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Dissertation
Identification and characterization of a novel bacterial virulence factor in Haemophilus influenzae
Doctor of Philosophy (Ph.D.), Drexel University
Jun 2012
DOI:
https://doi.org/10.17918/00007732
Abstract
Haemophilus influenzae (Hi) is part of the normal nasopharyngeal flora and is an opportunistic pathogen capable of causing serious human disease. The emergence of virulent, antibiotic-resistant nontypeable Hi (NTHi) emphasizes the importance of developing new vaccine targets. We searched the NTHi supragenome for distributed gene families encoding surface-exposed proteins with characteristics associated with immune evasion and virulence. This search identified an unannotated heterogeneous family containing Sel1-like repeats (SLRs) we named the vHiSLR. Clustering algorithms identified multiple vHiSLR families, each containing various numbers of SLRs per gene. Individual strains carry different numbers of genes that may belong to one or more families. An association study using a library of ~200 clinical strains demonstrated a significant link between SLR gene possession and virulence. vHiSLR virulence mechanisms were determined by: 1) creating vHiSLR knockouts (KO); 2) comparing the ability of the KO and wild type (WT) bacteria to infect and survive in macrophages in vitro; 3) utilizing the chinchilla otitis media invasive disease (OMID) model to, i) compare virulence between the WT and KO strains, and ii) determine their competitiveness during co-infection; and 4) utilizing an NTHi microarray for comparative transcriptome analyses. The KO strain showed a highly significant decrease in its ability to invade and survive in the human macrophages which could be complemented by insertion of a single vHiSLR gene. Using the chinchilla OMID model the mutant displayed a highly significant decrease in fitness in the presence of the WT, and in solo infections completely lost its ability to invade the brain. The singly complemented strain showed only a partial ability to compete with the WT in vivo suggesting that gene dosage is important. The transcriptional profile of the KO showed a highly significant change in the expression of multiple operons involved in anaerobiosis and virulence. The vHiSLRs are derepressed in KO's of the Al-2 operon suggesting that this repression is controlled by the Al-2 repressor. These findings demonstrate the vHiSLRs are important virulence factors in host cell invasion, persistence, and trafficking to nonmucosal sites, and are crucial for the ability of NTHi to respond to local oxygen levels.
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Details
- Title
- Identification and characterization of a novel bacterial virulence factor in Haemophilus influenzae
- Creators
- Jennifer M. Kress Bennett
- Contributors
- Garth D. Ehrlich (Advisor) - Drexel University, Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xii, 114 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 991021888782904721