Genetic variation of chlamydial Inc proteins Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/5h73q0174

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  • Genomic analysis is a new approach for the characterization and investigation of novel genes, gene clusters, the function of uncharacterized proteins, and genetic diversity in microorganisms. These approaches are important for the study of chlamydiae, a system in which several genomes have been sequenced but in which techniques for genetic manipulation are not available. The objective of this thesis is to combine computer-based analysis of chiamydial inclusion membrane proteins (Incs) with cellular and molecular biological analysis of the bacteria. Three different experimental lines of investigation were examined, focusing on Incs of C. trachomatis and C. pneumoniae. Chlamydiae are obligate intracellular bacteria that develop within a nonacidified membrane bound vacuole termed an inclusion. Putative Inc proteins of C. trachomatis and C. pneumoniae were identified from genomic analysis and a unique structural motif. Selected putative Inc proteins are shown to localize to the inclusion membrane. Chiamydia trachomatis variants with unusual multiple-lobed, nonfusogenic, inclusion were identified from a large scale serotyping study. Fluorescence microscopy showed that IncA, a chiamydial protein localized to the inclusion membrane, was undetectable on non-fusogenic inclusions of these variants. Sequence analysis of incA from non-fusogenic variant isolates revealed a defective incA in most of the variants. Some variants lack not only IncA on the inclusion membrane but also CT223p, an additional Inc protein. However, no correlation between the absence of CT223p and distinctive inclusion phenotype was identified. Nucleotide sequence analysis revealed sequence variations of C. trachomatis incA and CT223 in some variant and wild type isolates. Comparative analyses of the three recently published C. pneumoniae genomes have led to the identification of a novel gene cluster named the CPn1O54 gene family. Each member of this family encodes a polypeptide with a hydrophobic domain characteristic of proteins localized to the inclusion membrane. These studies provided evidence that gene variation might occur within this single collection of paralogous genes. Collectively, the variability within this gene family may modulate either phase or antigenic variation, and subsequent physiologic diversity, within a C. pneumoniae population. These studies demonstrate the genetic diversity of Inc proteins and candidate Inc proteins, within and among the different chiamydial species. This work sets the stage for further investigations of the structure and function of this set of proteins that are likely critical to chlamydial intracellular growth.
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