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The majority of our modern understanding of bacterial pathogenesis is based on the strategy that involves screening bacterial genomes for the presence of the genes encoding pathogenic factors, and analysis of these genes via forward and reverse genetics. Chlamydiae represent a unique group of pathogenic bacteria in which it is not feasible to apply genetic approaches that are currently used for other organisms. The obligate intracellular nature of these organisms also makes transfection with foreign DNA and subsequent selection for potentially successful transformants very challenging.
This thesis explores techniques that address the study of these organisms in the absence of a workable genetic system. The first goal of this study was to examine the role of a collection of unique chlamydial proteins, known as the Inc-proteins, in the host-pathogen interactions. Inc proteins are localized on the inclusion membrane,
and are exposed to the cytosolic surface of this membrane. Such localization brings Inc proteins into direct contact with the cytoplasm of the host cell. To study the influence of Inc proteins on host cell biology we used the method of transient expression of genes from a eukaryotic expression plasmid, followed by the analysis of the resulting host cell phenotype. These experiments were conducted in both infected and uninfected cells.
It was demonstrated that expression of Chlamydophila (Chlamydia) caviae IncA protects host cells from infection with C. caviae by blocking the development of chlamydiae inside of the host cells. This effect of C. caviae IncA was specific, because the expression of the homologous IncA protein from Chlamydia trachomatis has no effect on the development of C. trachomatis or C. caviae.
Using the same approach it was shown that Inc proteins CT223, CT224 and CT225 from C. trachomatis each were capable of affecting the cell cycle of the host cells by blocking cytokinesis. This is significant, as other investigators have reported that infection with C. trachomatis leads to a similar phenotype. Transient expression of CT223, CT224, and CT225 affect host cell cytokinesis on the same manner as C. trachomatis infection. A statistically significant proportion of cells producing any of these three Inc proteins had a multinuclear phenotype and multiple centrosomes, indicating that cell division was blocked late in the cell cycle. This effect was specific for these particular Inc proteins and was observed both in murine and human cultured cells.
Many scientists are trying to develop methods for chlamydial mutagenesis and transformation. However, these attempts have not yet been successful, and the
delivery of DNA into chlamydiae is not the only obstacle. Even if chlamydiae are transformed, the successful transformants have to be effectively selected and segregated. To address the challenge of isolating individual candidate chlamydial transformants, we developed a novel technique for the rapid and productive separation of microbiological clones of chlamydiae. This was accomplished using a new method that is based on a unique feature of live chlamydiae to accumulate and retain the fluorescent dye C6-NBD-ceramide. This labeling results in clearly identifiable chlamydial inclusions, and thus, clearly identifiable infected cells. These labeled, infected, cells can then be separated from uninfected cells on a fluorescence activated cell sorter. The approach was used to isolate clonal populations of prototype strains from Chlamydia trachomatis, Chlamydia caviae, and Chlamydia suis. Recent clinical isolates were also successfully cloned. The procedure is simple and rapid, with single cloning cycles being completed 24h post-culture of a sample. As will be discussed, this technique has applications in both research and clinical settings. |
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