IBIAPINA, Flávio Lúcio Pontes. Infecção genital por Clhamydia trachomatis em gestantes : prevalência e fatores associados. 2008. 73 f. Dissertação (Mestrado em Tocoginecologia) - Universidade Federal Ceará. Faculdade de Medicina, 2008. / Submitted by denise santos (denise.santos@ufc.br) on 2012-10-22T13:18:52Z No. of bitstreams: 1 2008_dis_flpibiapina.pdf: 217559 bytes, checksum: c74eced6e1e5dd1db0e1078e37ef23d1 (MD5) / Approved for entry into archive by Erika Fernandes(erikaleitefernandes@gmail.com) on 2012-10-26T15:11:52Z (GMT) No. of bitstreams: 1 2008_dis_flpibiapina.pdf: 217559 bytes, checksum: c74eced6e1e5dd1db0e1078e37ef23d1 (MD5) / Made available in DSpace on 2012-10-26T15:11:52Z (GMT). No. of bitstreams: 1 2008_dis_flpibiapina.pdf: 217559 bytes, checksum: c74eced6e1e5dd1db0e1078e37ef23d1 (MD5) Previous issue date: 2008 / To estimate the prevalence of Chlamydia trachomatis in pregnant women, comparing the positive group to the negative one in respect to socio-demographic factors, gynecologic history and exam, evaluating the risk factors associated to Chlamydia trachomatis genital infection. Subjects and methods: Hybrid capture test for Chlamydia trachomatis was performed in 446 pregnant women at Hospital Geral Dr César Cals, from the Health Secretary of the State of Ceará, from August, 2003 to May, 2004. Medium age in the group was 25.98 years and 19 weeks was the medium age of pregnancy. A structured questionnaire was applied, no matter the age of pregnancy, whether they were or not symptomatic, excluding those who had used antibiotics or any other substance into the vagina, during the previous fifteen days or who had kept sexual relationship until two days before the consultation, with a endocervical swab being performed, in order to have a hybrid capture test for the presence of Chlamydia trachomatis, as indicated by the manufacturer. Data were analysed by STATA 13.0, performed by means of the qui-square and logistic regression tests with descriptive and analytic presentation. Results: The prevalence of Chlamydia trachomatis among the pregnant women was 2.91%. Risk factors independently associated to Chlamydia trachomatis genital infection were history of pelvic pain or pelvic inflammatory disease, vulvar discharge and not using condom with an eventual sex partner. Respective odds ratio and confidence intervals were calculated to these variable. Conclusions: The positive group was younger, had smaller salaries and presented a greater frequency of divorced women, with less preservative use and more positive history of genital discharge and pelvic pain in the past. The OR to the presence of Chlamydia trachomatis genital infection was 1, 7 for those women not using condom and 0, 10 and 0, 17, respectively for a negative history of pelvic pain / PID, and the absence of vulvar discharge. / determinar a prevalência de infecção genital por Chlamydia trachomatis em gestantes, comparando o subgrupo com diagnóstico positivo com o de diagnóstico negativo quanto aos fatores bio-sócio-demográficos, história ginecológica e exame físico ginecológico, avaliando-se os fatores associados à presença de infecção genital por Chlamydia trachomatis. Sujeitos e métodos: submeteram-se ao teste de captura híbrida para Chlamydia trachomatis 446 gestantes do ambulatório de pré-natal do Hospital Geral Dr César Cals, da Secretaria Estadual de Saúde-Ceará, no período de Agosto de 2003 a Maio de 2004. A idade média do grupo foi de 25,98 anos, idade gestacional média de 19 semanas. Aplicou-se questionário diretamente às gestantes, independente da idade gestacional e de estarem sintomáticas ou não, excluindo-se aquelas que tivessem feito uso de antibióticos ou de qualquer substância química intravaginal nos quinze dias anteriores à coleta, ou que tivessem mantido relações sexuais nos dois dias anteriores à consulta de pré-natal, com coleta de swab endocervical para realização de teste de captura híbrida II, com material colhido em tubo com solução conservadora utilizando o sistema de micro placa, conforme procedimento descrito pelo fabricante. Os dados foram analisados utilizando o software STATA 13.0, procedendo-se análise descritiva e analítica através do teste de qui-quadrado e regressão logística, subtraindo-se variáveis. Resultados: A prevalência de Chlamydia trachomatis entre as gestantes foi de 2.91%. Identificou-se como fatores de risco independentemente associados à infecção genital por Chlamydia trachomatis a história de dor pélvica ou doença inflamatória pélvica, presença de corrimento vulvar ao exame físico e não uso de preservativo com parceiro eventual. Calculou-se o Odds-ratio (OR), para cada um destes fatores, com respectivos intervalos de confiança. Conclusões: O subgrupo com rastreamento positivo para Chlamydia trachomatis caracterizou-se por apresentar uma faixa etária e renda familiar menor que o subgrupo com sorologia negativa, além de apresentar maior frequencia de pacientes separadas, que usam menos preservativos com parceiros eventuais e com mais antecedentes de corrimento genital e dor pélvica. A OR para presença de infecção genital por Chlamydia trachomatis foi de 1,7 para aquelas que não usam preservativos e foi de 0,10 e 0,17, respectivamente, para ausência de dor pélvica/DIP e corrimento vulvar.

Chlamydia are Gram negative, obligate intracellular bacterial pathogens responsible for diseases that affect both animals and humans. Only two vaccines have been developed for Chlamydia, targeting C. felis and C. abortus, and development of antibiotic resistance and/or persistent infection forms has been documented for multiple species. Consequently, identification of new therapeutic targets is critical for prevention and treatment of chlamydial infections. These bacterial pathogens have a unique biphasic developmental cycle beginning with the infectious and environmentally stable elementary body, which enters a host cell and envelopes itself in the host membrane forming an inclusion. While residing in the inclusion, the EB transitions into the metabolically active and replicative reticulate body. The RB divides by binary fission before converting back into the EB and exiting the host cell by inclusion extrusion or cell lysis. The signals that initiate morphogenesis and the mechanism(s) mediating the transition between EB and RB forms are poorly understood. Eukaryote-like serine/threonine kinases (Hank’s type kinases) have recently been described to play major roles in cellular development and pathogenicity in prokaryotes. Chlamydia encode three Hank’s type kinase; Pkn1, PknD, and Pkn5. We hypothesize that these kinases control bacterial differentiation and metabolism by regulating protein activity via phosphorylation, making them potential targets for anti-chlamydial therapeutics. To aid in future efforts to elucidate the roles of these Hank’s type kinases in the physiology of C. trachomatis, my thesis developed protocols for the affinity purification of recombinant Pkn1 and Pkn5 and assessed the efficacy of a high-throughput kinase assay for Pkn1.

Made available in DSpace on 2016-08-29T15:34:59Z (GMT). No. of bitstreams: 1 tese_8350_Dissertação Renylena.pdf: 1497252 bytes, checksum: 3dde13e176aa101e14523497df431540 (MD5) Previous issue date: 2014-11-28 / O parto pretermo (PPT) é um dos principais determinantes da morbimortalidade neonatal, acarretando consequências adversas para saúde. As causas são multifatoriais, sendo a infecção intrauterina a razão mais provável para explicar a maioria destes desfechos. Acredita-se que a infecção por Chlamydia trachomatis (CT) também esteja envolvida no PPT e rotura prematura de membranas (ROPREMA). Objetivo: Determinar a prevalência de CT em parturientes e os possíveis fatores de risco relacionados com os casos de partos prematuros atendidos no Hospital Universitário Cassiano Antonio Moraes. Métodos: Estudo de corte transversal, realizado entre parturientes que apresentaram PPT em um Hospital Universitário em Vitória - ES, entre junho de 2012 e agosto de 2013. As participantes responderam a um questionário contendo dados sóciodemográficos, comportamentais e clínicos. Foi coletada uma amostra de urina para rastreio de CT usando reação em cadeia da polimerase. Resultados: A prevalência de PPT durante o período do estudo foi de 26%. Um total de 378 casos de PPT foram registrados, entre eles 323 mulheres foram testadas para o CT; quarenta e cinco (13,9%) tiveram um resultado positivo, sendo que 31,6% possuiam até 24 anos e as mulheres infectadas pela CT eram mais jovens do que as demais (p = 0,022). Um total de 76,2% eram casadas/em união estável, e CT foi mais frequente entre as solteiras (p = 0,018); 16,7% relataram primeira relação sexual com menos de 14 anos de idade. As causas de PPT foram materno-fetais em 40,9%, ROPREMA em 29,7% e trabalho de parto prematuro em 29,4%. Na análise multivariada, ser casada foi um fator de proteção [OR = 12:48 (IC 95%: 0,24-0,97)]. Nenhuma das demais características foram associadas com a infecção por CT. Conclusões: Este estudo evidencia uma alta prevalência de infecção por CT entre parturientes com PPT. Essa alta prevalência reforça a necessidade da definição de estratégias de rastreamento e assistência durante o pré-natal.

Made available in DSpace on 2014-06-12T15:49:33Z (GMT). No. of bitstreams: 2 arquivo1525_1.pdf: 3364712 bytes, checksum: 10443b95f96f92a8d3273aef9e3944b3 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2008 / A Chamydia trachomatis (C. trachomatis) é uma bactéria intracelular obrigatória, sexualmente transmitida, que causa doença inflamatória pélvica, gravidez ectópica e infertilidade. O papilomavírus humano (HPV) e a C. trachomatis são responsáveis por infecções sexualmente transmissíveis (ISTs) tipo-específicas, que infectam a pele, mucosas e o trato genital masculino e feminino, causando lesões, tanto benignas quanto malignas. A lectina ligadora de manose (MBL, Mannose-Binding Lectin) é uma proteína sérica da resposta imune inata capaz de ativar o sistema complemento e de se ligar a resíduos de carboidratos, inclusive a manose, em diferentes microrganismos. A deficiência ou polimorfismo gênico, dessa proteína, associa-se a um aumento da susceptibilidade a muitas doenças infecciosas. Os objetivos principais desse trabalho foram elaborar um protocolo para detecção da infecção por C. trachomatis e investigar a freqüência do polimorfismo no primeiro éxon do gene MBL2, correlacionando-os com a susceptibilidade à infecção por C. trachomatis, através da reação da PCR em Tempo Real (Real-Time Polimerase Chain Reaction), utilizando o SYBR® Green como fluoróforo. Para determinação do protocolo de identificação da C. Trachomatis foram utilizadas 98 amostras de secreção vaginal, oriundas de pacientes do Ambulatório Especializado da Mulher, da Prefeitura Municipal do Recife-PE, Brasil. O diagnóstico da infecção foi dado pela análise melting temperature assay. Aproximadamente, 14% (n=14) das amostras foram positivas para a infecção. Todas as amostras foram confirmadas através da análise em gel de agarose a 1%. Em relação à associação do polimorfismo do gene MBL2 entre os grupos infectados e saudáveis, os resultados demonstraram que não houve diferença estatística na freqüência dos alelos entre os grupos (64% vs. 74%) (36% vs. 26%), respectivamente, alelos A e 0, p-value = 0.3112), não sendo associado a um aumento na susceptibilidade à infecção pela C. trachomatis. Da mesma forma, quando comparadas às freqüências genotípicas, o genótipo OO , no grupo C. trachomatis-positivo, não mostrou diferença significativa em relação ao grupo controle, 6% vs. 3%, respectivamente. Além disso, apesar do genótipo AA , no grupo controle, apresentar uma freqüência maior em relação ao grupo C. trachomatis-positivo (47% vs. 33%), esta não foi significativa (p-value = 0.1430). Todas as amostras estavam em equilíbrio de Hardy-Weinberg (χ2=2,65). Conclui-se assim que com a metodologia proposta, pode-se facilmente detectar a presença de C. trachomatis, em amostras de secreção vaginal, e que a presença do alelo mutante O no gene da MBL2, não confere fator predisponente relevante para um aumento na susceptibilidade à infecção por C. trachomatis

The population for this study consisted of 4013 sexually active women seen for family planning. Culture for Chlamydia trachomatis yielded an isolation rate of 6.1%. Women aged 16 to 25 accounted for 81.7% of the C. trachomatis infections, while those younger than 16 or older than 35 accounted for only 2.4% of the infections. Of the 246 patients whose cultures were positive for C. trachomatis, 159 (65%) were asymptomatic. The incidence of C. trachomatis was 11.2% among those with symptoms but only 6.4% among the asymptomatic group. Among 63 patients with Neisseria gonorrhoeae (who were excluded from the study), 26 (41.3%) also were infected by C. trachomatis. There were no microbiologic drug failures with erythromycin or clindamycin. Of 56 patients who enrolled in the clindamycin arm of the protocol, 48 (85.7%) completed therapy and experienced microbiologic and clinical cures. In contrast, erythromycin therapy was completed by only 25 of 57 women (43.9%) enrolled. The number of side effect failures for erythromycin was 22 of 57 (38.6%). This was more than five times the number of side effect failures for clindamycin (4 of 56, or 7.1%).

Background: Chlamydia trachomatis is the commonest curable sexually transmitted infection in the UK. The prevalence is shared equally by men and women. A National Chlamydia Screening Programme (NCSP) has been introduced in England, supported by advances in testing technologies which enable non-invasive sampling methods to be used in non-healthcare settings. The NCSP tests nearly twice as many women as men and is more likely to test men in non-healthcare settings. Men are seen as an important, but difficult to reach group. Little is known about where men prefer to access testing and whether or not nontraditional settings, such as football clubs, are acceptable. Methods: 1) A national stratified random probability sample survey of men aged between 18 and 35 years resident in Great Britain, exploring attitudes to self-collected testing for Chlamydia, acceptability of venues to collect testing kits, health seeking and sexual risk behaviours. 2) Qualitative interviews with men who play amateur football. It explores the acceptability of three different, club-based, testing pathways; Health-care professional promoted; Peer-led promoted; and poster-led promoted. Results: Men are well engaged with existing health services and find selfcollected testing kits for Chlamydia highly acceptable. Healthcare settings are the most acceptable venues to access testing although sports settings are acceptable to a minority. Attitudes to testing in football clubs are influenced by factors relating to men’s characteristics, promoter characteristics and the impact of testing on time and effort involved. Conclusions: Whilst non-healthcare settings can be used to reach some men for Chlamydia testing, existing services are already well accessed and offer considerable opportunities to test more men. More should be done to ensure men are able to access testing within the context of daily living, without significantly impacting on the time needed to pursue their main interests.

The intracellular pathogen Chlamydia is the causative agent of millions of new infections per year transmitting diseases like trachoma, pelvic inflammatory disease or lymphogranuloma venereum. Undetected or recurrent infections caused by chlamydial persistence are especially likely to provoke severe pathologies. To ensure host cell survival and to facilitate long term infections Chlamydia induces anti-apoptotic pathways, mainly at the level of mitochondria, and restrains activity of pro-apoptotic proteins. Additionally, the pathogen seizes host energy, carbohydrates, amino acids, lipids and nucleotides to facilitate propagation of bacterial progeny and growth of the chlamydial inclusion. At the beginning of this study, Chlamydia-mediated apoptosis resistance to DNA damage induced by the topoisomerase inhibitor etoposide was investigated. In the course of this, a central cellular protein crucial for etoposide-mediated apoptosis, the tumour suppressor p53, was found to be downregulated during Chlamydia infections. Subsequently, different chlamydial strains and serovars were examined and p53 downregulation was ascertained to be a general feature during Chlamydia infections of human cells. Reduction of p53 protein level was established to be mediated by the PI3K-Akt signalling pathway, activation of the E3-ubiquitin ligase HDM2 and final degradation by the proteasome. Additionally, an intriguing discrepancy between infections of human and mouse cells was detected. Both activation of the PI3K-Akt pathway as well as degradation of p53 could not be observed in Chlamydia-infected mouse cells. Recently, production of reactive oxygen species (ROS) and damage to host cell DNA was reported to occur during Chlamydia infection. Thus, degradation of p53 strongly contributes to the anti-apoptotic environment crucial for chlamydial infection. To verify the importance of p53 degradation for chlamydial growth and development, p53 was stabilised and activated by the HDM2-inhibiting drug nutlin-3 and the DNA damage-inducing compound etoposide. Unexpectedly, chlamydial development was severely impaired and inclusion formation was defective. Completion of the chlamydial developmental cycle was prevented resulting in loss of infectivity. Intriguingly, removal of the p53 activating stimulus allowed formation of the bacterial inclusion and recovery of infectivity. A similar observation of growth recovery was made in infected cell lines deficient for p53. As bacterial growth and inclusion formation was strongly delayed in the presence of activated p53, p53-mediated inhibitory regulation of cellular metabolism was suspected to contribute to chlamydial growth defects. To verify this, glycolytic and pentose phosphate pathways were analysed revealing the importance of a functioning PPP for chlamydial growth. In addition, increased expression of glucose-6-phosphate dehydrogenase rescued chlamydial growth inhibition induced by activated p53. The rescuing effect was even more pronounced in p53-deficient cells treated with etoposide or nutlin-3 revealing additional p53-independent aspects of Chlamydia inhibition. Removal of ROS by anti-oxidant compounds was not sufficient to rescue chlamydial infectivity. Apparently, not only the anti-oxidant capacities of the PPP but also provision of precursors for nucleotide synthesis as well as contribution to DNA repair are important for successful chlamydial growth. Modulation of host cell signalling was previously reported for a number of pathogens. As formation of ROS and DNA damage are likely to occur during infections of intracellular bacteria, several strategies to manipulate the host and to inhibit induction of apoptosis were invented. Downregulation of the tumour suppressor p53 is a crucial point during development of Chlamydia, ensuring both host cell survival and metabolic support conducive to chlamydial growth. / Intrazellulär lebende Chlamydien führen jährlich zu Millionen an Neuinfektionen und lösen Krankheiten wie das Trachom, eine Entzündung des Auges, sowie entzündliche Beckenerkrankungen oder Lymphogranuloma venereum, eine venerische Lymphknotenentzündung, aus. Unentdeckte oder wiederkehrende Infektionen, ausgelöst durch chronisch persistierende Chlamydien, führen häufig zu schwerwiegenden Komplikationen. Um das Überleben der Wirtszelle und dauerhafte Infektionen zu ermöglichen, induzieren Chlamydien antiapoptotische Signalwege, hauptsächlich auf Höhe der Mitochondrien, und beeinträchtigen darüber hinaus die Aktivität proapoptotischer Proteine. Energie, Kohlenhydrate, Aminosäuren, Lipide und Nukleotide bezieht der Krankheitserreger vollständig aus der Wirtszelle. Erst dadurch wird sowohl die Vermehrung der Bakterien, als auch das Wachstum der chlamydialen Inklusion ermöglicht. Zu Beginn dieser Arbeit wurde die Chlamydien-vermittelte Resistenz gegenüber induziertem Zelltod nach Schädigung der DNA durch den Topoisomerase-Inhibitor Etoposid untersucht. Im Zuge dessen wurde entdeckt, dass der Tumorsuppressor p53, ein zentrales zelluläres Protein entscheidend für die Etoposid-induzierte Apoptose, während Chlamydien-Infektionen herunterreguliert wird. Nachdem verschiedene chlamydiale Stämme und Serovare untersucht wurden, konnte festgestellt werden, dass es sich bei der Herunterregulierung von p53 um ein allgemeines Merkmal chlamydialer Infektionen von humanen Zellen handelt. Die Reduzierung der Proteinmenge von p53 wird dabei durch den PI3K-Akt Signalweg, Aktivierung der E3-Ubiquitin-Ligase HDM2 und abschließendem Abbau durch das Proteasom vermittelt. Zusätzlich wurde ein interessanter Unterschied zwischen Infektionen humaner und muriner Zellen entdeckt. Sowohl Aktivierung des PI3K-Akt Weges, als auch der Abbau von p53 konnten in Chlamydien-infizierten Mauszellen nicht beobachtet werden. Kürzlich wurde darüber berichtet, dass während chlamydialer Infektionen reaktive Sauerstoffspezies produziert werden und die DNA der Wirtszelle geschädigt wird. Demnach trägt der Abbau von p53 entscheidend dazu bei, ein für chlamydiale Infektionen maßgebliches, anti-apoptotisch geprägtes Umfeld zu generieren. Um die Bedeutung des Abbaus von p53 für Wachstum und Entwicklung von Chlamydien zu ermessen, wurde p53 durch den HDM2-inhibierenden Wirkstoff Nutlin-3, sowie die DNA-Schäden induzierende Verbindung Etoposid stabilisiert bzw. aktiviert. Die Entwicklung der Chlamydien, sowie die Ausbildung der Inklusion wurden dadurch überraschenderweise stark beeinträchtigt bzw. waren fehlerhaft. Die Vollendung des chlamydialen Entwicklungszyklus wurde verhindert, was den Verlust der Infektivität nach sich zog. Interessanterweise erlaubte das Entfernen des p53-aktivierenden Stimulus die Ausbildung der bakteriellen Inklusion und die Wiedererlangung der Infektivität. Eine ähnliche Beobachtung konnte in Zelllinien mit einer p53-Defizienz gemacht werden. Da bakterielles Wachstum und Ausbildung der Inklusion durch aktiviertes p53 stark eingeschränkt war, wurde vermutet, dass p53-vermittelte Inhibierung des zellulären Metabolismus am fehlerhaften Wachstum der Chlamydien beteiligt ist. Analyse von Glykolyse und Pentosephosphatweg (PP-Weg) zeigten den Stellenwert eines funktionierenden PP-Wegs für das Wachstum der Chlamydien auf. Zusätzlich konnte durch Überexpression der Glucose-6-phosphat-Dehydrogenase das durch aktiviertes p53 gehemmte Wachstum der Chlamydien wiederhergestellt werden. Dieser Effekt war noch deutlicher in p53-defizienten Zellen, die mit Etoposid bzw. Nutlin-3 behandelt wurden. Demnach tragen auch p53-unabhängige Aspekte zur Einschränkung des chlamydialen Wachstums bei. Das Entfernen von reaktiven Sauerstoffspezies durch Antioxidationsmittel war jedoch nicht ausreichend zur Wiedererlangung der chlamydialen Infektivität. Demnach sind nicht nur die anti-oxidativen Eigenschaften des PP-Wegs sondern auch das Bereitstellen von Vorläufermolekülen für die Nukleotidsynthese, sowie dessen Beitrag zur DNA-Reparatur entscheidend für erfolgreiches Wachstum von Chlamydien. Veränderung der Signaltransduktion der Wirtszelle wurde bereits bei einigen Krankheitserregern nachgewiesen. Da reaktive Sauerstoffspezies und DNA Schäden häufig bei Infektionen intrazellulärer Bakterien auftreten, entstanden unterschiedliche Strategien, den Wirt zu manipulieren und das Einleiten des Zelltodes zu verhindern. Das Herunterregulieren des Tumorsuppressors p53 ist entscheidend während der Entwicklung von Chlamydien. Sowohl das Überleben der Wirtszelle, als auch die für chlamydiales Wachstum förderliche Unterstützung durch den Stoffwechsel werden dadurch gewährleistet.

Chlamydia trachomatis (Ctr), an obligate intracellular gram negative human pathogen, causes sexually transmitted diseases and acquired blindness in developing countries. The infectious elementary bodies (EB) of Ctr involved in adherence and invasion processes are critical for chlamydial infectivity and subsequent pathogenesis which requires cooperative interaction of several host cell factors. Few receptors have been known for this early event, yet the molecular mechanism of these receptors involvement throughout Ctr infection is not known. Chlamydial inclusion membrane serves as a signaling platform that coordinates Chlamydia-host cell interaction which encouraged me to look for host cell factors that associates with the inclusion membrane, using proteome analysis. The role of these factors in chlamydial replication was analyzed by RNA interference (RNAi) (in collaboration with AG Thomas Meyer). Interestingly, EphrinA2 receptor (EphA2), a cell surface tyrosine kinase receptor, implicated in many cancers, was identified as one of the potential candidates. Due to the presence of EphA2 in the Ctr inclusion proteome data, I investigated the role of EphA2 in Ctr infection. EphA2 was identified as a direct interacting receptor for adherence and entry of C. trachomatis. Pre-incubation of Ctr-EB with recombinant human EphA2, knockdown of EphA2 by siRNA, pretreatment of cells with anti-EphA2 antibodies or the tyrosine kinase inhibitor dasatinib significantly reduced Ctr infection. This marked reduction of Ctr infection was seen with both epithelial and endothelial cells used in this study. Ctr activates EphA2 upon infection and invades the cell together with the activated EphA2 receptor that interacts and activates PI3K survival signal, promoting chlamydial replication. EphA2 upregulation during infection is associated with Ctr inclusion membrane inside the cell and are prevented being translocated to the cell surface. Ephrins are natural ligands for Ephrin receptors that repress the activation of the PI3K/Akt pathway in a process called reverse signaling. Purified Ephrin-A1, a ligand of EphA2, strongly interferes with chlamydial infection and normal development, supporting the central role of these receptors in Chlamydia infection. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Ctr infection induces EphA2 upregulation and is mediated by activation of ERK signaling pathway. Interfering with EphA2 upregulation sensitizes Ctr-infected cells to apoptosis induced by tumor necrosis factor-alpha (TNF-α) suggesting the importance of intracellular EphA2 signaling. Collectively, these results revealed the first Ephrin receptor “EphA2” that functions in promoting chlamydial infection. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism how Chlamydia subverts the host cell and induces apoptosis resistance. By applying the natural ligand Ephrin-A1 and targeting EphA2 offers a promising new approach to interfere with Chlamydia infection. Thus, the work provides the evidence for a host cell surface tyrosine kinase receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate the chlamydial replication. / Chlamydia trachomatis (Ctr) ist ein obligat intrazellulär lebendes Gram negatives Bakterium, das Geschlechtskrankheiten verursachen kann. In Entwicklungsländern führt es zudem häufig zu erworbener Blindheit. Die infektiösen Elementarkörper (EB) sind für die Anheftung an die Wirtszelle sowie die Aufnahme von Ctr in die Wirtzelle verantwortlich. Dies ist ein wichtiger Schritt, da nur so die sich anschließende Krankheitsentwicklung stattfinden kann. Diese ist auch abhängig vom engen Zusammenspiel der Ctr Proteine mit den Wirtszellfaktoren. Obgleich dieser Schritt so wichtig ist, wurden erst wenige Wirtszellrezeptoren gefunden und welche Rolle diese Rezeptoren im weiteren Verlauf der Infektion spielen, ist noch nicht richtig verstanden. Die chlamydiale Inklusionsmembran fungiert als Signalplattform, die das Zusammenspiel von Chlamydien und Wirtszelle koordiniert. In dieser Arbeit wurden die Wirtszellproteine, die an der Inklusionsmembran lokalisiert sind, mit Hilfe einer Proteomanalyse identifiziert. Anschließend wurde die Rolle dieser Proteine bei der Chlamydienvermehrung in einem RNAi screen untersucht (in Zusammenarbeit mit der AG Thomas Meyer). Hier wurde überraschenderweise der EphrinA2 Rezeptor, eine sich auf der Oberfläche der Zellen befindliche Rezeptor Tyrosin Kinase, die vor allem mit Krebs in Verbindung gebracht wird, als ein potentieller Kandidat identifiziert. Da die Proteomdaten gezeigt haben, dass EphrinA2 an der Inklusionsmembran lokalisiert ist, wurde die Rolle von EphrinA2 während der Ctr Infektion hier näher untersucht. Es konnte gezeigt werden, dass EphrinA2 ein direkter Rezeptor für Ctr ist, der sowohl die Adhärenz als auch die Aufnahme von Ctr in die Wirtszelle bewerkstelligt. Vorinkubation von Ctr- EB mit rekombinantem menschlichen EphrinA2, das herunterregulieren von EphrinA2 mit Hilfe einer siRNA oder das Vorinkubieren der menschlichen Zelle mit Antikörpern gegen EphrinA2 oder dem Tyrosinkinase Inhibitor Dasatinib, reduzierten die Ctr Infektion signifikant. Diese drastische Reduktion der Ctr Infektion wurde sowohl in Epithelzellen als auch in Endothelzellen beobachtet. Ctr aktiviert EphrinA2 während der Infektion und invadiert die Wirtszelle zusammen mit dem aktivierten Rezeptor, dieser interagiert mit dem aktivierten PI3K Überlebenssignal, was die Replikation der Chlamydien ermöglicht. An der Inklusionsmembran akkumuliert EphrinA2, da der Transport von neuem Rezeptor zur Zellmembran unterbunden ist. Ephrine sind die natürlichen Liganden der Ephrinrezeptoren, sie unterdrücken die Aktivierung des PI3K/Akt Signalweges in einem Prozess, der reverse Signalübertragung genannt wird. Aufgereinigtes Ephrin-A1, ein Ligand des EphrinA2 Rezeptors, verhindert eine normale Chlamydieninfektion, was eine zentrale Rolle dieses Rezeptors weiterhin bestätigt. Die Überexpression von EphrinA2, erhöhte die PI3K Aktivierung und Ctr Infektion. Dies war nicht der Fall, wenn eine Mutante, der die intrazelluläre Domäne fehlt, überexprimiert wurde. Eine Ctr Infektion induziert die Hochregulierung von EphrinA2, welche durch die Aktivierung des ERK Signalwegs bewerkstelligt wird. Wenn die Hochregulierung von EphrinA2 verhindert wird, werden Ctr infizierte Zellen sensitiver für Apoptose induziert durch tumor necrosis factor-alpha (TNF-α), was ein weiter Hinweis für die Bedeutung der intrazellulären EphrinA2 Signalübermittlung ist. Insgesamt haben diese Ergebnisse den ersten Ephrin Rezeptor "EphA2" offenbart, der in der Förderung chlamydialer Infektionen fungiert. Hinzu kommt, dass die Bindung eines Oberflächenrezeptors an die Inklusionsmembran ein neuer Mechanismus ist, die Wirtszelle zu verändern und eine Apoptoseresistenz in der Zelle zu induzieren. Die Zugabe des natürlichen Liganden Ephrin-A1 eröffnet eine neue vielversprechende Möglichkeit Chlamydieninfektionen zu bekämpfen. Daher liefert diese Arbeit erste Hinweise, das eine Wirtszelltyrosinkinase, die sich an der Zelloberfläche befindet, notwendig ist für die Invasion und die intrazelluläre Signalübermittlung, welche für die chlamydiale Replikation notwendig ist, essentiell ist.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine.

Although both are caused by Chlamydia trachomatis, Lymphogranuloma Venereum (LGV) presents differently from the infections caused by Oculogenital (OG) strains. The endothelium of blood and lymph vessels allows passage of cells to the site of infection. Endothelial cells also secrete chemokines and cell adhesion molecules which act as attractants and binding sites for various cellular immune components. Since LGV biovar affect the lymphoid tissue we studied the effect of C. trachomatis on endothelial cells. Human umbilical vein endothelial cells (HUVEC) were infected with C. trachomatis LGV serovars L1, L2, L3 and the OG strain E at multiplicity of infection (MOI) of 1 and incubated for 24 hours. Stimulation of Interleukin-8 (IL-8) and monocyte chemokine protein-1 (MCP-1) chemokines and the intercellular adhesion molecule -1 (ICAM-1) were quantified by enzyme linked immunosorbent assays (ELISA). Transendothelial migration of neutrophils and monocytes was carried out in transwells. The lactate dehydrogenase (LDH) release assay was used to measure cell necrosis. Apoptotic cell death was analysed using the BioVisionTM CaspGLOW Fluorescein Caspase Staining Kit and DeadEndTM Colorimetric Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) system with the C. trachomatis Culture Confirmation kit as a counter stain. All Chlamydia trachomatis serovars (L1, L2, L3 and E) successfully infected and replicated in HUVEC after 24 hours of infection. Only L3 stimulated significantly higher production of IL-8, MCP-1 and ICAM-1 by HUVEC as compared to the negative control and mock-infected cells. However, the remaining LGV serovars (L1 and L2) and the OG serovar E showed no significant difference in the stimulation of IL-8, MCP-1 and ICAM-1 when compared to the controls. Comparison of LGV and OG serovars showed no significant difference between these two biovars in inducing production of IL-8 and MCP-1, but L3 stimulated ICAM-1 at a significantly higher level than E. There was no significant difference in the number of migrated neutrophils between untreated HUVEC, mock infected HUVEC and HUVEC infected with Chlamydia serovars. L2 and L3 had significally higher amount of migrated monocytes than the controls with L3 being the highest. L3 was the only serovar that had a significant level of cell death by necrosis. Apototic cells were observed in both uninfected and infected HUVEC which is due to normal cell turn over. None of the infected cells showed TUNEL positive nuclei. It can be concluded that L3 is more virulent than the other serovars during the first 24 hours of infection. Infection with C. trachomatis serovars does not seem to cause any cell death by apoptosis 24 hours post infection. The only cell death that occurs is by necrosis and only on serovar L3 infected cells. / Thesis (M.Med.)-University of KwaZulu-Natal, Durban, 2011.