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Dissertation
Characterization of autoinducer-2 quorum sensing and the Lsr locus in Haemophilus influenzae
Doctor of Philosophy (Ph.D.), Drexel University
Jul 2012
DOI:
https://doi.org/10.17918/00008700
Abstract
The Lsr (LuxS-regulated) locus encodes genes that bind, import and process autoinducer-2 (AI-2), which is a quorum sensing (QS) molecule produced in a wide range of bacterial species. This operon was previously unknown among the non-typeable Haemophilus influenzae (NTHi) but through whole genome sequencing and comparative genomic hybridizations (CGH) I detected it in 43/187 (23%) of strains. A mutant was constructed from strain CZ4126/02 which lacks all nine Lsr genes. In minimal media the Lsr-KO showed a slight but consistent growth defect that was not observed in complex media. Using the Vibrio harveyi BB170 bioassay, AI-2 kinetics in WT and Lsr-KO strains were characterized. Production and uptake of AI-2 was determined to be highly dependent on media conditions (similar to growth) and in defined media strain CZ4126/02 was able to take up AI-2 in early stationary phase but the Lsr-KO was not. RNA samples were collected pre and post AI-2 uptake to determine what role the Lsr locus plays in global gene expression patterns. The NTHi supragenome chip, previously used for CGH, was established as a tool for transcriptional analyses by thorough replicate testing and development of an analysis software pipeline. Two major families of virulence factors were found to be under the control of the Lsr locus: the vHiSLR and Hgp (hemoglobin-binding proteins). Surprisingly, the data suggest that the regulation of these two virulence factors is independent of the AI-2 uptake event. Additional differentially regulated genes showed strong overlap with the regulon controlled by the nitrate/nitrite-controlled NarPQ two-component system. vHiSLR and Hgp proteins were also found to be differentially regulated between the WT and Lsr-KO strains by 2D-DIGE proteome comparisons. These results demonstrate that the Lsr operon is linked to major virulence factors (vHiSLR, Hgp) and a large anaerobic switch making it highly relevant in pathogenesis.
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Details
- Title
- Characterization of autoinducer-2 quorum sensing and the Lsr locus in Haemophilus influenzae
- Creators
- Benjamin Armund Janto
- 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
- x, 209 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 991021888902804721