The role of chlamydial inclusion membrane proteins in host-pathogen interaction and the development of novel methods for studying chlamydial biology

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

Cytokinesis is blocked in mammalian cells transfected with Chlamydia trachomatis gene CT223

<p>Abstract</p> <p>Background</p> <p>The chlamydiae alter many aspects of host cell biology, including the division process, but the molecular biology of these alterations remains poorly characterized. Chlamydial inclusion membrane proteins (Incs) are likely candidates for direct interactions with host cell cytosolic proteins, as they are secreted to the inclusion membrane and exposed to the cytosol. The <it>inc </it>gene <it>CT223 </it>is one of a sequential set of orfs that encode or are predicted to encode Inc proteins. CT223p is localized to the inclusion membrane in all tested <it>C. trachomatis </it>serovars.</p> <p>Results</p> <p>A plasmid transfection approach was used to examine the function of the product of <it>CT223 </it>and other Inc proteins within uninfected mammalian cells. Fluorescence microscopy was used to demonstrate that <it>CT223</it>, and, to a lesser extent, adjacent <it>inc </it>genes, are capable of blocking host cell cytokinesis and facilitating centromere supranumeracy defects seen by others in chlamydiae-infected cells. Both phenotypes were associated with transfection of plasmids encoding the carboxy-terminal tail of CT223p, a region of the protein that is likely exposed to the cytosol in infected cells.</p> <p>Conclusion</p> <p>These studies suggest that certain Inc proteins block cytokinesis in <it>C. trachomatis</it>-infected cells. These results are consistent with the work of others showing chlamydial inhibition of host cell cytokinesis.</p

Biochemical characterization of diverse Stat5b-binding enhancers that mediate growth hormone-activated insulin-like growth factor-I gene transcription.

Many of the biological effects of growth hormone (GH) are mediated by insulin-like growth factor I (IGF-I), a 70-amino acid secreted peptide whose gene expression is rapidly induced by GH via the Stat5b transcription factor. We previously identified multiple evolutionarily conserved GH-activated chromosomal binding domains for Stat5b within the rat Igf1 locus, and proposed that they could regulate IGF-I gene activity. Here we investigate the biochemical and functional characteristics of these putative long-range transcriptional enhancers. Each element contained 2 or 3 individual Stat5b recognition sequences that could bind Stat5b in vitro, but with affinities that varied over a >100-fold range. Full transcriptional responsiveness to GH required that all Stat5b sites be intact within an individual enhancer. Replacement of a single lower-affinity Stat5b sequence with a higher-affinity one increased in vitro binding of Stat5b, and boosted transcriptional potency of the entire element to GH. As enhanced transcriptional activity involved changes in only one or two nucleotides within an enhancer DNA segment, there appears to be remarkable specificity and sensitivity in the ability of Stat5b to transform DNA binding activity into transcriptional function. Stat5b was able to stimulate the transcriptional activity of two enhancers in the absence of GH, indicating that individual Stat5b-regulated elements possess distinct functional features. We conclude that combinatorial interplay among multiple Stat5b-binding response elements with distinguishable biochemical properties is responsible for highly regulated control of IGF-I gene activity by GH

Development of Secondary Inclusions in Cells Infected by Chlamydia trachomatis

The chlamydiae are obligate intracellular bacteria that occupy a nonacidified vacuole (the inclusion) during their entire developmental cycle. These bacteria produce a set of proteins (Inc proteins) that localize to the surface of the inclusion within infected cells. Chlamydia trachomatis IncA is also commonly found in long fibers that extend away from the inclusion. We used standard and confocal immunofluorescence microscopy to demonstrate that these fibers extend to newly developed inclusions, termed secondary inclusions, within infected cells. Secondary inclusions observed at early time points postinfection were devoid of chlamydial reticulate bodies. Later in the developmental cycle, secondary inclusions containing variable numbers of reticulate bodies were common. Reticulate bodies were also observed within the IncA-laden fibers connecting primary and secondary inclusions. Quantitative differences in secondary inclusion formation were found among clinical isolates, and these differences were associated with serovar. Isolates of serovar G consistently produced secondary inclusions at the highest frequency (P < 0.0001). Similar quantitative studies demonstrated that secondary inclusion formation was associated with segregation of inclusions to daughter cells following cytokinesis. We conclude that the production of secondary inclusions via IncA-laden fibers allows chlamydiae to generate an expanded intracellular niche in which they can grow and may provide a means for continuous infection within progeny cells following cell division

Assessing binding affinity of Stat 5b for individual DNA sites.

<p>Quantitative DNA-protein binding was assessed by gel-mobility shift experiments as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050278#s2" target="_blank">Materials and Methods</a>’ using varying concentrations of Cy5.5-labeled double-stranded oligonucleotides for R58, R34, or R35, and 1 µg of nuclear protein from Cos-7 cells transfected with expression plasmids for the mouse GH receptor and wild-type rat Stat5b, and incubated with rat GH [40 nM] for 1 h. DNA binding was quantified with a LiCoR Odyssey infrared scanner and v3.0 analysis software, and results were plotted as shown. <u>Left panels</u>: representative results from individual experiments using nuclear proteins from cells expressing the mouse GH receptor and wild-type Stat5b after GH treatment. FP = unbound probe. The arrow indicates protein-DNA complexes. <u>Right panels</u>: binding curves with Kds listed (mean ± S.E., n = 3 experiments).</p

Stat5b binding elements in the rat <i>Igf1</i> locus confer GH-responsiveness to <i>Igf1</i> promoter 2 in promoter-reporter assays. A

<p>. Diagram of the rat <i>Igf1</i> locus showing seven conserved Stat5b binding elements, R2–4, R8–9, R13, R34–35, R53–54, R57–59, and R60–61. Each element is depicted as a gray oval and the six <i>Igf1</i> exons are shown as black boxes. <b>B</b>. <u>Top</u> - schematic of luciferase reporter plasmids containing rat <i>Igf1</i> promoter 2 (P2) and exon 2, and individual Stat5b binding elements (Enhancer). <u>Bottom</u> - Results of luciferase activity assays in Cos-7 cells transiently transfected with expression plasmids encoding the mouse GH receptor and rat Stat5b and reporter plasmids containing each putative enhancer region diagramed to the <u>left</u> (with each Stat5b site indicated as a white oval, and R13.5 as a gray curved shape), after incubation of cells with vehicle (dark bars) or rat GH [40 nM] (light bars) for 18 h. The graph presents results of 4 independent experiments for each promoter plasmid (mean ± S.E.; *, p<0.01; **, p<0.001 <i>vs.</i> R34–35 without GH [unpaired t-test]). Other p values are indicated, and compare ± GH treatment [paired t-test]. Luciferase counts for R34–35 without GH ranged from 3.5 to 5.5×10³ relative light units/sec.</p

DNA Sequences of Oligonucleotide Probes [core Stat5b binding site underlined].

<p>DNA Sequences of Oligonucleotide Probes [core Stat5b binding site underlined].</p

Stat5b binding sites are required to confer GH-responsiveness to <i>Igf1</i> promoter 2 in promoter-reporter assays.

<p>Results of luciferase assays in Cos-7 cells transiently transfected with reporter plasmids containing <i>Igf1</i> P2 and exon 2, plus wild type (WT) or mutated versions of individual Stat5b binding elements, and expression plasmids encoding the mouse GH receptor and rat Stat5b, and incubated with vehicle (dark bars) or rat GH [40 nM] (light bars) for 18 h. KO signifies knockout of a Stat5b binding site, with DKO representing double knockout and TKO, triple knockout. See ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050278#s2" target="_blank">Materials and Methods</a>’ for details. Bars represent the mean ± S.E. of 4–10 independent experiments (*, p<0.007; **, p<0.0007; #, p<0.017; ##, p<0.0017; ^∧, p<0.013 <i>vs.</i> WT with GH [unpaired t-test]). In each graph, relative luciferase values obtained using the WT Stat5b element in the absence of GH were set to 1. <b>A</b>. R2–4. <b>B</b>. R13. <b>C</b>. R34–35. <b>D</b>. R53–54. <b>E</b>. R57–59. <b>F</b>. R60–61.</p