Chlamydia trachomatis is a bimorphic, obligate intracellular parasite that develops entirely within a membrane-bound vacuole. Chlamydiae manipulate eukaryotic hosts by translocating effector proteins into the host cell. Chlamydia genomes encode a type III secretion system (T3SS) which is a likely mechanism for delivery of these effectors. The infectious chlamydial elementary body (EB) is metabolically inactive yet possesses a functional T3S apparatus capable of translocating effector proteins into the host cell to facilitate invasion and other early-cycle events. For example, the T3S-dependent chlamydial substrate Translocated actin-recruiting phosphoprotein (Tarp) is present in infected cells as early as 5 minutes post infection, and it is likely that EBs translocate other proteins in a T3S-dependent manner. Evidence is presented herein that the C. trachomatis protein CT694 represents an early-cycle-associated effector protein. CT694 is secreted by the heterologous Yersinia T3SS and immunodetection studies of infected HeLa cultures indicate that CT694-specific signal accumulates directly adjacent to, but not completely overlapping with EBs during invasion. Yeast two hybrid analyses revealed an interaction of CT694 with the repeat region and C-terminus of human AHNAK and the C-terminus of AHNAK2. Immunolocalization studies of CT694 ectopically expressed in HeLa cells were consistent with an interaction with endogenous AHNAK. In addition, expression of CT694 in HeLa cells resulted in alterations in the detection of stress fibers that correlated with the ability of CT694 to interact with AHNAK. Domain differentiation studies indicated that the C-terminus of CT694 is required to interact with AHNAK and a domain within the N-terminus localizes CT694 to the plasma membrane and triggers aberrant morphological changes in HeLa cells. CT694 function is conserved in other C. trachomatis serovars and C. muridarum, but not by similarly positioned genes of C. pneumoniae or C. caviae. These data indicate that CT694 is a novel T3S-dependent substrate unique to C. trachomatis, and that its interaction with host proteins such as AHNAK may be important for aspects of invasion or development unique to this species.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2009. / Härtill 4 uppsatser.

Background: Annual screening of sexually active women aged 15 -24 years for Chlamydia trachomatis has been widely advocated. Goal: Examine relationships between physician characteristics and reported rates of sexual history taking, testing patients for chlamydia, and contact tracing activities taken. Study Design: Two hundred and forty one physicians in Nova Scotia completed a mailed questionnaire. Results: Contextual factors associated with the patient visit, demographic characteristics of the physician, and their beliefs, attitudes and perceptions were associated with rates of sexual history taking, and of annually testing for chlamydia. Further, physicians in Nova Scotia did not perform as well as might be expected with respect to contact tracing activities. Conclusion: The frequency of sexual history taking and patient testing were below recommended levels. Both modifiable and non-modifiable characteristics held by physicians were associated with these reported rates. Physicians may be failing to adequately assess and manage patients infected with chlamydia.

The study mainly focuses on the role of dendritic cells (DCs)，especially DC subsets, in modulating intracellular bacterial infections and the modulating effect of these infections on allergic responses. Specifically, it investigated the molecular mechanisms of 1) mycobacterial infection, 2) chlamydia infection and 3) the effect of these infections on allergic reactions. The results demonstrated that Bacillus Calmette-Guérin (BCG) vaccination promoted the expansion of CD8+DCs, which showed a more mature phenotype and produced more IL-12 compared to CD8-DCs. In vivo BCG-primed CD8+DC, but not CD8-DC, was the dominant DC subset in inducing protective immunity against BCG infection. Further, the results showed that adoptive transfer of different DC subsets from BCG-infected mice inhibited OVA-induced allergic reactions through immune deviation and immune regulation mechanisms. In addition, the results showed that CD8+DCs primed by Chlamydia, another intracellular bacterial pathogen, inhibited the allergic responses through IL-10 dependent pathway. Further, the data demonstrated that IL-10 inhibited the expansion of protective Th17 cells following Chlamydia infection. DCs from IL-10 KO mice showed significantly higher ICOS ligand (ICOS-L) expression compared to WT mice. Th17 from Chlamydia infected mice, but not Th1 cells, expressed high levels of ICOS. Blockade of ICOS/ICOSL signaling virtually abolished the Th17 promoting effect of DCs from IL-10 KO mice but had no significant effect on Th1 cells. This study provided new knowledge on the immune regulation of DC, especially its subsets, which may be helpful for the rational development of new preventive or therapeutic methods to tuberculosis / Chlamydia infections and allergic diseases.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections worldwide and it has been linked to increased risks of cervical cancer. Our study aims to understand the mechanisms by which chlamydia infection contributes to higher risks of tumorigenesis. We discovered in over 90% of infected human epithelial cells that chlamydia inclusions localized to the host cell centre during mitosis and prevented the completion of cleavage furrow ingression. Infected cells that failed to divide into two daughter cells re-entered interphase as tetraploid multinuclear cells, which are known initiators of tumorigenesis. In order to address whether chlamydia actively sought the host cell centre during mitosis, we created vacuoles using internalized latex beads and our findings indicated that chlamydia inclusions localized to the host cell centre much more frequently than similarly sized vacuoles. In addition, we demonstrated that metabolically inactive chlamydia inclusions localized to the cell centre less frequently than inclusions under normal conditions. Together, these results suggested that chlamydia actively localized to the host cell centre to efficiently block host cell mitosis. We took advantage of the microtubule-displacing capability of the C. trachomatis inclusions and demonstrated that astral microtubules could promote furrow initiation independent of the central spindle microtubules. Microdomains on chlamydia inclusions play important roles in inclusion localization and chlamydia replication. We discovered that these structures were surprisingly resistant to bacterial protein synthesis and host Src family kinase inhibitors. Finally, we demonstrated that multinuclear cells that resulted from unsuccessful mitosis contained significantly higher Golgi content, an important nutrient source for chlamydia. Our results indicated that C. trachomatis in multinuclear cells intercepted Golgi-derived lipids faster than in mononuclear cells. Together, my results reveal that C. trachomatis inclusions robustly localize to the cell centre to block host cell mitosis in order to acquire host Golgi nutrients more quickly in multinucleated cells.

The study mainly focuses on the role of dendritic cells (DCs)，especially DC subsets, in modulating intracellular bacterial infections and the modulating effect of these infections on allergic responses. Specifically, it investigated the molecular mechanisms of 1) mycobacterial infection, 2) chlamydia infection and 3) the effect of these infections on allergic reactions. The results demonstrated that Bacillus Calmette-Guérin (BCG) vaccination promoted the expansion of CD8+DCs, which showed a more mature phenotype and produced more IL-12 compared to CD8-DCs. In vivo BCG-primed CD8+DC, but not CD8-DC, was the dominant DC subset in inducing protective immunity against BCG infection. Further, the results showed that adoptive transfer of different DC subsets from BCG-infected mice inhibited OVA-induced allergic reactions through immune deviation and immune regulation mechanisms. In addition, the results showed that CD8+DCs primed by Chlamydia, another intracellular bacterial pathogen, inhibited the allergic responses through IL-10 dependent pathway. Further, the data demonstrated that IL-10 inhibited the expansion of protective Th17 cells following Chlamydia infection. DCs from IL-10 KO mice showed significantly higher ICOS ligand (ICOS-L) expression compared to WT mice. Th17 from Chlamydia infected mice, but not Th1 cells, expressed high levels of ICOS. Blockade of ICOS/ICOSL signaling virtually abolished the Th17 promoting effect of DCs from IL-10 KO mice but had no significant effect on Th1 cells. This study provided new knowledge on the immune regulation of DC, especially its subsets, which may be helpful for the rational development of new preventive or therapeutic methods to tuberculosis / Chlamydia infections and allergic diseases.

Charité, Universiẗat-Med., Diss--Berlin, 2005.

Universiẗat, Diss--Ulm, 2005.

Konstanz, Univ., Diss., 2007.

Proefschrift Universiteit van Amsterdam. / Met samenvatting in het Nederlands.