes encoding hydroxamate production. H. influenzae produces hydroxamates in high iron concentrations, and hydroxamate production by H. influenzae is highly conserved along clonal lines of virulent type b strains. To investigate this unique iron-related characteristic of H. influenzae a genetic analysis of defined mutants will be performed to identify the structural and regulatory genes responsible for hydroxamate production. These genes will be cloned, mapped with restriction enzymes, and regulatory regions will be sequenced to determine whether the """"""""iron box"""""""" operator found in other bacteria is present in H. influenzae. The hydroxamate have been purified, and the biological role of hydroxamate production will be analyzed by 1) determining the survival of isogenic strains in the presence of purified neutrophils, and 2) comparing the virulence of the isogenic strains; and 3) determining the ability of H. influenzae hydroxamates to interfere with the production of hydroxyl radical by iron sequestration. The molecular structure of the hydroxamates will be determined by mass spectroscopy and nuclear magnetic resonance.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI029611-03
Application #
3144487
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1990-12-01
Project End
1995-11-30
Budget Start
1992-12-01
Budget End
1993-11-30
Support Year
3
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Allegheny University of Health Sciences
Department
Type
Schools of Medicine
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19129
Hempel, Randy J; Morton, Daniel J; Seale, Thomas W et al. (2013) The role of the RNA chaperone Hfq in Haemophilus influenzae pathogenesis. BMC Microbiol 13:134
Whitby, Paul W; VanWagoner, Timothy M; Seale, Thomas W et al. (2013) Comparison of transcription of the Haemophilus influenzae iron/heme modulon genes in vitro and in vivo in the chinchilla middle ear. BMC Genomics 14:925
Morton, Daniel J; Hempel, Randy J; Whitby, Paul W et al. (2012) An invasive Haemophilus haemolyticus isolate. J Clin Microbiol 50:1502-3
Whitby, Paul W; Morton, Daniel J; Vanwagoner, Timothy M et al. (2012) Haemophilus influenzae OxyR: characterization of its regulation, regulon and role in fitness. PLoS One 7:e50588
Morton, Daniel J; Hempel, Randy J; Seale, Thomas W et al. (2012) A functional tonB gene is required for both virulence and competitive fitness in a chinchilla model of Haemophilus influenzae otitis media. BMC Res Notes 5:327
Whitby, Paul W; VanWagoner, Timothy M; Morton, Daniel J et al. (2012) Signature-tagging of a bacterial isolate demonstrates phenotypic variability of the progeny in vivo in the absence of defined mutations. J Microbiol Methods 91:336-40
Whitby, Paul W; Seale, Thomas W; Morton, Daniel J et al. (2010) Characterization of the Haemophilus influenzae tehB gene and its role in virulence. Microbiology 156:1188-200
Morton, Daniel J; Turman, Elizabeth J; Hensley, Patrick D et al. (2010) Identification of a siderophore utilization locus in nontypeable Haemophilus influenzae. BMC Microbiol 10:113
Morton, Daniel J; Seale, Thomas W; Vanwagoner, Timothy M et al. (2009) The dppBCDF gene cluster of Haemophilus influenzae: Role in heme utilization. BMC Res Notes 2:166
Whitby, Paul W; Seale, Thomas W; VanWagoner, Timothy M et al. (2009) The iron/heme regulated genes of Haemophilus influenzae: comparative transcriptional profiling as a tool to define the species core modulon. BMC Genomics 10:6

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