<p>Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted infections, employs a type III secretion (T3S) system to deliver effector proteins into host epithelial cells to establish a replicative vacuole. Although the temporal manner in which effectors are secreted is important for the proper manipulation of host cell functions, the mechanism remains a mystery. In this study, we provide several lines of evidence that T3S chaperones may impart coherence to effector secretion. In addition, we identified a new early T3S effector in Chlamydia. Aside from the phosphoprotein TARP, a Chlamydia effector that promotes actin re-arrangements, very few factors mediating bacterial entry and early inclusion establishment have been characterized. By defining proteins that associate with the three most abundant T3S chaperones, Slc1, Scc2 and Mcsc in invasive C. trachomatis elementary bodies (EB) by immunoprecipitation coupled with mass spectrometry, we identified Ct875, a new Slc1 client protein and T3S effector, which we renamed TepP (Translocated early phosphoprotein). We provide evidence that T3S effectors form stable complexes with Scl1 in vitro and that Slc1 enhances their T3S-dependent secretion in a heterologous Yersinia T3S system. We demonstrate that TepP is translocated early during bacterial entry into epithelial cells and is phosphorylated at tyrosine residues by host kinases. However, TepP phosphorylation occurs later than TARP, which together with the finding that Slc1 preferentially engages TARP in EBs leads us to postulate that these effectors are translocated into the host cell at different stages during C. trachomatis invasion. TepP co-immunoprecipitated with the scaffolding proteins CrkI-II during infection and Crk was recruited to nascent inclusions. Importantly, C. trachomatis mutants lacking TepP failed to recruit CrkI-II to inclusions, providing genetic confirmation of a direct role for this effector in the recruitment of a host factor. Finally, endocervical epithelial cells infected with a tepP mutant showed altered expression of a subset of genes associated with innate immune responses and lack of C. trachomatis-induced morphological changes. We propose a model wherein TepP acts downstream of TARP to recruit scaffolding proteins at entry sites to initiate and amplify signaling cascades important for the regulation of innate immune responses to Chlamydia.</p> / Dissertation

<p>The obligate intracellular pathogen, Chlamydia trachomatis, is becoming an ever greater public health threat worldwide. Despite aggressive public health awareness campaigns and treatment with antibiotics, chlamydial infections continue to be the most frequently reported sexually transmitted infection in the United States and the cause of 3% of worldwide blindness. While research into understanding various mechanisms of chlamydial pathogenesis is ongoing, efforts to identify critical protein targets are hampered by the lack of facile genetic manipulation systems available for Chlamydia. Without the ability to perform genetic studies, researchers have employed chemical biology tools to close the gap in understanding how Chlamydia survives and thrives in the host cell.</p><p>Chlamydial protease-like activity factor (CPAF) has been identified as a central virulence factor in chlamydial pathogenesis. Several studies have indicated a role for CPAF-mediated degradation of host proteins in the late stages of infection. CPAF is hypothesized to interfere with myriad host cell processes, including inflammation, cell proliferation, cytoskeletal development, and immunity presentation. However, recent studies have called into question the methods used to previously identify bona fide in vivo CPAF targets, as CPAF has been shown to retain proteolytic activity even in the presence of broad spectrum protease inhibitors. As a result of these new finding, there is a renewed call to carefully identify CPAF substrates using methods that ensure total inhibition of post-lysis proteolysis.</p><p>This dissertation aims to clarify the role of CPAF in chlamydial pathogenesis and to identify mechanisms by which CPAF exhibits substrate specificity. Because enzymes can manifest specificity through kinetic mechanisms, sequence recognition, secondary site substrate binding, or protein structure level specificity, multiple methods of biochemical characterization were employed to distinguish between these modes of specificity. </p><p>Optimized HPLC-based and fluorescence quenching assays were developed and used to investigate the chemical and kinetic mechanism of CPAF proteolysis, as well as to characterize CPAF resistance to broad spectrum protease inhibitors. Peptide library proteomics were designed to probe active site sequence recognition of specific amino acids. Bioinformatic approaches were used to recognize and annotate a cryptic PDZ-like domain in CPAF, which bears strong structural similarity to human epithelial tight junction proteins. Using a new endocervical cellular model of infection, a recently developed C. trachomatis mutant lacking CPAF activity was investigated. Mass spectrometry proteomics analysis was employed to detect differential cleavage of host proteins in endocervical cells infected with CPAF+ and CPAF- strains of C. trachomatis. Lastly, methods for N-terminal labeling and enrichment were adapted for further identifying CPAF substrates in a cellular infection model. The subtiligase system for biotinylation of N-terminal amines was adapted for integration with C. trachomatis infection assays and downstream mass spectrometry proteomics. Ultimately, the dissertation offers clarification of the role of CPAF in chlamydial infection and provides chemical biology tools for further study of protease function in bacterial pathogenesis.</p> / Dissertation

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

A mouse in vitro co-culture model between endometrial epithelial cells (EEC) and peripheral blood lymphocytes and monocytes (PBLM) immune cells was established, and Ct lipopolysaccharide (LPS) was added to the cells to mimic Ct bacterial infection and to stimulate an immune response. This model enabled us to study the cross-talk between EEC and PBLM and the physiological changes that occur in the endometrium upon Ct LPS stimulation. Results showed that EEC-PBLM co-culture and Ct LPS stimulation caused changes in transepithelial resistance (TER) as well as the expression and function of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel of epithelial cells. CFTR gene transcription was up-regulated at early hours after Ct LPS stimulation, while its channel activity was down-regulated at later hours. These results suggested the possible involvement of CFTR acting as a receptor for the internalization of Ct, which may ultimately lead to the disappearance of CFTR on the apical membrane. The EEC-PBLM co-culture showed that cross-talk was important for host defense in the endometrium. Direct cross-talk by cell-cell contact between EEC and PBLM was vital for immune cell survival as well as strengthening epithelials' barrier function. (Abstract shortened by UMI.) / Chlamydia trachomatis (Ct) infection is one of the most prevalent causes for sexually transmitted disease (STD) in the female reproductive tract. Ct is unique in that it is a bacterium, but infects and replicates like a virus inside a host cell. Ct infection can lead to a variety of reproductive diseases, such as pelvic inflammatory diseases (PID), tubule scarring, salpingitis, endometriosis, ectopic pregnancy and infertility. Effective immune defense in the uterus is necessary to eliminate these bacteria and to ensure optimal uterine environment for sperm motility, fertilization, and embryo implantation to occur. The immune system of the endometrium responds to Ct infection by the recruitment of many types of leukocytes, such as T-lymphocytes, B-lymphocytes, monocytes, macrophages and neutrophils, to the site of infection. Cross-talk between endometrial epithelial cells and immune cells may alter the activities of epithelial cells causing changes in channels function and anion secretion. / Ho Alice. / "August 2005." / Adviser: Chan Hsiao Chang. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3532. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 155-167). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2015-02-12T17:38:37Z No. of bitstreams: 2 Dissertação - Rafael Navarrete Fernandez - 2004.pdf: 737923 bytes, checksum: 9f9c7b498798d7f0e65f37b958ef6d70 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2015-02-12T17:38:59Z (GMT) No. of bitstreams: 2 Dissertação - Rafael Navarrete Fernandez - 2004.pdf: 737923 bytes, checksum: 9f9c7b498798d7f0e65f37b958ef6d70 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-02-12T17:39:02Z (GMT). No. of bitstreams: 2 Dissertação - Rafael Navarrete Fernandez - 2004.pdf: 737923 bytes, checksum: 9f9c7b498798d7f0e65f37b958ef6d70 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2004-04-20 / Introduction: Chlamydia trachomatis is the bacteria responsible for the most prevalent sexually transmitted disease worldwide. Most of the infections in men and women is asymptomatic and when undiagnosed and untreated may reach the joints causing not only arthritis, but also other acknowledged complications related to the female reproductive system. Objective: To investigate C. trachomatis DNA in the urine and synovial fluid from patients with spondyloarthropathies (SpA) and rheumatoid arthritis (RA) and evaluate serum anti-C. trachomatisIgG and IgM antibodies. Methods: The population consisted of 15 patients with spondyloarthropathies, being nine with undifferentiated spondyloarthropathy (US) and six with reactive arthritis (ReA) (group I), and 15 patients with rheumatoid arthritis (RA) (group II). The chlamydial DNA was assessed in synovial fluid and urine samples of all patients by Amplicor PCR. The anti-chlamydial IgG and IgM antibodies were quantified through indirect imunofluorescence (IIF), while 15 patients of group I were typed for HLA-B27 by the use of flow citometry. Social demographical data and all information on sexual behavior and presence of symptoms were collected through a (questionnaire in the form of) an interview. Results: C. trachomatis DNA was found in only one synovial fluid sample from patient with ReA (6,7%). In two patients with RA, chlamydial DNA was identified in the urine sample (13,3%). The anti-chlamydial IgG antibodies were present in eight patients of the population studied; being three patients from group I (20%), and five from group II (33,3%). The greatest titer of this antibody 1/256 was associated with the presence of chlamydial DNA in a patient from group II. The IgM antibody was not detected in any of the samples from both groups. Four individuals from group II (26,7%) were HLA-B27 positive and its presence was 16 related to sacroiliitis. Conclusion: The results in this study show that in patients with spondyloarthropathies and rheumatoid arthritis, presenting a picture of articular activity one might not exclude C. trachomatisas the triggering agent. / Introdução: A Chlamydia trachomatis é a bactéria responsável pela doença sexualmente transmissível mais prevalente no mundo. A maioria das infecções em homens e mulheres é assintomática e, quando não diagnosticada e tratada, pode alcançar as articulações e causar artrite, isto sem mencionar outras complicações conhecidas relacionadas ao aparelho reprodutor feminino. Objetivos: Pesquisar o DNA de C. trachomatis no líquido sinovial e urina, em pacientes com espondiloartropatias e artrite reumatóide (AR) e avaliar a presença de anticorpos séricos IgG e IgM anti-C. trachomatis nestes dois grupos de doenças. Identificar o antígeno HLA-B27 em pacientes com espondiloartropatias. Metodologia: A população do estudo consistiu de 15 pacientes com espondiloartropatias: nove com espondiloartropatia indiferenciada (EI) e seis com artrite reativa (ARe) (grupo I) e 15 pacientes com AR (grupo II). O DNA clamidial foi pesquisado em amostras de líquido sinovial e urina de todos eles empregandose a PCR (Amplicor Roche). Os anticorpos IgG e IgM anti-clamidiais foram quantificados por imunofluorescência indireta (IFI), enquanto o HLA-B27 foi tipado em 15 pacientes do grupo I por citometria de fluxo. Os dados sócio-demográficos, de comportamento sexual e presença de sintomas foram obtidos através de questionário na forma de entrevista. Resultados: O DNA da C. trachomatis foi evidenciado apenas em uma amostra de líquido sinovial do grupo I (6,7%), sendo o paciente portador de ARe. Em dois pacientes com AR, o DNA de clamidial foi identificado na urina (13,3%). Os anticorpos IgG anti-clamidiais estavam presentes em oito pacientes da população estudada, três do grupo I (20%) e cinco do grupo II (33,3%). O maior título desse anticorpo (1/256) associou-se com a presença do DNA clamidial na urina de um paciente do grupo II. O anticorpo IgM não foi detectado em nenhuma amostra 14 dos dois grupos. O antígeno HLA-B27 foi positivo em quatro indivíduos do grupo II (26,7%) e sua presença relacionou-se com sacroiliite. Conclusões: Os resultados desse estudo indicam que em portadores de espondiloartropatias e artrite reumatóide, com quadro articular em atividade, a C. trachomatisnão pode ser excluída como agente desencadeador.

The purpose of this study was to investigate the association between Chlamydia trachomatis reinfection rates of Puerto Rican adolescents and failure to follow the retesting protocol, failure of sexual partners to receive treatment, and failure to participate in the sexual orientation program about risk factors. Secondary data analysis, from a historical prospective study from the Health Department of Puerto Rico, was used in this study. Data analysis was restricted to adolescents 15 to 19-years-old who had a positive chlamydia result and reinfection pattern since January 2008 through December 2012. Multiple logistic regression analyses were run to predict Chlamydia trachomatis reinfection. Results showed a statistically significant association association between Chlamydia trachomatis reinfection and not having followed the retesting protocol (OR=1.243, 95% CI 1.089-2.930, p-value 0.038). A statistically significant association association was found between Chlamydia trachomatis reinfection and sexual partners having not received treatment (OR=1.713, 95% CI 0.761-2.024, p-value 0.029). A statistically significant association was found between Chlamydia trachomatis reinfection and having not participated in the Puerto Rico Department of Health's sexual orientation program (OR=1.243, 95% CI 0.762-2.026, p-value 0.034). The contribution to social change is identifying factors significantly associated with Chlamydia trachomatis reinfection. Study findings provide useful guidance for clinicians and public health professionals on how to reduce Chlamydia trachomatis reinfection rates among at risk Puerto Rican adolescents.

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. / Graduation date: 2003

Amino acid biosynthesis is an essential process in living organisms. Certain amino acids can be synthesized by some organisms but not by others. L-Lysine is one of the essential amino acids that bacteria can synthesize but humans cannot. This is somewhat inconvenient for humans as much of their L-lysine must come from their diet. However, the lack of the lysine biosynthetic pathway in humans makes the bacterial enzymes within the pathway attractive drug targets. Recently, a novel lysine biosynthetic pathway was discovered in plants, Chlamydiae and some archaea. It is called the diaminopimelate aminotransferase (DAP-AT) pathway. In this pathway, LL-DAP-AT plays a key role by directly converting L-tetrahydrodipicolinate to LL-DAP in a single step. This is a quite interesting characteristic of LL-DAP-AT as the above conversion takes three sequential enzymatic steps in the previously known lysine biosynthetic pathways. Due to its absence in humans, LL-DAP-AT would be an attractive target for the development of novel antibiotics. In order to understand the catalytic mechanism and substrate recognition of LL-DAP-AT, the structural characterization of LL-DAP-AT is of paramount importance. In this thesis, the overall architecture of LL-DAP-AT and its substrate recognition mechanism revealed by the crystal structures of LL-DAP-AT from Arabidopsis thaliana and Chlamydia trachomatis will be discussed. The crystal structure of the native LL-DAP-AT from A. thaliana (AtDAP-AT) presented in this thesis is the first structure of LL-DAP-AT to be determined. This structure revealed that LL-DAP-AT forms a functional homodimer and belongs to the type I fold family of PLP dependent aminotransferases. The subsequent determination of the substrate-bound AtDAP-AT structure showed how the two substrates, (LL-DAP and L-Glu) significantly different in size, are recognized by the same set of residues without significant conformational changes in the backbone structure. In addition, the LL-DAP-bound AtDAP-AT structure shows that the C-amino group of LL-DAP is recognized stereospecifically by the active site residues that are unique to the family of LL-DAP-AT enzymes. Lastly, the chlamydial LL-DAP-AT presented in this thesis shows a new open conformation for LL-DAP-AT. The implications of the conformational flexibility of CtDAP-AT on the differences in substrate specificities among LL-DAP-AT are discussed.

A general aim was to combine molecular typing methods with clinical background information to increase epidemiological knowledge about Chlamydia trachomatis infections. An outbreak of Lymfogranuloma venereum (LGV), caused by a more invasive variant of C. trachomatis, was reported from the Netherlands in 2003 among men who have sex with men (MSM). All Chlamydia positive specimens from a venereal disease clinic for MSM in Stockholm during one year were genotyped. No spread of LGV was found, apart from three symptomatic cases. The same ompA genotypes were found among MSM in Melbourne, but the genotype distribution was different compared to findings among the heterosexual population in Sweden. The standard method for genotyping of Chlamydia is ompA-sequencing, but it has low resolution because one genotype predominates. A multilocus sequence typing (MLST) system based on five targets was developed. In a sample of 47 specimens, 32 variants were found with MLST, but only 12 variants with ompA-sequencing. The polymorphisms in the hctB gene, one MLST target, are caused by an element of 108 bp that is present in two to four repetitions and in different variants. Although the DNA-binding function of Hc2 that is encoded by hctB has been studied, our findings of a considerable size variation show that new studies are needed. In 2006, specimens with a 377 bp deletion in the cryptic plasmid covering the target region for diagnostic test systems from Abbott and Roche were discovered in Sweden. Applying MLST to these specimens indicated that there was a single clone, denoted nvCT. The proportion of nvCT in all detected Chlamydia cases was higher (20% to 65%) in counties using Abbott/Roche compared to counties using the BectonDickinson test system (7% to 20%). The proportions of nvCT converge in counties with high or low levels when detection systems were adjusted to detect nvCT.

Thesis (M. Med. Sc.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves 35-42).