Neisseria gonorrhoeae is known to survive in the host through the extensive variation of surface-exposed structures, including the type IV pilus. N. gonorrhoeae attaches to the human urogential epithelium via its type IV pilus, which is composed of pilin monomers encoded by the pilE gene. Pilin antigenic variation (Av) is defined as the high frequency change of amino acid residues in the pilin protein. During pilin Av a portion of a silent pilin locus (pilS) replaces part of the expressed pilin locus (pilE) producing a recombinant pilin that is efficiently expressed on the cell surface. The aim of this work is to examine the mechanisms involved in pilin Av.
A semi-quantitative real-time RT–PCR assay was designed to measure pilin Av. This assay employs hybridization probe sets that quantify subpopulations of pilin transcripts carrying a different silent pilin copy sequence and one set that detects total pilE transcript levels. A mixture of a DNA standard carrying the silent copy being detected and a clone encoding the starting pilE sequence provided amplification curves that closely matched the amplification curves of the experimental data and allowed an analysis of the contribution of different silent pilin copies to variation. The results of this SQ-PCR Av assay were verified using DNA sequence analysis. Both assays showed that with a starting pilE sequence, a subset of the silent pilin copies recombine into pilE at a detectable level. When an isogenic pilE sequence variant was examined, a new subset of silent copy sequences was detected recombining into pilE and the frequency of variation was increased. A sequencing assay showed that the frequency of pilin Av was increased in iron-starved cultures versus iron-replete conditions. The SQ-PCR Av assay and the sequencing Av assay were used to confirm that pilin Av occurs primarily through intracellular recombination at 20 h, but suggests that there is a role for transformation in pilin Av.
Lastly, a genetic screen was developed to determine the factors involved in the initiation of pilin Av. In this screen two novel open reading frames were identified which may be involved in pilin Av.
|Advisor:||Seifert, H. S.|
|Commitee:||Cianciotto, Nicholas, Engman, David, Hauser, Alan, Satchell, Karla F.|
|Department:||Integrated Graduate Program in the Life Sciences|
|School Location:||United States -- Illinois|
|Source:||DAI-B 70/04, Dissertation Abstracts International|
|Subjects:||Molecular biology, Microbiology|
|Keywords:||Antigenic variation, Neisseria gonorrhoeae, Pili, Real time RT-PCR, Recombination, Transformation|
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