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Dissertation
Cloning and characterization of the haemocin locus of Haemophilus influenzae and assessment of its role in virulence
Doctor of Philosophy (Ph.D.), Allegheny University of the Health Sciences
Aug 1997
DOI:
https://doi.org/10.17918/00009346
Abstract
The bacteriocin haemocin (HMC) is produced by most type b strains of Haemophilus influenzae, including strains determined to be genetically diverse, and is toxic to virtually all non-type b strains of H. influenzae, both encapsulated and nonencapsulated. We prepared a protein preparation enriched for HMC that was useful in subsequent cloning efforts. Complementation of a HMC-deficient H. influenzae mutant resulted in the identification of a recombinant plasmid containing several ORFs involved in HMC expression. Subsequent deletional and site-directed mutagenesis of this plasmid identified the HMC immunity gene, hmcI. This gene encodes a protein similar in size to a number of other characterized bacteriocin immunity proteins, although it has little homology to other nucleotide sequences in available databases. Cloning of DNA flanking hmcI in the H. influenzae chromosome led to the identification of several other genes involved in HMC production and transport. Examination of the deduced amino acid sequences of these genes revealed several features common to class II bacteriocins of certain gram-positive bacteria. Specifically, the HMC structural gene encodes the characteristic class II double-glycine leader peptide. Interestingly, the HMC proteolytic processor and transporter are encoded by different genes rather than a single gene, as with all other class II transporter systems described to date. While subcloning of the locus into a HMC- Escherichia coli resulted in HMC immune transformants, they did not produce HMC, suggesting that additional genes are required for HMC production. The effects of a mutation in the HMC structural gene, hmcA, were examined in vitro and in vivo by using cell culture and the H. influenzae infant rat model of infection. Loss of HMC production did not have an apparent effect on cytotoxicity, but significantly delayed the onset of bacteremia in infant rats.
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Details
- Title
- Cloning and characterization of the haemocin locus of Haemophilus influenzae and assessment of its role in virulence
- Creators
- Yvette Marie Murley
- Awarding Institution
- Allegheny University of the Health Sciences
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Allegheny University of the Health Sciences; Philadelphia, Pennsylvania
- Number of pages
- ix, 92 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Microbiology and Immunology [Historical]; Allegheny University of the Health Sciences (1996-1998); School of Medicine (1996-1998)
- Other Identifier
- 991021888897504721